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a2) United States Patent Gang et al. US 12,548,243 B2 US012548243B2 (10) Patent No.: (54) 3D-ORGANIZED NANOMATERIALS THROUGH DNA-PRESCRIBED AND VALENCE-CONTROLLED MATERIAL (71) Applicant: THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK, New York, NY (US) (72) Inventors: Oleg Gang, Setauket, NY (US); Ye Tian, Nanjing (CN); Jason S. Kahn, New York, NY (US); Yan Xiong, New York, NY (US): Brian Minevich, New York, NY (US); Sanat K. Kumar, New York, NY (US) (73) THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK, New York, NY (US) Assignee: Notice: Subject to any disclaimer, the term of this patent is extended or adjusted under 35 U.S.C. 154(b) by 310 days. @1) (22) Appl. No.: 17/070,643 Filed: Oct. 14, 2020 (65) Prior Publication Data US 2021/0264663 Al Aug. 26, 2021 Related U.S. Application Data Provisional application No. 62/914,792, filed on Oct. 14, 2019. (60) (51) Int. Ch. GIOB 15/10 BB2Y 30/00 CR 168 GO6T 17/00 400 (2019.01) (2011.01) (2018.01) (2006.01) (45) Date of Patent: Feb. 10, 2026 (52) US. CL CPC... . GO6T 17/00 (2013.01); C12 1/68 (2013.01); G16B 15/10 (2019.02), B82Y 30/00 (2013.01) (58) Field of Classification Search CPC ........ GO6T 17/00; C12Q 1/68; G16B 15/10; B82Y 30/00; C12N 15/11 See application file for complete search history. (56) References Cited U.S. PATENT DOCUMENTS 2005/0009079 AL 2016/0176988 AL 1/2005 Anders et al. 6/2016 Zhang et al. FOREIGN PATENT DOCUMENTS WO — WO 2005/034205 A2 4/2005 WO WO 2009/149091 Al — 12/2009 WO = WO 2016/109911 Al 7/2016 OTHER PUBLICATIONS Tian et al., Lattice engineering through nanoparticle-DNA frame- works, Nature Materials, Feb. 2016, 15, 654-661 (Year: 2016).* (Continued) Primary Examiner — Anne M. Gussow Assistant Examiner — Allisan E Schloop (74) Attorney, Agent, or Firm — Baker Botts L.L.P. (57) ABSTRACT The present subject matter relates to a voxel and methods of organizing an object into a three-dimensional (3D) array using the voxel. The voxel can include a plurality of frames including at least one single stranded (ss) DNA motif with at least one free base, wherein the at least one ssDNA motif hybridizes with a complementary strand fragment of other frames. 10 Claims, 78 Drawing Sheets Specification includes a Sequence Listing. Frame with valiace vy Mytyeist Bead
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US 12,548,243 B2 Page 2 (56) References Cited OTHER PUBLICATIONS Wang et al., A DNA Crystal Designed to Contain Two Molecules per Asymmetric Unit, Journal of the American Chemical Society, Oct. 2010, 132, 15471-15473 (Year: 2010).* Agarwal et al., “Mesophase behaviour of polyhedral particles.” Nat Mater 10:230-235 (2011). Baimuratov et al., “Quantum-dot supercrystals for future nanophoton- ics,” Scientific Reports 3:1727 (2013) 9 pgs. Chang et al., “High near-infrared photoluminescence quantum efficiency from PbS nanocrystals in polymer films,” Synthetic Metals 148:257-261 (2005). Chen et al., “Synthesis from DNA of a molecule with the connec- tivity of a cube,” Nature 350:631-633 (1991). Church et al. “Next-Generation Digital Information Storage in DNA,” Science 337:1628 (2012) 2 pgs. Damasceno et al., “Predictive Self-Assembly of Polyhedra into Complex Structures,” Science 337(6093):453-457 (2012). 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Lee et al., “A mechanical metamaterial made from a DNA hydrogel,” Nat. Nanotechnol. 7:816-820 (2012). Liu et al., “Diamond family of nanoparticle superlattices,” Science 351:582-586 (2016). Lu et al., “Superlattices assembled through shape-induced direc tional binding,” Nature Communications 6:6912 (2015) 10 pgs. Macfarlane et al., “Assembly and organization processes in DNA- directed colloidal crystallization,” PNAS 106(26): 10493-10498 (2009). Macfarlane et al., “Nanoparticle superlattice engineering with DNA,” Science 334:204-208 (2011). Wertheim, “Fluids with Highly Directional Attractive Forces. III. Multiple Attraction Sites,” J. Stat. Phys 42(3/4):459-476 (1986). Murray et al., “Synthesis and Characterization of Monodisperse Nanocrystals and Close-Packed Nanocrystal Assemblies,” Annual Review of Materials Science 30:545-610 (2000). Niemeyer et al., “DNA-Directed Assembly of Bienzymic Com- plexes from In Vivo Biotinylated NAD(P)H:FMN Oxidoreductase and Luciferase,” ChemBioChem, 02-03:242-245 (2002). Nykypanchuk et al., “DNA-guided crystallization of colloidal nanoparticles,” Nature 451:549-552 (2008). Oliver, J. Quantum Dots: Global Market Growth and Future Com- mercial Prospects, BCC Research NANO27E (2016). Pal et al., “DNA Directed Self-Assembly of Anisotropic Plasmonic Nanostructures,” Journal of the American Chemical Society 133(44):17606-17609 (2011). Park et al., “DNA-programmable nanoparticle crystallization,” Nature 451:553-556 (2008), PI: Hao Yan, Awardee Organization: Arizona State University, “DNA Directed Self: Assembly of Multicomponent Nanoarchitectures,” NSF Grant #: 0545652 (2006). PI: Thom LaBean, Awardee Organization: North Carolina State University, “Collaborative Research: Photonic and Electronic Devices Based on Self-Assembling DNA Templates,” NSF Grant #: 1608847 (2016) Qietal., “DNA-directed self-assembly of shape-controlled hydrogels,” Nature Communications 4:2275-2285 (2013). Rogers et al., “Direct measurements of DNA-mediated colloidal interactions and their quantitative modeling,” PNAS 108(38):15687- 15692 (2011). Rothemund et al., “Folding DNA to create nanoscale shapes and patterns,” Nature 440:297-302 (2006). Seeman, “DNA in a material world,” Nature 421:427-431 (2003). Seeman et al., “Nucleic Acid Junctions and Lattices,” J. Theor. Biol., 99:237-247 (1982). Sharma et al., “DNA-Tile-Directed Self-Assembly of Quantum Dots info two-Dimensional Nanopatterns,” Angewandte Chemie 47(28):5157-5159 (2008). Talapin et al., “Quasicrystalline order in self-assembled binary nanoparticle superlattices,” Nature 46 1:964-967 (2009) [with Eng- lish abstract]. Tang et al., “DNA-Directed Self-Assembly of Graphene Oxide with Applications to Ultrasensitive Oligonucleotide Assay,” ACS Nano 5(5):3817-3822 (2011). Tian et al., “Lattice Engineering via Nanoparticle-DNA Frame- works,” Nat. Mater. 15:654-661 (2016). Travesset, “Binary nanoparticle superlattices of soft-particle sys- tems,” Proc. Natl. Acad. Sci. USA 112:9563-9567 (2015). Varilly et al., “A general theory of DNA-mediated and other valence-limited colloidal interactions,” The Journal of Chemical Physics 137:094108 (2012) 16 pes. Vo et al., “Stoichiometric control of DNA-grafted colloid self- assembly,” Proc. Natl. Acad. Sci. USA 112:4982-4987 (2015). Wang ef al., “An Organic Semiconductor Organized into 3D DNA Arrays by ‘Bottom-Up’ Rational Design,” Angew. Chem. Int. Edit. 56:6445 -6448 (2017). Wilner et al., “Enzyme cascades activated on topologically pro- grammed DNA scaffolds,” Nature Nanotechnology 4:249-249 (2009). Wood et al., “Colloidal quantum dot light-emitting devices,” Nano Reviews 1:5202 (2010) 7 pgs. Yager et al., “Periodic lattices of arbitrary nano-objects: modeling and applications for self-assembled systems,” Journal of Applied Crystallography 47:118-129 (2014). Zhang et al., “3D DNA Origami Crystals,” Advanced Materials 30:1800273 (2018) 7 pgs. Zhao et al., “Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion,” Nature Commu- nications 7:10619 (2016) 9 pgs. Zheng et al., “Two-Dimensional Nanoparticle Arrays Show the Organizational Power of Robust DNA Motifs,” Nano Letters 6(7):1502- 1504 (2006). Zheng et al., “From Molecular to Macroscopic via the Rational Design of a Self-Assembled 3D DNA Crystal,” Nature 461:74-77 (2009). * cited by examiner
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US 12,548,243 B2 1 3D-ORGANIZED NANOMATERIALS THROUGH DNA-PRESCRIBED AND VALENCE-CONTROLLED MATERIAL CROSS-REFERENCE TO RELATED APPLICATION This application claims priority to U.S. Provisional Patent Application No. 62/914,792, which was filed on Oct. 14, 2019, the entire contents of which are incorporated by reference herein. GRANT INFORMATION This invention was made with government support under DE-SC0008772 and DE-SC0012704 awarded by the Department of Energy. The government has certain rights in the invention. SEQUENCE LISTING: A Sequence Listing conforming to the rules of WIPO Standard ST.25 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via the USPTO patent electronic filing system in ASCII formatted text. The electronic document, created on Sep. 5, 2024, is entitled “070050.6413_ST25”, and is 143,688 bytes in size. BACKGROUND The organization of nanoscale objects of various types into well-defined 3D arrays can transform nanoscale-syn- thesis into targeted material fabrication. Self-assembly of nanoscale objects into a system organization (lattice) can use both inter-object interactions and an object’s specific shape in order to achieve a particular system organization. DNA can be a polymer capable of defining structural organization at small scales while being precisely programmable, mean- ing that it can target specific particles and materials to use in the self-assembly process. However, certain methods cannot achieve desired lattices independent of specific nanoscale objects because of a coupling between the properties of the particles used and the resulting lattice. Even certain techniques using DNA cannot decouple particles from the assembly structure. As a result, such techniques require unique solutions, since each result- ing structure relies on the interactions between the 5 assembled particles and the underlying organizational struc- ture. Therefore, there is a need for self-assembly platforms that can be created independent of the nanoscale objects con- tained in the resulting lattice and which can assemble lattices using nanoscale objects of different natures. SUMMARY The disclosed subject matter provides techniques for organizing an object into a three-dimensional (3D) array using a voxel. The 3D array can contain an organic, an inorganic, a protein, an enzyme, or combinations thereof inside of the voxel. In certain embodiments, the disclosed subject matter provides a voxel that can include a plurality of frames including at least one single-stranded (ss) DNA motif with 0 a 30 2 at least one free base. In non-limiting embodiments, the ssDNA motif can hybridize with a complementary strand fragment of other frames. In certain embodiments, the plurality of frames can include stand-alone DNA frames, polyhedral frames, or a combination thereof. In non-limiting embodiments, the plu- rality of frames can form a tetrahedra framework, an octa- hedra framework, a cubic framework, or a combination thereof. In certain embodiments, the voxel can include at least one object in the voxel. In non-limiting embodiments, the object can include a gold nanoparticle, a streptavidin, a protein, a quantum dot (QD), an enzyme, or a combination thereof. In some embodiments, the object can include a metallic par- ticle, a semiconductor particle, a protein superlattice, or combinations thereof. In non-limiting embodiments, the object can be a functionalized object. In certain embodiments, the voxel can form a three- dimensional (3D) origami lattice. The 3D origami lattice can be a body-centered-cubic (BCC) lattice, a simple cubic (SC) lattice, or a cubic diamond lattice. In non-limiting embodi- ments, the voxel can be a DNA-prescribed voxel. In some embodiments, the voxel can be a valence-controlled voxel. The disclosed subject matter provides a method for orga- nizing an object into a three-dimensional (3D) array. The method can include creating a voxel that can include a plurality of frames by hybridizing at least one single- stranded (ss) DNA motif with at least one complementary strand fragment of the plurality of frames and inserting the object into the voxel by mixing the object and the voxel in a predetermined ratio. In non-limiting embodiments, the predetermined ratio between the object and the voxel is from about 1:1 to about 30:1. In certain embodiments, the plurality of frames can include stand-alone DNA frames, polyhedral frames, or a combination thereof. In non-limiting embodiments, the plu- rality of frames can form a tetrahedra framework, an octa- 4p hedra framework, a cubic framework, or a combination B thereof. In certain embodiments, the method can further include annealing the voxel to form a lattice. The lattice can be a three-dimensional (3D) origami lattice. In non-limiting embodiments, the 3D origami lattice can be a body-cen- tered-cubic (BCC) lattice, a simple cubic (SC) lattice, or a cubic diamond lattice. In certain embodiments, the method can further include functionalizing the object. In non-limiting embodiments, the functionalized object can be an inorganic object, a bio- organic object, an enzyme, and combinations thereof. The presently disclosed subject matter will be further described below. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 provides schematics of the DNA material voxels platform for assembly of 3D lattices from inorganic (nan- oparticles) and bioorganic (proteins) nano-objects with DNA frames in accordance with the presently disclosed subject matter. FIGS. 2A-2C provide an example assembly of octahedra frames into DNA lattice. FIG. 2A shows an example ssDNA encoded octahedral frames with complementary strands (left) that are mixed to form a DNA lattice (right) and a unit cell (center) with three faces of the unit cell. FIG. 2B provides a graph showing 2D SAXS pattern and correspond-
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US 12,548,243 B2 3 ing structure factor. FIG. 2C shows an example negative stained TEM image for DNA lattice formed by DNA octa- hedra. FIGS. 3A-3G show an example simple cubic (SC) lattice of gold nanoparticles (AuNP) assembled using material voxels based on DNA octahedra. FIG. 3A shows an example assembly of caged nanoparticles into SC lattice. FIG. 3B provides an example TEM image for AuNPs encaged by octahedra. FIG. 3C provides an example Z-contract annular- dark field Cryo-STEM imaging for nanoparticle caged 3D superlattice (R1). FIG. 3D provides an example assembly by route 2, R2. FIG. 3E provides a graph showing SAXS pattern and the corresponding structure factor S(q) for lattices of caged AUNP. FIG. 3F shows an example assembly of a binary system of octahedral frames with one frame empty and another one hosting AuNP. FIG. 3G provides a graph showing SAXS pattern and the related S(q) for the binary lattice shown in FIG. 3F. FIGS. 4A-41 provide an example assembly of nanopar- ticles from different materials and frames of different geom- etries into SC, BCC and cubic diamond superlattices. FIG. 4A shows a graph showing experimental and calculated structure factors, S(q), for QDs caged superlattice. FIG. 4 B shows an illustration of SC superlattice formed by QDs encaged into octahedra. FIG. 4C provides a graph showing experimental and calculated S(q) for the superlattice of cube encaged AuNP. FIG. 4D provides a schematic of BCC lattice of a vertex-driven assembly of cubes with encaged AuNPs. FIG. 4E provides a graph showing experimental and calcu- lated S(q) for the superlattice of tetrahedron encaged AuNP. FIG. 4F shows a schematic of cubic diamond lattice of a vertex-driven assembly of tetrahedra with encaged AuNPs. FIGS. 4G-4] provide graphs showing lattice free energy theoretical prediction per frame for octahedral (4G), cubic (4H) and tetrahedral (41) systems. FIGS. 5A-5F provide graphs and images showing the structure and enzymatic activity of assembled 3D designed lattices (SC) of material voxels with proteins. FIG. 5A is an illustration of the encaging of 6 streptavidin inside the DNA octahedra. FIG. 5B provides images showing a reference- based 2D class average as obtained from the cryo-TEM single-particle analysis. FIG. 5C provides an image showing cryo-TEM derived 3D density maps of the octahedron encaged proteins. FIG. 5D shows a confocal fluorescent microscopy image of formed crystallites with dye-labeled encaged proteins. FIG. 5E provides a graph showing experi- mental structure factors S(q) for empty DNA 3D lattice and for DNA lattice with 6 proteins placed in a DNA octahedra 5 cage, as shown in FIG. 4A. FIG. 5F provides an illustration of the assembly of octahedra with encaged 6 streptavidin into 3D superlattice. FIGS. 6A-6D provide images and graphs showing optical and enzymatic functions of a 3D lattices assembled respec- tively from DNA material voxels with quantum dots and enzymes. FIG. 6A shows fluorescence images demonstrat- ing the assembled SC lattice from material voxels with QD525 and QD705. FIG. 6B shows fluorescence profiles over a range of sample analysis areas. FIG. 6C shows three different conditions (shown as I, II, III) examined to deter- mine the effect of lattice packing and aggregation on the output of an enzymatic cascade. FIG. 6D illustrates a graph showing the production of the fluorescent product resorufin. FIG. 7 provides a diagram showing an example structure of octahedral DNA frames including bases of a poly T part from the vertex (m, n) and complementary bases (1). a w 8 w 8 40 4 4 FIG. 8 provides a diagram showing an example structure of cubic DNA frames including bases of a poly T part from the vertex (m, n) and complementary bases (1). FIG. 9 provides a graph showing melting temperature measured by dynamic light scattering for octahedral DNA lattice. FIG. 10 provides negative stained TEM images of assembled 3D octahedral DNA lattices. FIG. 11 provides a graph showing structure factor for simple cubic lattice formed by octahedral DNA with encaged gold NP of systems 1-8 (Top to bottom) in Table 1. FIG, 12 provides q, values for the simple cubic systems formed by octahedral DNA that cages gold nanoparticles. FIG. 13 provides negative stained TEM images of cubic frames. FIG. 14 provides negative stained TEM images of 10 nm gold nanoparticle inside DNA cubic frames. FIG. 15 provides negative stained TEM image of tetra- hedral structures. FIGS. 16A-16C provide octahedron with one streptavidin inside of the DNA cage. FIG. 16A shows a model of protein caged octahedron. FIG. 16B shows a cryo-EM image for one streptavidin inserted octahedral particles. FIG. 16C shows the reference-based 2D class average for one protein inserted octahedra as obtained from the cryo-TEM single- particle analysis. FIG. 17A provides an illustration of the assembly of one-streptavidin caged octahedra (OB and OR) into a 3D lattice. FIG. 17B provides a graph showing I(q) vs q curve for the DNA lattice with and without one protein in a cage. FIG. 18 provides confocal Fluorescent Microscopy images of 6 streptavidin, labeled with Cy3 dye, encaged by DNA octahedra, and assembled into a lattice. FIGS. 19A-19D provides images showing fluorescence microscopy of QD superlattices. FIG. 19A shows a large area fluorescence microscopy image of QD-525 superlattice using a 525 nm filter. FIG. 19B shows a large area fluores- cence microscopy image of QD-525 superlattice using a 690 nm filter. FIG. 19C shows a small area fluorescence micros- copy image of a single QD-525 superlattice using a 525 nm filter. FIG. 19D shows a small area fluorescence microscopy image of a single QD-525 superlattice using a 705 nm filter. FIGS. 20A and 20B provide fluorescence lifetime point scan measurements of QD-525 superlattice. FIG. 20A pro- vides a small area fluorescence microscopy image ofa single QD-525 superlattice using a 525 nm band-pass filter. FIG. 20B provides fluorescence lifetime measurements for the QD-525 superlattice using multiple point scans. FIGS. 21A-21D provide fluorescence microscopy of QD superlattices. FIG. 21A shows a large area fluorescence microscopy image of QD-705 superlattice using a 525 nm band-pass filter. FIG. 21B shows a large area fluorescence microscopy image of QD-705 superlattice using a 690 nm band-pass filter. FIG. 21C provides a small area fluorescence microscopy image of QD-705 superlattice using a 525 nm band-pass filter. FIG. 21D shows a small area fluorescence microscopy image of QD-705 superlattice using a 690 nm band-pass filter. FIGS. 22A and 22B provide fluorescence lifetime point scan measurements of QD-705 superlattice. FIG. 22A shows a small area fluorescence microscopy image of a single QD-705 superlattice using a 525 nm band-pass filter. FIG. 22B shows fluorescence lifetime measurements for the QD-705 superlattice using multiple point scans. FIGS. 23A-23D provide fluorescence microscopy of QD superlattices. FIG. 23A shows a large area fluorescence microscopy image of QD-525 and 705 superlattices using a
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US 12,548,243 B2 5 525 nm band-pass filter. FIG. 23B shows a large area fluorescence microscopy image of QD-525 and 705 super- lattices using a 690 nm filter. FIG. 23C shows a small area fluorescence microscopy image of QD-525 and 705 super- lattices using a 525 nm band-pass filter. FIG. 23D shows a small area fluorescence microscopy image of QD-525 and 705 superlattices using a 690 nm band-pass filter. FIGS. 24A and 24B provide fluorescence lifetime point scan measurements of QD-525 and 705 superlattice. FIG. 24A shows a small area fluorescence microscopy image of a single QD-525 and 705 superlattices using 525 nm and 690 nm band-pass filters. FIG. 24B shows fluorescence lifetime measurements for the QD-525 and 705 superlattice using multiple point scans. FIGS. 25A-25] provide fluorescence microscopy images of different area and corresponding spectra for QD-525 superlattice, using three band-pass filters; Detector 1: 500- 550 nm (FIGS. 25A, 25D, and 25G), Detector 2: 555-675 nm, (FIGS. 25B, 25E, and 25H), Detector 3: 680-730 nm (FIGS. 25C, 25F, and 251). FIG. 25J provides a graph showing the mean intensity of the images. FIGS. 26A-26J provide fluorescence microscopy images of different area and corresponding spectra for QD-705 superlattice, using three band-pass filters; Detector 1: 500- 550 nm (FIGS. 26A, 26D, and 26G), Detector 2: 555-675 om, (FIGS. 26B, 26E, and 26H), Detector 3: 680-730 nm (FIGS. 26C, 26F, and 261). FIG. 26] provides a graph showing the mean intensity of the images. FIGS. 27A-27J provide fluorescence microscopy images of different area and corresponding spectra for QD-525 and 705 superlattices, using three band-pass filters; Detector 1: 500-550 nm (FIGS. 27A, 27D, and 27G), Detector 2: 555-675 nm, (FIGS. 27B, 27E, and 27H), Detector 3: 680-730 nm (FIGS. 27C, 27F, and 271). FIG. 27J provides a graph showing the mean intensity of the images. FIG. 28 provides a graph showing the initial reaction of DNA origami lattice containing GOx-HRP pair (Sample I), using (i) 150 mM glucose, and (ii) 300 mM glucose as initial substrate concentrations. FIGS. 29A-29D provides images showing polycrystalline domains within single isolated self-assembly. FIG. 294 shows a projection image (Scale bar: 500 nm) and its Fourier transform. FIG. 29B shows three Fourier masks that are related to the three domains that have different relative orientations. FIG. 29C shows an inverse Fourier transform of the Fourier masked results that illustrate where the masked domain is concentrated. FIG. 29D shows a com- bined image. FIG. 30 provides a negative stained TEM image of octahedral DNA origami structures. FIG. 31 provides a diagram of the DNA octahedron model. FIG. 32 provides a diagram showing the DNA octahedra arranged in a simple cubic lattice. FIG. 33 provides a graph showing the measured scattering intensity of a suspension of un-aggregated free DNA octa- hedra, and objects assembled into a superlattice. FIG. 34 provides a plot of the structure factor S(q) for experimental data and model for a cubic superlattice of DNA octahedra. FIG. 35 provides diagrams showing the projected scat- tering of the DNA octahedra versus the DNA octahedra. FIG. 36 provides a plot of the structure factor S(q) for experimental data and modeled result, compared with the simulated structure factor of the same DNA octahedra. a 8 w 40 4 5 60 B 6 FIG. 37 provides a diagram showing a model represen- tation of the DNA octahedra with the quantum dots and 6 streptavidin used for SAXS modeling. FIG. 38 provides a diagram showing an octahedron model with 6 proteins (streptavidin) encaged in shown position, used for SAXS modeling. FIG. 39 provides a graph showing structure factors S(q) of an enlarged image of SAXS data for octahedral DNA lattice with and without 6 streptavidin inside the octahedral cages. FIG. 40 provides a diagram of an example model of one tetrahedron. FIG. 41 provides a plot of the computed form factor for DNA tetrahedra, q° power law, and the scattering data obtained from a lattice of tetrahedra. FIG, 42 provides a model for the cubic superlattice of DNA tetrahedra. FIG. 43 provides a plot of the structure factor of the scattering obtained from DNA tetrahedra combined into a lattice compared with different models. FIG. 44 provides a plot of the data and fits of gold spheres assembled in an SC lattice by octahedral system caged with nanoparticles. FIG. 45 provides a plot of the data and fits of gold spheres assembled in an FCC lattice by an octahedral system, while half of the octahedra were filled with nanoparticles. FIG. 46 provides a plot of the data and fits of gold spheres assembled in a BCC lattice by a cubic system. FIG. 47 provides a plot of the data and fits of gold spheres assembled in a diamond lattice. FIG. 48 provides a plot showing an example fit of the form factor of the BCC lattice by the cubic system. FIGS. 49A and 49B show diagrams of singlet patchy particles. FIG. 49A shows a single patch particle. FIG. 49B shows an example of patchy particle bonding. FIGS. 50A-50C provide diagrams of patchy particle designs. FIG. 50A shows patch particle design in Octahe- dron, FIG. 50B shows patch particle design in Cube, and FIG. 50C shows patch particle design in a tetrahedron. FIGS. 51A and 51B provide diagrams of nanoparticle cluster self-assembly directed by the valence-programmable DNA mesh frame. FIG. 51A shows diagrams of a nanopar- ticle cluster coordinated by a sphere-like frame structure. FIG. 51B shows diagrams of designed DNA mesh frame origami and pentakis icosidodecahedron for programming designed nanoparticle cluster architectures. FIGS. 52A-52F provide diagrams and graphs showing structures of nanoparticle (NP) cluster architectures assembled by programmed mesh frame. FIG. 52A shows designed DNA mesh frames with different valence numbers. FIG. 52B shows NPs decorated with DNA that are assembled into clusters. FIG. 52C shows negative-stained TEM images of assembled NP clusters based on mesh frames with different valence modes. FIG. 52D shows NP cluster population histograms. FIG, 52E shows a recon- structed 3D structure of the triangular bipyramid (TBP) cluster from cryo-TEM based tomography (top) and designed center-to-center distances between NPs (bottom). FIG. 52F shows a reconstructed 3D structure of the octahe- dral cluster by cryo-TEM tomography (top) and designed NP distances (bottom). FIGS. 53A and 53B provide graphs showing in-liquid structures of NP cluster architectures revealed by in situ SAXS. FIG. 53A shows an extracted structure factor S(q) from in situ SAXS for designed clusters with the different mesh frame valence numbers (from bottom to top): two,
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US 12,548,243 B2 7 three, four, five, and six, respectively. FIG. 53B shows interparticle distances derived from SAXS (squares) for NP clusters. FIGS. 54A-54G provide diagrams and graphs showing the design and structural and optical characterization of the spherical helix NP cluster. FIG. 544 shows a schematic of the spherical helix NP cluster. FIG. 54B shows negative- stained TEM images of spherical helix clusters (left) and population analysis of assembled spherical helix clusters (tight). FIG. 54C shows designed NP positions of the spherical helix cluster on a sphere-like mesh frame (left) and particles on the equatorial plane of the sphere-like structure (right). FIG. 54D shows a spherical helix cluster recon- structed from cryo-TEM tomography. FIG. 54E shows cen- ter-to-center distances between adjacent NPs, D, and center- to-center distance between sphere-like mesh frame. FIG. 54F shows a computed CD spectrum and absorption spec- trum for spherical helix clusters. FIG. 54G shows an experi- mental CD spectrum, Lorentzian fit, and absorption spec- trum for spherical helix clusters. FIGS. 55A-55E provide diagrams and graphs showing designs and characterization of multitype NPs clusters assembled with a mesh frame of directional and polychro- matic valence modes. FIG. 55A shows mesh frames pro- 2 grammed with a valence of TBP geometry and encoded sites at selected vertices, labeled from 1 to 5, results in polychro- matic valence: (I-II) two-color valence. (III-V) three-color valence. FIG. 55B shows multitype NP clusters. FIG. 55C shows negative-stained TEM images of NP hetero-clusters. 3 FIG. 55D shows population histograms for corresponding multitype NP clusters. FIG. 55E shows a cryo-TEM tomog- raphy reconstructed cluster (left) and designed interparticle distances (right). FIG. 56 shows multi-dimensional assembly (1D, 2D, and 3 3D) including a DNA nano-chamber with differentiated polychromatic bonds. FIGS. 57A and 57B provide a diagram showing multi- dimensional assemblies and their building blocks. FIG. 574 shows building blocks with different types of bonds. FIG. 57B shows schematics of programmable assembly of DNA nanochambers (DNCs). FIGS. 58A-58G provide images and diagrams showing the construction of DNA nanochambers and their ability to carry AuNPs. FIG. 58A shows the formation of DNC by folding an M13 scaffold with 224 blunt staples. FIG. 58B shows agarose gel electrophoresis analysis of DNCs. FIG. 58C shows a negatively stained TEM image of DNCs. FIG. 58D shows a cryo-TEM image of DNCs. FIG. 58E shows schematics of encapsulating AuNPs in the cavity or on the 50 sidewall of DNCs by anchoring strands. FIGS. 58F and 58G show TEM images of DNCs bound with (FIG. 58F) 10 nm. AuNP in the cavity, and (FIG. 58G) 20 nm AuNP on the bundle of one sidewall. FIGS. 59A-59F show images and diagrams showing 1D assembly of DNA nanochambers. FIGS. 59A-59C show TEM images of (FIG. 59A) empty 1D chains, (FIG. 59B) 1D alternating chains alternatively filled with 10 nm AuNPs, and (FIG. 59C) 1D homochains fully filled with 10 nm AuNPs. FIG. 59D shows a schematic for creating 1D 60 polymer chain with a controlled monomer sequence of ABCD, where A, B, C, and D are four different kinds of DNCs. FIG. 59E shows TEM images of linear 1D chains consisting of two 10 nm AuNPs-filled and two empty DNCs in an alternating manner. FIG. 59F shows TEM images of 65 helical 1D chains bound 20 nm AuNPs on the sidewall of DNCs in a helical architecture. S a 8 8 40 8 FIGS. 60A-60L provide images and graphs showing 2D assemblies of DNA nanochambers. FIGS. 60A-60D show schematics and TEM images of 2D arrays assembled from two complementary DNCs with the varying number of sticky ends within the XY-plane: (FIG. 60A) 4 sticky ends, (FIG. 60B) 8 sticky ends, (FIG. 60C) sticky ends, and (FIG. 60D) 16 sticky ends. FIG. 60E provides a graph showing a statistical analysis of average 2D array size with increasing sticky ends, based on both experimental results and com- putational simulation. FIG. 60F-60H shows Monte Carlo simulation snapshots of 2D arrays created by variation of binding strength: (FIG. 60F) 8 bonds, (FIG. 60G) 12 bonds, and (FIG. 60H) 16 bonds. FIG. 601 shows a TEM image of 2D square lattices fully filled with 10 nm AuNPs. FIG. 60] provides a graph showing a SAXS pattern of 2D square lattices filled with 10 nm AuNPs. FIG. 60K provides a chess-board-like 2D lat- tices alternatively filled with 10 nm AuNPs. FIG. 60L. provides a graph showing a SAXS pattern of chess-board- like 2D lattices alternatively filled with 10 nm AuNPs. FIGS. 61A-61H provide images and diagram showing 3D assemblies of DNA nanochambers. FIG. 61A shows four types of DNA nanochambers (DNCs) with different binding modes. FIG. 61B provides a graph showing structure factor, S(q), for assembly filled with AuNPs for different designs. FIG. 61C shows a TEM image of elongated 3D structures formed by empty DNCs. FIGS. 61D and 61E show TEM images of (FIG. 61D) AuNPs-filled and (FIG. 61E) empty 3D assembly for certain DNCs. FIGS. 61F-61H show Monte Carlo simulation snapshots of various 3D assembled struc- tures created by variation of relative binding energies in XY-plane versus Z-axis: (FIG. 61F) fiber-like (Z-dominant) domains of 3D assembled DNCs with strong longitudinal bonds, (FIG. 61G) cubic-like domains of 3D assembled DNCs with balanced longitudinal and lateral bonds, and (FIG. 61H) pancake-like (XY-dominant) domains of 3D assembled DNCs with strong lateral bonds. It is to be understood that both the foregoing general description and the following detailed description are exem- plary and are intended to provide further explanation of the disclosed subject matter. DETAILED DESCRIPTION The disclosed subject matter provides techniques for organizing an object into a three-dimensional (3D) array using a voxel. The disclosed subject matter can be used for inorganic and bio-organic components of different intrinsic properties and shapes for organizing the components into the 3D array. The disclosed subject matter can also be used for creating light-emitting 3D arrays with diffraction-limited spectral purity, 3D enzymatic arrays, metamaterials, infor- mation storage devices, and combinations thereof. In certain embodiments, the disclosed subject matter provides a voxel that can include a plurality of frames. As shown in FIG. 1, the voxel 101 can be a block that can have a valence determined by the vertices of the frame 102. The voxel 101 can be an elementary volumetric block carrying material property and its binding characteristics fully deter- mined by frame shape that can lead to a 3D nanomaterial lattice 103. In non-limiting embodiments, the voxel can be a valence-controlled voxel. In some embodiments, the voxel can be a DNA-prescribed voxel. A DNA voxel can include a designed DNA object of specific shape (e.g., polyhedral frames, or container-like object) that can carry nanoscale cargo (e.g., nanoparticles, proteins, and enzymes). The DNA voxel can be empty or carry a nanoscale cargo. A valence-
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US 12,548,243 B2 9 controlled voxel can have a prescribed number of binding regions at the prescribed location (e.g., at vertices). It can be in any desired location. In this way, a voxel valence can provide number and directionality for specific connectivity with other voxels. The location of binding regions can be full designed and even re-programmed for the same DNA object. In certain embodiments, the plurality of frames can include at least one single-stranded (ss) DNA motif with at least one free base. The ss DNA motif can hybridize with a complementary strand fragment of other frames to form an inter-frame hybridization. For example, the plurality of frames can form an inter-frame hybridization via vertex-to- vertex hybridization. The hybridized frames can provide improved control over interactions encoding, energetics, and flexibility of interframe linkages. In non-limiting embodi- ments, the frame can possess a spatially determined valence (v) defined by its shape. The inter-frame hybridization can form an assembly with the ability to form an ordered lattice, whose type can be determined by the frame valence. In some embodiments, the plurality of frames can include stand- alone DNA frames, polyhedral frames, or a combination thereof. In non-limiting embodiments, the plurality of frames can include a frame where certain faces can be solid walls or spherical DNA objects with arbitrarily program- mable valences. In certain embodiments, the plurality of frames can form a tetrahedra framework, an octahedra framework, a cubic framework, or a combination thereof. For example, the plurality of frames can be hybridized to form a tetrahedra framework, an octahedra framework, a cubic framework, or a combination thereof. In certain embodiments, the voxel can form a three- dimensional (3D) origami lattice. For example, the voxel can form a body-centered-cubic (BCC) lattice, a simple cubic (SC) lattice, or a cubic diamond lattice. In non- limiting embodiments, the 3D origami lattices can be formed from octahedral frames containing twelve 6-helix- bundle edges. The lattices formed from the octahedral frame can have a valence of 6 (v=6). The edge length of the lattices formed from the octahedral frame can be less than about 30 nm. In some embodiments, the edge length of the lattices formed from the octahedral frame can be from about 20m, to about 100 nm. In non-limiting embodiments, the voxel can form a 2D lattices with different square patterns includ- ing honeycomb, stacked hexagonal, and/or tetragonal lat- tices. Each octahedron vertex (OB) can incorporate single- stranded (ss) DNA motifs with at least one free base. This construction can allow the hybridization of nucleotides with complementary strand fragments on another octahedron 5 (OR). The vertex-driven 6-fold octahedral valency can yield ordered assemblies through vertex-to-vertex hybridization of frames. For example, the octahedral frame assemblies (e.g., with 22 complementary bases, 22 bases in first respec- tive polyT domains, and 8 bases in second respective polyT domains) can form a well-ordered simple cubic (SC) lattice with 15 orders of resolution-limited Bragg peaks. In non- limiting embodiments, the size of the SC lattice can be up to about 200 jum, up to about 150 jum, up to about 100 um, up to about 50 jum, up to about 25 um, up to about 10 ym, up to about 5 jum, up to about 3 jum, up to about 2 jum, or up to about 1 um. For example, the size of the SC lattice can be from about 150 jum to about 200 um or from about 3 um to about 5 uum. In certain embodiments, the voxel can include at least one object 104. The object 104 can include a metallic particle, a semiconductor particle, a protein superlattice, or combina- S a w 8 w 40 4 10 tions thereof. In non-limiting embodiments, the object can have various shapes. For example, an inorganic object can have a cube, rod, octahedra, and/or spherical shape. Proteins and enzymes can be included in the voxel regardless of their shapes. In certain embodiments, the object can be inserted into the voxel before assembling the voxel. For example, gold nan- oparticles (AUNP) can be encaged inside the octahedra before these material voxels self-assemble. The AuNPs can be positioned in the middle of each octahedron by hybrid- ization with its internal strands. Then mix OB and OR frames (with encaged AuNPs) and anneal to form a tem- plated 3D AuNP array. In certain embodiments, the object can be inserted into the voxel after assembling the voxel. For example, objects (e.g., AuNP) can be inserted into the voxel by adding the objects into a solution of formed, empty frame lattices over the octahedra at predetermined ratio. Annealing of the mixed solution can cause diffusion/distribution of at least one object into the ordered scaffold. In certain embodiments, the ssDNA length of frames and/or inter-octahedra distance can be modified. In non- limiting embodiments, the ssDNA can be designable. For example, the range of the ssDNA length can be from about 5 to about 150 bases. In certain embodiments, properties of the voxel can be modified with different valence. Various lattice symmetries can be formed by adjusting the valence of the voxel. For example, cubic frames (e.g., 8 valence) including twelve 6-helix bundle edges (6HB) with edge lengths about -29 nm can be assembled to a well-ordered body-centered-cubic (BCC) lattice. In non-limiting embodiments, two kinds of cubes (ie, CB and CR). CB and CR are the same cubic DNA frame with encoded vertices for inter-voxel connec- tions. CB and CR have complementary ssDNA strands at the vertices. Each CB vertex can include three DNA strands complementary to the three CR vertex strands. Both cube populations can have another set of DNA sticky-ends at each vertex that point towards the cube center for binding with ssDNA of AuNPs for encaging of the AuNPs. This encap- sulation can allow restoring the cube shape from their skewed empty forms to a normal, undistorted form. Cube assembly can be carried out by mixing CB and CR with encaged NPs, followed by annealing. The well-ordered BCC lattice can be in-line with a vertex-to-vertex hybridization of cubic frames. In certain embodiments, the voxel can form a diamond lattice. For example, voxels with 4 valences can be hybrid- ized between vertices of tetrahedral DNA frames, for which each edge can include a 10HB with length ~36 nm. Four internal DNA strands can bind to an object (e.g., AuNPs). Equal amounts of two kinds of tetrahedra possessing complementary sets of 6 sticky-end sequences at their vertices can be mixed and annealed to form the diamond lattice. Each tetrahedron can bind with four tetrahedra through its vertices. In certain embodiments, at least one object can include a gold nano particle, a streptavidin, a protein, a quantum dot (QD), an enzyme, or a combination thereof. In non-limiting, embodiments, the object can be inorganic materials, and/or the voxel can form a 3D protein arrays. For example, a streptavidin, a protein containing four high-aflinity sites for biotin binding, can be inserted in the voxel. The voxel can include a plurality of hosting sites (e.g., 6 hosting sites) for an organic object (e.g., streptavidin). For example, there can be one hosting site at every vertex of a frame (e.g., v=6). In non-limiting embodiments, each site can include at least one
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US 12,548,243 B2 1 ssDNA (e.g., four ssDNA) attached to the corresponding edges that form the vertex. Each streptavidin can bind to biotinylated DNA that is complementary to the edge-at- tached strands. In some embodiments, the voxel including an organic object can form a 3D origami lattice. For example, OB and OR frames with streptavidin and bioti- nylated DNA can be mixed and annealed. In certain embodiments, the object can be functionalized. For example, the gold particles can be functionalized with thiolated oligonucleotides. The object can be mixed with the oligonucleotides in a predetermined ratio (e.g., 1:300). Quantum can be functionalized by mixing with excess biotinylated DNA (x10 molar excess). Streptavidin can be functionalized by mixing with biotinylated DNA to fabricate the streptavidin/DNA complex based on the specific binding of biotin and streptavidin. Enzymes can be functionalized by mixing with activated oligonucleotides in a predetermined ratio (e.g., 1:5). In certain embodiments, the voxel can be used for optical applications and catalytic applications. For example, the voxel can be combined with quantum dots for the optical applications. Multiple distinct binding sites (e.g., 8) can be programed within the interior region of octahedron DNA origami using orthogonal, interior ssDNA overhangs. The OB and OR octahedra can each encoded to host a specific quantum dot (QD), which can have certain fluorescence emissions (e.g., 525 or 705 nm) resulting in two optically distinctive types of DNA material voxels. ssDNA-modified quantum dots can then be annealed into empty, fully- assembled material voxels lattices designed to either be half-filled by a single QD or fully-filled by two types of QD’s at predetermined ration (e.g., 1:1 ratio). In certain embodiments, a cascaded enzyme network organized within the disclosed 3D lattice can be used for the catalytic applications (e.g., 3D biomolecular arrays). Enzymes can be inserted into active high-density 3D arrays using material voxel, and further be manipulated and enhanced cascade reactions using such 3D packaging of enzymes. For example, ssDNA-modified glucose oxidase (GOx) and horseradish peroxide (HRP) can bind, respec- tively, to a single interior hosting site within each OB and OR octahedra, forming two enzymatic types of material voxels, which can be co-assembled in the disclosed lattice. The assembled lattice architecture can provide an increased activity of the enzymatic cascade, high packing density, and co-localization of catalytic components. The disclosed subject matter provides a method for orga- nizing an object into a three-dimensional (3D) array. The method includes creating a voxel including a plurality of frames by hybridizing at least one single stranded (ss) DNA 5 motif with at least one complementary strand fragment of the plurality of frames and inserting the object into the voxel by mixing the object and the voxel in a predetermined ratio. In certain embodiment, the voxel can be designed to form octahedral, cubic, tetrahedral origami frames. In non-limit- ing embodiments, each edge of the octahedral and cubic frames can include a 6HB with length of 84 base pairs. For each 6HB, both ends can have one single-stranded DNA sticky end for binding with another frame. In some embodi- ments, for an octahedron, there can be four DNA sticky ends per vertex. For a cube lattice, there can be three DNA sticky ends per vertex. In non-limiting embodiments, each edge of the tetrahedron can include a 10HB, and both ends can have two single-stranded DNA sticky ends for binding with another frame in each 10HB. There can be six DNA sticky ends per vertex. In some embodiments, the DNA origami frames can be folded by mixing scaffold DNA, designed S a w 8 w 40 & B 12 staple oligonucleotide, and sticky end strands extending from the vertices in a buffer/salt solution. The mixed solu- tion can be annealed to obtain the target DNA structure. In certain embodiments, the object can be encaged in the voxel by mixing the object and the voxel in a predetermined ratio. In non-limiting embodiments, the predetermined ratio between the object and the voxel can be from about 1:1 to about 30:1. For example, in order to insert nanoparticles/ streptavidin inside the DNA cage, nanoparticle-DNA or streptavidin-DNA complex can be mixed with DNA frames at a ratio of about 1.5:1 or about 2:1, respectively. For the system of six streptavidin proteins caged within octahedra, the ratio of streptavidin-DNA complex and DNA frame can be about 15:1. In certain embodiments, the mixed solution can be annealed by cooling to encage the object (e.g., particles or proteins) inside the DNA frame. These material-filled DNA frames can be then used to synthesize a lattice. In order to load QDs into the lattice, two complimentary octahedron DNA origami can be annealed without guest particles. Assembled DNA origami crystals can be mixed with the functionalized QDs, and a secondary annealing can be performed. In certain embodiments, DNA-functionalized enzymes can be mixed with either formed octahedra DNA lattice, octahedra DNA lattice in order to create the 3D enzyme array. In certain embodiments, the method can further include functionalizing the object. For example, the gold particles can be functionalized with thiolated oligonucleotides. The object can be mixed with the oligonucleotides in a prede- termined ratio (e.g., 1:300). Quantum can be functionalized by mixing with excess biotinylated DNA (x10 molar excess). Streptavidin can be functionalized by mixing with biotinylated DNA to fabricate the streptavidin/DNA com- plex based on the specific binding of biotin and streptavidin. Enzymes can be functionalized by mixing with activated oligonucleotides in a predetermined ratio (e.g., 1:5). In certain embodiments, the disclosed subject matter provides nanoparticle-based clusters that can allow harvest- ing of collective and emergent properties, with applications ranging from optics and sensing to information processing and catalysis. For example, the disclosed subject matter can provide cluster architectures that can be formed using com- ponents with programmable valence. The cluster assemblies can be formed by employing a three-dimensional (3D) DNA meshframe with spatial symmetry as a site-programmable scaffold, which can be prescribed with desired valence modes and affinity types. In non-limiting embodiments, the meshframe can be a versatile platform for the coordination of nanoparticles into desired cluster architectures. Target positions and types of bonds can be prescribed. The meshframe can offer designability over different valence modes using the underlying symmetry frame, including various subset symmetries, arbitrarily prescribed helix-like valence, and valence with different types of affinities. In some embodiments, the structures of assembled 3D clusters can be verified by electron microscopy imaging, cryo-EM tomography, or in-situ X-ray scattering methods. In certain embodiments, the disclosed subject matter provides methods for creating a DNA nanochamber (DNC). The DNC can be a hollow cuboid nano-object with differ- entiated bonds. The bonds of the DNC can be prescribed and encoded along its three orthogonal axes. For example, by differentiating the bonds, one-(1D), two-(2D), and/or three- (3D) dimensional organized arrays can be formed. Through different binding modes, the DNC can be sequence encoded
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US 12,548,243 B2 13 nanoscale heteropolymers, helical polymers, 2D lattices, or mesoscale 3D nanostructures with internal order. In non- limiting embodiments, the DNC can host a nanoscale cargo and be integrated with functional nano-objects and/or their organization in larger-scale systems. In certain embodiments, the disclosed subject matter can also be used for creating light-emitting 3D arrays with diffraction-limited spectral purity and the disclosed 3D enzymatic arrays. EXAMPLES, Example 1: Ordered Three-Dimensional Nanomaterials Using DNA-Prescribed and Valence-Controlled Material Voxels Design and folding of DNA origami frames: Octahedral, cubic and tetrahedral DNA origami frames were designed by caDNAno software. In the design, each edge of the octahe- 5 dral and cubic frames was composed of a 6HB with length of 84 base pairs. For each 6HB, both ends have one single-stranded DNA sticky end for binding with another frame. For an octahedron, there are four DNA sticky ends per vertex, while for a cube there are three. Each edge of the tetrahedron was composed of a 10HB. For each 10HB, both ends have two single-stranded DNA sticky ends for binding with another frame. Thus, there are six DNA sticky ends per vertex. DNA origami frames were folded by mixing 10 nM M13mp18 scaffold DNA (Bayou Biolabs, LLC), 74 nM of each designed staple oligonucleotide and 100 nM of each of the sticky end strands extending from the vertices in a buffer/salt solution containing 1 mM EDTA, 12.5 mM magnesium chloride and 40 mM tris acetate with a pH around 8.0. The mixed solution was then annealed slowly by cooling from 90° C. to room temperature over 20 h to obtain the target DNA structure. Details of the interframe bonds and designs of the complementary DNA sequences can be found listed in Table 1 (for the octahedron) and Table 2 (for the cube). Sys: Sys- Sys: Sys Sys- Sys- Sys- Sys tem tem tem tem tem tem tem tem 1 2 3 4 5 6 7 8 m 7 120 1200 22002~« 38D I 8 8 8 8 8 8 8 8 n 7 7 12 12 32 2 2 32 Table 1. Example systems of octahedral DNA frames, labeled 1 to 8, with different lengths of inter-vertex linking motifs used for assembly of empty DNA lattices and for lattices with encaged NPs. m bases is a poly T part from OB vertex, n bases is a poly T part from the OR vertex. Hybridization between these strands occurs with 1 (=8) complementary bases. System System System System 1 2 3 4 m 7 12 32 32 1 8 8 8 8 a 7 12 12 32 Table 2. Example systems, labeled 1 to 4, for cubic DNA frames with different lengths of linking motifs used for assembly of empty DNA lattices and for the lattices with a 8 w 50 35 60 14 encaged NPs. PolyT part with m bases attached to the vertex of CB, and polyT part with n bases is attached to the CR vertex. Hybridization between these strands occurs with | (=8) complementary bases. DNA functionalization of nanomaterials: Gold nanopar- ticles-Thiolated oligonucleotides, purchased from Integrated DNA Technologies (IDT), were first reduced by tris[2- carboxyethyl]phosphine (TCEP) and then purified by a size exclusion column (G-25, GE Healthcare) to remove small molecules. Spherical AUNP with a diameter of ~10 nm (Ted Pella Inc.) were then mixed with the oligonucleotides in a ratio of 1:300, followed by buffering the solution to obtain 10 mM phosphate buffer (pH 7) after 2 h. Following buffer addition, sodium chloride was slowly added into the solution until a final concentration of 0.3 M was reached. The solution was then aged at room temperature for at least 12 h. Excessive reagents were removed by centrifuging the solution four times and washing with 10 mM phosphate buffer with 0.1 M sodium chloride. Quantum dots: Three different streptavidin-coated QDs (cadmium selenide/zine sulfide) were purchased from Thermo Fisher Scientific with emissions of 525 nm, 605 nm. and 725 nm. Each was mixed with excess biotinylated DNA (x10 molar excess) and aged over 12 h in the dark at room temperature (1xTAE buffer, 12.5 mM magnesium chloride) to obtain QD/DNA complexes. Streptavidin: A solution of streptavidin-Cy3 conjugate was purchased from Sigma Aldrich. Biotinylated DNA was purchased from IDT, which was then mixed with streptavi- din-CY3 and aged over 12 h in the dark to fabricate the streptavidin/DNA complex based on the specific binding of biotin and streptavidin. Enzymes: GOx was purchased from Sigma Aldrich. HRP was purchased from Thermo Fisher Scientific. Enzymes were first mixed with Sulfo-EMCS in a ratio of 20:1 (Sulfo-EMCS:enzyme) in PBS buffer (pH 7.4) for 1.5 h at room temperature, and then purified by a Amicon 30k cutoff filter. Thiolated oligonucleotides (IDT) were reduced by TCEP (1,000:1) in PBS buffer. The excess TCEP was removed by a size exclusion column (G-25, GE Healthcare). Next, sulfo-EMCS-treated enzymes were mixed with acti- vated oligonucleotides with a ratio of 1:5 in PBS buffer and rotated at 4° C. overnight. The DNA attached enzymes were then purified by Amicon cutoff filter (50 k for GOx, 30 k for HRP). The number of DNA attached to the enzyme was quantified by ultravioletvisible light spectroscopy. Thermal annealing of DNA origami frames: For empty DNA lattices, OB and OR were mixed in a 1:1 ratio (same method as for other origami topologies). For single ‘cargo’ inserted DNA lattices, OB, OR and ‘cargo’ complex were mixed with a ratio of 1:1:2 (same method as for other shapes). For the system of six streptavidin proteins, the ratio was 1:1:15. The mixed solution was then carefully annealed by cooling from 50° C. to room temperature at a rate of 0.2° C. h-1 to obtain the lattice. Encaging materials in DNA frames and lattice. In order to insert nanoparticles/streptavidin inside the DNA cage, nan- oparticle-DNA or streptavidin-DNA complex were mixed with DNA frames at a ratio of 1.5:1 or 2:1, respectively. For the system of six streptavidin proteins caged within octahe- dra, the ratio of streptavidin-DNA complex and DNA frame was 15:1. The mixed solution was then slowly annealed by cooling from 50° C. to room temperature with rate of 0.2° C. h-1 to encage the particles or proteins inside the DNA frame. These material-filled DNA frames were then used to synthesize a lattice.
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US 12,548,243 B2 15 In order to load QDs into the lattice, two complimentary octahedron DNA origami were annealed, following the lattice annealing protocol described previously, without any guest particles. Assembled DNA origami crystals were then mixed with the functionalized QDs and underwent a short (~4 h) secondary annealing protocol. In order to create the enzymatic systems, DNA-function- alized enzymes were mixed with either formed octahedra DNA lattice (10 nM), octahedra DNA lattice melted for 1 h at 52° C. and immediately placed on ice (to form an amorphous aggregate), or free octahedra containing enzyme binding sites. These solutions were mixed in TE buffer (pH 7.5) containing 12.5 mM magnesium chloride. Because of the contribution of TAE buffer from the DNA samples, the final pH was measured to be pH 8.0. The sample was gently shaken at room temperature for 24 h. Glucose and Amplex Red were added at final concentrations of 150 mM and 200 M, respectively, with additional magnesium chloride to ensure the final concentration was maintained at 12.5 mM. Then 100 ul reactions were measured in a 96-well plate using a Tecan Infinite plate reader, with four repeats per- formed for each sample. Dynamic light scattering: The dynamic light-scattering measurements were conducted using a Malvern Zetasizer ZS instrument at the backscattering angle of 173°. It was equipped with a laser source (633 nm) and a backscattering detector. For measuring the melting temperatures of each nanoparticle-frame assembly, the samples were cooled (Ix TAE buffer with 12.5 mM magnesium chloride) slowly in the chamber of the machine from 50° C. to room tempera- ture. The dependence of measured aggregate size versus temperature was obtained. TEM and cryo-EM. The carbon-coated grids were glow discharged in a 0.39 mbar air atmosphere for 1 min using PELCO easiGlow (Ted Pella, Inc.). Before EM grid prepa- ration, the sample was assessed for homogeneity by nega- tive-stain electron microscopy. Then 3 1] of sample solution was applied to glow discharged carbon-coated lacey grids, incubated for 2 min at 10° C. and 95% humidity, blotted for 3s and then plunged the sample into liquid ethane using a FEI Vitrobot IV. The grids were loaded into FEI Talos Arctica electron microscope operated at 200 kV high tension and collected images with EPU under low-dose mode at a magnification of x92,000 and a pixel size of 1.55 A. A Falcon III direct electron detector was used under linear mode for image recording with an under-focus range from 1.5 to 3.5 m. The dose rate was 20 electrons per A2 per second and total exposure time was 2 s. The total dose was divided into a 39-frame video and each frame was exposed for 0.05 s. Cryo-STEM. Cryo-samples for imaging under STEM were prepared similar to that described above but blotted for only 0.5 s before plunging into the liquid nitrogen-cooled liquid ethane. The samples were then transferred to a cryo- genic holder and imaged under a field-emission TEM (JEOL 2100F). The TEM was operated at 200 keV in the annular dark field scanning TEM mode. SAXS. The SAXS measurements were conducted at the Coherent Hard X-ray and Complex Materials Scattering beamlines. The 2D scattering data were collected on area detectors placed downstream of the sample. Self-assembly relies on a combination of inter-object interactions and an object’s specific shape to achieve a system organization. The significant progress in designing shells and shapes of nanoscale objects provides a play- ground for exploring and understanding these relationships and mapping out the resulting phases. Despite significant a w 8 w 8 40 4 5 16 progress in this field, it can be challenging to create desired organizations from specific nano-objects due to an intrinsic problem (e.g., a coupling between the properties of the objects and the resulting organization). Thus, from a mate- rial design point of view, nearly each system requires its own solution. The problem of forming ordered structures is even greater for biomolecules due their complex shapes and distributions of surface groups. While for the two-dimen- sional (2D) assemblies, surface patterning can be often used for scaffolding nano-objects, there is no viable option for building three-dimensional (3D) ordered arrays with the desired organizations. DNA can be a polymer capable of define structural organization at small scales. Moreover, in this context, DNA is a precisely programmable material appropriate for direct- ing 3D particle organization. Particles can be bound with single-stranded DNA chains and assembled using base- pairing of complementary sequences. This approach, how- ever, cannot decouple particle properties, for example, its shell, size and shape, from the assembly structure. The problem of organizing biomolecules is even greater. Thus, an outstanding challenge is to establish platform approaches for assembly of nanoscale objects of different natures in 3D. Here, 3D DNA frameworks were used to form from polyhedral DNA frames for organizing nano-objects that are contained inside frames. The versatility of this approach can extend to catalytic, biological and inorganic matrices, meta- materials, and information storage devices. One of the key difficulties for realizing this strategy was revealing the relationship between the design of frame and interframe connectivity and formation of the 3D ordered framework. A successful approach can be using DNA strand sets forming tensegrity triangles to precisely assemble 3D DNA molecu- lar crystals. These crystals can incorporate organic semicon- ductors or DNA devices. However, the lattice type can be limited to that motif and the small guest species (<7 nm). DNA origami in the format of a tensegrity triangle was used to reproduce this type of lattice through stacking interactions with guest nanoparticles. Here, DNA origami frames of different shapes were used to both host desired nano-objects and access different lattice symmetries through vertex-to- vertex hybridization of frames. Unlike stacking, hybridiza- tion permits a great control over interactions encoding, energetics and flexibility of interframe linkages. Co-assem- bly of lattices of spherical nanoparticles and DNA frames of different shapes depend on the intricate balance of maxi- mizing a number of particle-to-frame hybridizations and minimizing interframe interactions. Thus, formation of the lattice depends on the particle properties. In contrast, a 3D ordered lattice can be fully assembled purely from DNA frames with shapes of Platonic solids, such as a tetrahedron, octahedron and cube. Here, Polyhedral frames were capable of interframe hybridization via vertex-to-vertex hybridization (FIG. 1). Thus, the DNA frame 101 possesses a spatially determined valence (v) defined by its shape. Interframe hybridization results in assembly with the ability to form an ordered lattice 102, whose type is determined by the frame valence. The frame can be empty or enclose nano-objects 103 (e.g. nanoparticles or proteins), correspondingly resulting in the formation of an empty DNA framework or arrays of pre- cisely positioned nano-objects. This strategy allows to use the same assembly process for creating arrays of very different types of nano-objects (inorganic nanoparticles and proteins), since the assembly approach intrinsically decouples formation of the lattice 102 from the nano-objects 103 and their specific materials identity (FIG. 1, left). The
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US 12,548,243 B2 17 DNA frame 101 with nano-object 103 inside (FIG. 1, middle) represents a building block for the formation of a3D lattice 102 (FIG. 1, right). This block has a specific valence determined by the vertices of the DNA frame and carries a specific nanomaterial object inside (i.e., DNA material voxel 104). Instead of the optical signature associated with pixel terminology, a DNA material voxel 104 is an elementary volumetric block carrying material property and its binding characteristics are fully determined by the frame shape 101 that leads to a 3D nanomaterial lattice 102. 3D DNA origami lattices were formed from octahedral frames (valence, v6, edge length is about 29 nm) contain- ing twelve 6-helix-bundle edges (6HB), designed with caD- NAno. Each octahedron vertex (OB) incorporates single- stranded (ss) DNA motifs withm+1 bases (FIG. 2A). FIG. 2A shows an example ssDNA encoded octahedral frames OB and OR (left) with complementary strands that are mixed to form a DNA lattice (right) and an unit cell (center) with three faces of the unit cell (dots). FIG. 2B provides a graph showing 2D SAXS pattern (inset) and corresponding structure factor, S(q), for 3DDNA lattice and a fit modelled using a SC lattice of DNA octahedra. FIG. 2C shows an example negative stained TEM image for DNA lattice formed by DNA octahedra. This construction allows for hybridization of 1 nucleotides with complementary strand fragments (1+n free bases, in FIG. 2A) on another octahe- dron (OR) (Table 1). Motifs m and n define the number of bases in respective polyT domains, while motif 1 refers to the number of complementary bases in the binding region between the two sequences. The vertex-driven sixfold octa- hedral valency can yield ordered assemblies through vertex- to-vertex hybridization of frames, which probed by small angle X-ray scattering (SAXS) and understand by compar- ing to a validated model accounting for the nano-object’s form factor, DNA bundle arrangement and the lattice type. For the octahedral frame assemblies (I=8, m=22, n=22; no encapsulated nano-objects), the 2D SAXS pattern and the corresponding structure factor, S(q) (where q is the scatter- ing vector), reveal a remarkably well-ordered simple cubic (SC) lattice with about 15 orders of resolution-limited Bragg, peaks (FIG. 2B). Negative stained transmission electron microscopy (TEM) (FIG. 1C and FIG. 10) reveals domain sizes of 3-5 jum. While formation of a stand-alone DNA framework offers proof-of-concept for this design approach, more relevant is to template nano-object organization into 3D arrays. Thus, 10 nm gold nanoparticles (AUNP) were first sequestered inside DNA octahedra before the material voxels self- assembled (FIG. 3A, R1). AuNPs are positioned in the middle of each octahedron by hybridization with its internal 5 strands (FIG. 3B). Then, OB and OR frames (with encaged AuNPs) can be mixed and annealed to form a templated 3D AuNP array. In non-limiting embodiments, AuNP arrays can also be formed post facto (FIG. 3D, R2). Nanoparticles were added into a solution of formed, empty frame lattices at 2.5 times excess over the octahedra. Annealing allows for the diffusion/distribution of particles into the ordered scaffold. The resulting S(q) (FIG. 3E) is in close agreement with route 1. Thus, both routes successfully create the same 3D nanopar-ticle array. To explore the effect of intervertex linkage designs, the ssDNA length was modi- fied and thus the interoctahedra distance d (Table 1 and FIGS. 11 and 12). Well-ordered SC lattices were observed for all but the smallest d values, possibly due to destabilizing electrostatic and steric effects at close separations. To further emphasize this approach’s versatility, one of the two types of cages can be selected to prefill with w 40 4 65 18 nanoparticles while leaving the other empty. Thus, even within the restriction of identical DNA cage scaffolds, a totally different nanoparticle superlattice results (FIG. 3F). Approximately 20 recognizable SAXS peaks (FIG. 3G, black curve), where the peak position ratios q/ql=1: root (4/3): root (8/3): root (11/3:2) . . . correspond to a face- centered-cubic lattice with a lattice constant of 81.2 nm. Detailed S(q) modelling confirms (FIG. 3G, red curve) that the nanaoparticles are located precisely in every second octahedron in the frame lattice (FIG. 2). An advantage of this assembly method can be that nano- objects of different kinds can be organized in 3D ordered arrays in a similar manner. For example, encaged quantum dots (QD, cadmium selenide, 5 nm core, FIG. 4A, inset) were assembled using material voxels based on the same type of DNA octahedra. Modelling shows excellent agree- ment between the calculated and measured SC S(q) (FIG. 4A, structure illustrated in FIG. 4B). This versatile lattice formation strategy can be expanded to create other symmetries using DNA material voxels with different valence values. Cubic frames (v=8) composed of twelve 6HB with edge lengths similar (~29 nm) to the octahedral frames were assembled (FIG. 13). Two kinds of cubes (CB and CR) were used, where each CB vertex contained three DNA strands complementary to the three CR vertex strands (FIG. 4C). Both cube populations have another set of DNA sticky ends at each vertex that point towards the cube centre for binding with ssDNA of 10 nm AuNPs. TEM images (FIG. 4A and FIG. 14) show that the AuNPs are encaged with high fidelity; this encapsulation presumably helps to restore cube shape from their skewed empty forms to a normal, undistorted form. Cube assembly was carried out by mixing CB and CR with encaged nan- oparticles, followed by annealing as described above. The experimental data and model S(q) (FIG. 4A, curve), reveal a well-ordered body-centered-cubic (BCC) lattice, which is in-line with a vertex-to-vertex hybridization of cubic frames. Considering the nearest interparticle distance (66.2 nm), each CB is connected to CRs at eight vertices yielding the BCC arrangement. Next assembly of material voxels with v=4 using hybrid- ization between vertices of tetrahedral DNA frames was tested for which each edge is composed of a 10HB with length ~36 nm (FIG. 15) and four internal DNA strands bind 10 nm AuNPs (FIG. 4C). Equal amounts of two kinds of tetrahedra possessing complementary sets of six sticky end sequences at their vertices (TR) and (TB) in FIG. 4E were mixed and annealed. SAXS (FIG. 4E) revealed a well- ordered diamond type lattice with ~15 observable scattering peaks (black and red curves). Each tetrahedron binds with four tetrahedra through its vertices (FIG. 4F). This organi- zation is different from particles that were only arranged in the lattice under the restriction of the specific size ratio of a frame footprint to a particle. Here, formation of diamond lattice in respect to frames was detected, thus, any objects encaged in the frames can form a diamond lattice. The correlation between the DNA frame valence and the resulting lattice was identified. Conventional theories for DNA-mediated self-assembly operate within the limit of high grafting densities and isotropic interaction site distri- bution. In the presently disclosed model, however, interac- tion sites are localized to small regions, resulting in ‘patchy’ interframe attractions. Wertheim’ s thermodynamic perturba- tion theory was modified for associating particles to properly account for this patchiness. The resulting free energies yield the most favorable ground state crystal structure for a particular frame. Cubic frames preferentially self-assemble
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US 12,548,243 B2 19 into a BCC structure, whereas the octahedral frames pack into a SC lattice; diamond lattices are favored for tetrahedral frames. The preference for these morphologies can be explained by decomposing the AG into its enthalpic and entropic components, AG=AH-TAS, where AG is the change in Gibbs free energy, AH the change in enthalpy and TAS the change in entropy; enthalpy dominates were iden- tified. Further, the size and interaction range of these patches depends on the degree of frame truncation; the degree of truncation can also be used to modulate the self-assembled morphologies. These results suggest that truncation tuning can provide an additional powerful handle for expanding this approach to wider ranges of crystal symmetries. While this theoretical understanding underscores the Opportunities in a crystal lattice design, the DNA material voxel assembly strategy was expanded by moving beyond inorganic AUNPs and QDs as templated nano-objects to organic materials, by creating ordered 3D protein arrays. As an illustration, streptavidin, a protein containing four high- affinity sites for biotin binding, was used. Six hosting sites were designed for streptavidin, one at each vertex of an octahedral DNA frame (v=6), with each site containing four ssDNA attached to the four corresponding edges that form the vertex (FIG. 5A). Thus, each streptavidin can bind to biotinylated DNA that is complementary to the edge-at- tached strands. FIGS. 16 and 17 provide data for a second organization whereby DNA cages attach a single streptavi- din. FIG. 16A shows a model of protein caged octahedron. Star shape is the streptavidin, while four balls represents four biotins. FIG. 16B shows a representative cryo-EM image for one streptavidin inserted octahedral particles. Circles are examples of particles selected for further analy- sis. Scale bar: 100 nm. FIG. 6C shows a reference-based 2D class average for one protein inserted octahedra as obtained from cryo-TEM single particle analysis. Arrow showed the density of protein inside the octahedron. Scale bar: 20 nm. FIG. 17A shows a diagram of assembly of one-streptavidin caged octahedra (OB and OR) into 3D lattice. FIG. 17B shows I(q) vs q for the DNA lattice with and without one protein in a cage. The protein-filled material voxels were characterized with cryo-EM using single particle analysis. 2D class averages of raw particle images are nearly identical to the corresponding reconstructed 3D density model, demonstrating the correct assembly of six proteins in the octahedral cage (FIG. 5B). The cryo-EM 3D map (resolution ~23 A), allowing for the visualization of the precise protein positions (FIG. 5C), shows that the octahedral edge is ~30 nm long, in agreement with the disclosed design. The streptavidin lattice structure shows that the streptavidin proteins are well-docked into six 5 globular densities located at the inner space of vertices. These measurements also indicate full protein occupancy inside cages and high stability of cage-streptavidin com- plexes. Subsequently, OB and OR frames were mixed with streptavidin and biotinylated DNA (labeled with Cyanine 3), followed by annealing. The assembled pink-colored aggre- gates were examined using Confocal Fluorescence Micros- copy and found fluorescent ~3-5-am-sized square shapes (FIG. 4D and FIG. 18). Control experiments mixing OB, OR and labeled strepta- vidin, but without biotinylated DNA, showed no fluorescent aggregates, confirming that protein incorporation into DNA frameworks requires specific binding. SAXS of the encaged streptavidin lattice (FIG. 5E) shows the same symmetry and peak positions as for the corre- sponding empty DNA cage lattice; moreover, the similar S a w 8 w 60 B 20 electron density of DNA bundles and streptavidin leads to roughly similar peak intensities for both structures. How- ever, small differences were observed in two highlighted regions of S(q) (FIG. 5E). Detailed modeling, accounting for the geometry, locations and density of DNA cages and the six encaged streptavidin proteins (FIGS. 4C and 4F) matches the overall shape of the experimental S(q) for both empty and protein-filled ordered DNA frameworks and moreover, explains the observed differences in S(q). Clearly, protein arrays are templated by an ordered 3D array of octahedral DNA cages. The confirmation and characterization of different 3D lattices, organizing both inorganic and organic materials, demonstrates the versatility and universality of this DNA material voxel assembly approach. This platform was used to synthesize two different material organizations with prop- erties relevant to dramatically different applications-optical using a combination of QDs and catalytic using a cascaded enzyme network organized within a 3D lattice. The exploitation of colloidal QD properties can lead to a rapidly growing market for QD-based devices and even more specifically, QD optoelectronics. The tunability of a cadmium selenide core shell QD emission has made them an ideal candidate for QD light-emitting diodes, which have characteristic high color purity over a color gamut that is far larger than has traditionally been incorporated into display technologies. Such devices are formed from layers or films of QDs, yet it has the photoluminescence quantum yield, PL that is reduced by one to two orders of magnitude in such formats in a field where higher display brightness is the desired device characteristic. The 3D organization of QDs with a controlled, larger separation distance can improve the yPL by reducing energy transfer between QDs. Further- more, precise control over the structural (unit cell, lattice parameters and so on) and material properties of 3D QD organizations can allow for the engineering of desired QD superlattice photo-optical responses. Eight distinct binding sites were programmed within the interior region of octahedron DNA origami through the use of orthogonal, interior ssDNA overhangs. The OB and OR octahedra were each encoded to host a specific cadmium selenide QD, possessing fluorescence emissions of either 525 or 705 nm, thus resulting in two optically distinctive types of DNA material voxels. ssDNA-modified QDs were then annealed into empty, fully assembled material voxel (v=6) lattices designed to either be half-filled by a single QD or fully-filled by two types of QDs at a 1:1 ratio (charac- terization in FIGS. 19-27). Fluorescence microscopy imag- ing and spectra measurements were performed on the QD superlattices, demonstrating a ‘perfect mixing’ of the two fluorescence emissions in superlattices encoded to bind both QDs in equal ratios (FIG. 6A). This ‘perfect mixing’ refers to the inability to identify an emission signal from a non- equal QD mixture over a wide range of scan areas down to minimal diffraction-limited scales, (FIG. 6B and FIGS. 27A-27J) and thus presents a 3D optical organization with subdiffraction spectral stability. Furthermore, the fluores- cence lifetime (t) of the mixed QD superlattices (FIGS. 24A-24B) was within 15% of superlattices of only one QD for both 1525 and 1705 (FIGS. 20A, 20B, 22A, and 22B), indicating a minimal energy transfer a vast improvement over directly layered, thin-film systems. Next, the catalytic functionality of 3D biomolecular arrays were demonstrated. While the effects of enzyme colocalization in simple geometries and one-dimensional or 2D scaffolds were shown, this 3D format provides both significantly denser enzyme packing and a different spatial
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US 12,548,243 B2 21 architecture. ssDNA-modified glucose oxidase (GOx) and horseradish peroxide (HRP), were bind, respectively, to a single interior hosting site within each OB and OR octahe- dra, forming two enzymatic types of material voxels (v-6), which are co-assembled in a lattice as discussed above. The GOx-HRP pair can be used as a model enzymatic cascade and tracked by the synthesis of fluorescent resorufin from the precursor Amplex Red upon glucose addition (reaction overview and sample layout shown in FIG. 6C). Equal amounts of origami and enzyme were utilized in each experimental setup to ensure accurate comparisons of activ- ity. The 3D architecture (I) yields a nearly 300% increase in initial reaction velocities (V) over the solution format con- taining the same concentration of enzyme encaged in free octahedra (III), as shown in FIG. 6D. The reaction is undertaken at a concentration of glucose above that needed to achieve Vmax, as demonstrated by FIG. 28. In order to determine the role of DNA lattice organization itself versus a high local density of origami and enzymes, an associated origami aggregate was formed. This synthesis involved dissociating a lattice prior to enzyme incorporation by heating to 52° C., holding for 1 h and then immediately placing the tube on ice to induce the formation of an amorphous assembly. This formation procedure stands in contrast to the controlled, five-day anneal. Sample II in FIG. 6D demonstrates that aggregate assem- bly induces higher activity than free origami, but not to the extent of an ordered lattice: furthermore, the significantly larger sample variability for amorphous aggregates reflects an intrinsic variability of the disordered states. These results indicate that the lattice architecture contributes to increased activity of the enzymatic cascade, rather than solely to aggregation into higher local concentrations. Multiple con- tributions can play a role in this higher activity, including high packing density, colocalization of catalytic components and local environment effects of the DNA framework. This work shows an ability to structure enzymes into active high-density 3D arrays using a material voxel strategy and the possibility of manipulating and enhancing cascade reac- tions using 3D packaging of enzymes. In summary, the assembly platform was shown for cre- ating 3D lattices from nanomaterials of different natures, both inorganic and bio-organic, as well as stand-alone DNA origami frames. The disclosed methods integrate DNA frames with a prescribed valence and material nano-objects into a material voxel. This allows the definition of a lattice symmetry and a lattice composition through the material voxel design and enables nanomaterials with novel optical and chemical properties. The presented strategy offers a 5 powerful pathway for the rational assembly of 3D ordered nanomaterials from desired nano-objects for a broad range of applications. Example 2: Characterization of DNA-Prescribed and Valence-Controlled Material Voxels Fabrication of empty DNA lattice and ‘cargo’-inserted DNA lattices: For empty DNA lattices, OB and OR were mixed with a ratio of 1:1 (same method for other shapes). For single ‘cargo’ inserted DNA lattices, the ratio for OB, OR and ‘cargo’ complex were mixed with a ratio around 1:1:2 (same method for other shapes). For the system of 6 streptavidins, the ratio was around 1:1:15. The mixed solu- tion was then carefully annealed by cooling from 50° C. to room temperature with a rate of 0.2° C/h to obtain the lattice. FIG. 7 provides a diagram showing an example a 40 4 3: 2 s B 22 structure of octahedral DNA frames. m bases is a poly T part from OB vertex, n bases is a poly T part from the OR vertex. Hybridization between these strands occurs with 1 comple- mentary bases. FIG. 8 provides a diagram showing another example structure of cubic DNA frames including bases of a poly T part from the vertex (m, n) and complementary bases (1). Melting temperature determination: The melting tempera- ture of the octahedral 3D DNA lattice was obtained by Dynamic Light Scattering (DLS). The mixed sample solu- tion was cooled slowly in the sample chamber from 50° C. to room temperature and measured the in situ size of the assemblies to obtain the dependence of the size of aggre- gates versus temperature. FIG. 9 provides a graph showing example melting temperature measured by dynamic light scattering for octahedral DNA lattice. Encaging quantum dots inside DNA lattice for mixed QD lattice: Two complimentary octahedron DNA origami are annealed, following the lattice annealing protocol described previously, without any guest particles. Assembled DNA origami crystals were then mixed with the functionalized QD’s and undergo a short (~4 hour) secondary annealing protocol. In detail, the QD’s were mixed with lattice at a 3x molar excess, for example (QDA:OB) was (3:1). The sec- ondary annealing protocol included the following proce- dure: 25° C. for 10 sec, 35° C. for 10 sec, 40° C. for 1 min, 45° C. for 10 min, then a ramp of -5° C./hr was applied to the samples for the next 4 hours, bringing the final tempera- ture to 25°C. Fluorescence microscopy and measurements: Confocal imaging and micro-PL spectrum measurements were con- ducted on Leica SP5 TCS laser-scanning confocal imaging/ hyperspectral imaging microscope mounted with a 63x, 1.2 NA water immersion objective. A CW argon laser (458 nm) was applied to excite the sandwiched sample, and three photomultiplier (PMT) channels were selected for simulta- neous PL imaging of three different colors (500-550 nm, $55-576 nm, and 680-730 nm) accompanied by a transmit- ted light detector (TLD) for transmission imaging. Hyper- spectral imaging was performed using a single PMT channel in the spectrum range of 500-749 nm (bandwidth 5 nm, detection step size 3 nm). The micro-PL spectra were extracted from hyperspectral images in various regions of interest (ROIs). The hyperspectral imaging can be slightly affected by the detection efficiency of the PMT channel due to the large wavelength range, but it is reasonable to compare spectra from various ROIs. Time-resolved confocal fluorescence-lifetime imaging microscopy (FLIM) measurements were performed on a home-built inverted microscope (Olympus IX8 1) with raster scanning stage mounted with a 60x, 1.2 NA water immer- sion objective. The sample solutions (typically 0.8 pL) were sandwiched by hydrophobic coverslips and excited by a pulsed diode-pumped solid-state laser system (LHD-440 PicoQuant, wavelength 440 nm, full width at half maximum 90 ps) operated at 10 MHz repetition rate with an average power of 500 nW. The photoluminescence (PL) was col- lected in the epi-illumination scheme, spectrally separated from the excitation laser light by a dichroic mirror (Sem- rock, DiO-495) and spatially filtered by a 100 um pinhole. Then the PL was split by another dichroic mirror (Semrock, DiO-605). The transmitted and reflected PL were filtered by 690/40 and 525/50 band-pass filters (Semrock), respectively, and imaged onto two single-photon-counting avalanche photodiodes (MPD PicoQuant) coupled to a time-analyzer
Page 92
US 12,548,243 B2
23
(PicoHarp 300, PicoQuant). Data acquisition and data analy-
sis were performed with the Symphotime 64 analysis soft-
ware (PicoQuant).
DNA functionalization of enzymes: Glucose oxidase
(GOx) was purchased from Sigma Aldrich. Horseradish
peroxidase (HRP) was purchased from Thermo Fisher Sci-
entific. Enzymes were first mixed with Sulfo-EMCS in ratio
of 20:1 (Sulfo-EMCS:enzyme) in PBS buffer (pH 7.4) for
1.5 hours at room temperature, and then purified by a
Amicon 30 k cutoff filter. Thiolated oligonucleotides (IDT),
(i and ii, Table 3) were reduced by TCEP (1000:1) in PBS
buffer. The excess TCEP was removed by size exclusion
column (G-25, GE Healthcare). Next, sulfo-EMCS-treated
enzymes were mixed with activated oligonucleotides with a
ratio of 1:5 in PBS buffer and rotated at 4° C. overnight. The
DNA-attached enzymes were then purified by Amicon cutoff
filter (50 k for GOx, 30 k for HRP). The number of DNA
attachment on enzymes was quantified by UV-Vis. Follow-
ing is the method for calculating DNA attachment on
enzyme (GOx):
Ayzgq(DNA-attached GOX)=€2¢9(DNA)C(DNA}+€ 260
(GOx)C(GOx) qa)
Ags DNA-attached GOx)=€459(GOx)C(GOx) Q)
The DNA attachment ratios were calculated to be approxi-
mately 1.0 for GOx and 1.8 for HRP.
Encaging enzymes inside DNA lattice—experimental set-
ups and methods: DNA-attached enzymes were mixed with
octahedral DNA lattice (10 nM), octahedral DNA lattice
melted for 1 hour at 52° C. and immediately placed on ice,
free octahedra containing enzyme binding sites, and free
octahedra with no enzyme binding sites in a ratio of 1:1 in
TE buffer (pH 7.5) containing 12.5 mM MgCl. Due to
contribution of TAE buffer from the DNA samples, the final
pH was measured to be pH 8.0. The sample was gently
shaken at room temperature for 24 hours. Glucose and
Amplex Red were added at final concentrations of 150 mM
and 200 uM, respectively, with additional MgCl, to ensure
final concentration was maintained at 12.5 mM. 100 pL
reactions were measured in a 96-well plate using a Tecan
Infite plate reader, with four repeats performed for each
sample.
Sample preparation: The carbon-coated grids were glow
discharged in a 0.39 mbar air atmosphere for 1 min by using
PELCO easiGlow (Ted Pella, Inc.). Before EM grid prepa-
ration, the sample was assessed for homogeneity by nega-
tive-stain electron microscopy. 3 ul of sample solution was
applied to glow-discharged carbon-coated lacey grids, incu-
bated for 2 min at 10° C. and 95% humidity, blotted for 3 s_5
then plunged into liquid ethane using an FEI Vitrobot IV.
The grids were loaded into a FEI Talos Arctica electron
microscope operated at 200 kV high tension and collected
images semi-automatically with EPU under low-dose mode
at a magnification of x92,000 and a pixel size of 1.55 A.A
Falcon III direct electron detector was used under linear
mode for image recording with an under-focus range from
1.5 to 3.5 m. The dose rate was 20 electrons per A? per
second and total exposure time was 2 seconds. The total dose
was divided into a 39-frame movie and each frame was
exposed for 0.05 s.
Image processing and 3D reconstruction: For octahedral
DNA, 2155 raw movie micrographs were collected. For
octahedral DNA with one streptavidin in center 1122 raw
movie micrographs were collected. For octahedral DNA
with six streptavidins in corners, 615 raw movie micro-
graphs were collected. The movie frames were first aligned
S
a
w
8
40
4
24
and superimposed by the program Motioncorr 2.0. Contrast
transfer function parameters of each aligned micrograph
were calculated using the program CTFFIND4. Subsequent
procedures, including particle auto picking, 2D classifica-
tion, 3D classification, 3D refinement, and density map
post-processing were performed using Relion-2.0°. Tem-
plate for automatic picking was firstly generated from a 2D
average of about ~1,000 particles from different views.
Automatic particle selection was performed for the entire
data set. Then the particles obtained from automatic picking
were assessed and removed the bad particles. 2D classifi-
cation of all good particles was performed and particles in
unrecognizable classes by visual inspection were removed.
Particles in good classes were used for further 3D classifi-
cation, and the best model was chosen for further 3D
refinement. The resolution of the final maps was estimated
by the gold-standard Fourier shell correlation, at the corre-
lation cutoff value of 0.143. The crystal structure of strepta-
vidin (PDB code 4YVB) was docked as a single rigid body
into the EM density in the Chimera program. Structural
figures and movies were prepared in Chimera.
Cryo-samples for imaging under STEM were prepared
similar to that described above but blotted for only 0.5s
before plunging into the liquid nitrogen-cooled liquid eth-
ane. The samples were then transferred to a cryogenic holder
and imaged under a field-emission transmission electron
microscope (TEM) (JEOL 2100F). The TEM was operated
at 200 keV in the annular dark-field scanning TEM mode
(ADE-STEM).
Analysis of polycrystalline domains: To identify
polycrystalline domains, three Fourier masks that are related
to three different crystalline domains that have different
relative crystallographic orientations were used (FIGS. 29A-
29D). The Fourier masks were found by windowed Fourier
transfer. This process can be automated using independent
component analysis/multivariate curve resolution; however,
for simplicity, three domains that have distinctive Fourier
space patterns were manually extracted. Inverse Fourier
transform of the Fourier space masked results illustrates
where the crystalline sub-domain is concentrated.
To identify the orientation of the subdomains, windowed
FFT was used to find relatively pure phase and indexed the
reflections and zone axes. The lattices have been sheared due
to cryo sample prep: during sample prep, the water film is
finite think and the surface can exert force on the embedded
the structures. Using this method, a couple low index
orientations was deconvolved. The most inner green set of
reflections are the {100} reflections. The corresponding
domains are projected along <001> orientations. The reflec-
tions in the “second shell” are the {110} reflections. The
corresponding domains are projected along <111>. The set
of reflections are the {111} reflections. The corresponding
domains are projected along <110> orientations.
Small Angle X-ray Scattering (SAXS): The SAXS mea-
surements were conducted at the Coherent Hard X-ray
(CHX) and Complex Materials Scattering (CMS) beamlines.
The 2D scattering data were collected on area detectors
placed downstream of the sample. Information on the beam-
line setups is found in Table 3
TABLE 3
The specific characteristics of the CMS and CHX beamlines.
Beamline cHX CMS
Photon energy (keV) 9.65 13.5
Horizontal x Vertical Beam size 10 x 12 100 x 100
(um x yum)Page 93
US 12,548,243 B2
25
TABLE 3-continued
The specific characteristics of the CMS and CHX beamlines.
Beamline CHX CMS
Approximate Flux (photons/s) Io! 10"!
Sample-Detector Distance {m) 5.00 5.038
Detector Manufacturer Dectris Dectris
Detector Model Eiger 4M Pilatus 300k
Detector Pixel Size (um) 15 I?
Area images were integrated into a one-dimensional (1D)
I(q) scattering curve as a function of the scattering vector q,
where
q= sn(5)
with and 6 being the wavelength of the incident X-rays and
the full scattering angle, respectively. The resultant 1D
curves spanned roughly 0.04 nm’ to 1 nm’ with a reso-
Jution of 0.002 nm7!. The structure factor S(q) was obtained
by dividing I(q) by the corresponding particle form factor
P(q).
In this work, modeling of the presented analysis was
implemented using the ScatterSim software package, a
python package that implements a scattering formalism for
superlattices. This formalism can generically model the
scattering from arbitrary anisotropic nano-objects within the
unit cell of a regular superlattice. This library was extended
to perform the modeling described below.
SAXS theory: The scattered intensity I(q) of any object in
solution versus q is related to the Fourier transform of the
arrangement of its electrons:
Kg=ClF(g)l? 3)
Fogle? "a? @
where an overall scale factor dependent upon parameters
specific to the experimental setup, such as solid angle (pixel
size/detector distance), integration time, illuminated volume
and beam fiux is considered by the constant C. Here, p’
refers to an effective electron density which is described
later. Typically, this sum is broken up over its constituents,
(objects). Nano-objects as solids with uniform density were
modeled. The samples considered here are gold, CdSe, and
protein spheres, as well as DNA duplex bundles which was
modeled as cylinders. For some small volume that is rep-
resentative of the density of the larger scale element (homo-
geneous), the effective electron density is defined as:
1 6)
At low angles, f, is equal to the number of electrons per
atom and f,' is an energy dependent correction term arising
from quantum mechanical effects of the binding energy the
electrons bound to their atoms. the q dependence in f, was
neglected, since this is pronounced only at atomistic length
scales (wide-angle scattering), and not relevant to the
nanoscale measurements (small-angle scattering) consid-
ered here.
25
35
40
45
0
55
65
26
These effective electron densities result in an overall scale
factor for the scattered field of each object.
For the accuracies needed here, one can neglect dispersion
effects and assume that the sample density is roughly the
same as the mass density for simple elements of the periodic
table (an average of | electron every 2 AMU). In this case,
the electron density can be substituted with the mass density.
This results in an overall scale factor for equation (2), which
is a degree of freedom for the fits presented here. The latter
are easier to determine. From here on, p' will refer to the
sample density and not the electron density.
Scattering with a solvent: When a solvent is present in the
sample, it has the effect of reducing the electron density
contrast. The scattering of a sample and solvent can be
rewritten as:
FDA, Psa dP Hy, Prot dP )
where V,,, and V,,, are the volume over the sample and
solvent, respectively. Since the scattering of a uniform
sample is zero except at q=0, the solvent integral to integrate
over the full scattering volume V,,,,; Was Written:
FUG. qe0=h, Paaiei a
PegPsal? "dF =
7
0)
and the sum of the scattering of sample and solution is then
the difference of effective electron densities:
san-Psa Od?
FD =Svien co)
The effect of the solvent is to screen the scattering of the
sample, by reducing the density contrast. Thus, the scatter-
ing strength of samples of varying electron densities in
solution depends not on their absolute electron density, but
effective electron density difference. For cases of one type of
object of the same effective electron density in solution, this
is simply an overall scale factor and will be ignored. For
cases of multiple types of objects with different electron
densities each, this can be taken into account.
Form factor of a sphere: The form factor of a sphere is
well known and is:
an
ep fs ®
Faptorol@ R, p’) = srr
, sin(gR) — gReos(qR)
(Ry
Form Factor of a cylinder: The cylinder form factor for a
cylinder of height h and radius r is defined as:
Siler)
(10)
Foglq) = Bsinetgshn/2)—
ay
where J; is the first order of the Bessel function of the first
kind.
Form factor of composite objects: More complex non-
overlapping objects can be modeled adding objects whose
form factor is known, shifted by some Ar and rotated by
rotation matrix R. Importantly, this can be done at the level
of the scattering amplitudes (and not the intensity) so that
interference between sub-components is correctly captured.Page 94
US 12,548,243 B2 27 From (2), it is easily seen that a rotation and shift of an object amounts to a rotation and phase shift in its form factor amplitude: FGARAP)=FG-RY 4”. wy Thus addition of objects to form composite object consists of the following sum: FomposielDZjectieenF (GER Vea () where Ty and R, are the shift and rotation transformation of each object, respectively. This is taken into account by the ScatterSim library. Polydispersity: Finally, object polydispersity can be intro- duced. Since any sample contains a probability distribution in its parameters, the form factor can be averaged over these parameters. The probability distribution chosen here is the Gaussian probability distribution. The form factor moments, dependent on some parameter € with average value € and standard deviation 6: <Fig)> =/Ple€o)Fiq.edh (4) and <IF(Qh?> =[PUDIF(Qh)Pdh (5) where neo? (16) PGR 0) =0 is the Gaussian distribution. Note that this operation can be performed multiple times over multiple parameters ¢, (for example, cylinder radius, and orientation) so long as they are uncorrelated. In the ScatterSim library, this is computed by numerically integrating over a list of objects, or randomly creating objects whose parameters are sampled from their respective probability distributions and averaging their resultant form factor amplitudes F(q) or form factor inten- sities [F(q)?. For notational brevity, the symbol E can signify an average over one or many of these uncorrelated parameters. Form factor intensity: a collection of objects freely float- ing in solution will then yield an intensity: Kg)P@)=s\FQ)”>, (dy) where P(q) is known as the isotropic form factor intensity. The scattered intensity was calculated by the ScatterSim library. The scattered intensity of a periodic lattice is mod- eled by: TKg\=eZ (VG GQ4P@I-B@OG@) where c is an overall scale factors, P(q) is the form factor intensity of the composite object comprised of all objects in the lattice (assuming their relative positional order and orientation preserved), and 1g) 19 ZO= 0) Lom P Da e . 2 Pa (FATA) expL2niCxjh-+ yjk-+ 29D] LQ ~ gu) is known as the lattice factor, where L(q-q,,,.) is a peak shape function. The structure and symmetry of the lattice is taken into account by properly sampling over their corre- 20 25 30 45 60 65 28 sponding Miller indices q,,,,. G(q) is the Debye-Waller factor arising from thermal vibrations in the lattice, defined as: Giger Ferm, where a denotes the lattice size and 6,,,,,, the rms displace- ment of the elements in the lattice. For a simple cubic lattice of length a, it is convenient to re-write this as fractional displacement of the lattice length (20) opy = . TDyms Finally, B(q) is defined as: KEP Ql) (For), B@= and arises from any polydispersity in parameters E. The effect of G(q) for increasing thermal vibrations (or static positional disorder) is to reduce the ordered scattering from Z,(q) and increase the diffuse scattering from unordered elements. The effect of B(q) is the same except that the decreasing trend is not an exponential decay, but rather a more complex trend that depends on the length scales of the parameters €. In the cases involved here, B(q) is not con- sidered. Structure factor: The structure factor is defined as the ratio of the scattered intensity [(q) from the lattice to the form factor of the underlying sub-element, as if it was dispersed in solution. Since for the most part the latter is not easily obtained, it is estimated as described in further sections. Both the structure factor for the respective data and calcu- lated models are then normalized by this factor. SAXS modeling results: The data was modeled using the ScatterSim library using the theory described above. This model has been validated, and successfully used to deter- mine the structures of superlattices. This model accounts for the lattice symmetry and unit cell size, particle shape and various types of disorder such as vibrations, finite grain size and polydispersity. The unit cell size and shape define the peak positions, and the particle shape and disorder strongly affect the relative peak heights. For some of the systems modeled here, the particle shape is especially important as the particle anisotropy modulates the peak heights in a non-intuitive fashion. The ScatterSim library was extended® to allow an easier method to compound more complex nano objects that will be presented here. Scattering of an octahedron: The DNA octahedron is the central scaffold used in the nano-engineered superlattices presented here. It is comprised of 12 bundles of 6 cylinders formed into an octahedron shape (FIG. 30). It was modeled by 12 cylinders representing each edge defined by the cylinder radius rp,,4. cylinder height hp, and DNA octa- hedron edge length L,,,, (FIG. 31). It was found that modeling a 6-cylinder bundle with 1 larger cylinder was sufficient, with no appreciable difference in computed mod- els as compared to modeling each DNA duplex within the bundle as a distinct cylinder. This simplification was chosen to reduce computation times. The DNA octahedron model: First, a superlattice of DNA octahedra was measured, assembled in a cubic lattice of lattice spacing apy, (FIG. 32). The scattering contrast of DNA is approximately an order-of-magnitude weaker than gold and so the scattering is 100 times weaker. To put this
Page 95
US 12,548,243 B2 29 in perspective, in order to obtain a measurable scattering signal at the CMS beamline, integration times of 100 s were necessary, as compared to only seconds being required for the superlattices containing gold nanoparticles. Both a sus- pension of freely floating un-aggregated DNA octahedra, and DNA octahedra assembled into a superlattice were measured to obtain P(q) and I(q), respectively. Due to the background scattering dominating the sample in the free- floating DNA octahedra case, a control measurement of the buffer solution without the sample was also measured, and subtracted from it. The results can be seen in FIG. 33. One can see that roughly the shape of the form factor P(q) aligns with the superlattice scattering I(q). This suggests that the DNA freely floating in solution retain the same shape as those bound in the superlattice. The model for the DNA octahedra: As mentioned previ- ously, the ScatterSim model was modified to allow for the computation of the scattering of more complex nano objects. The octahedron was made by combining cylinders together, as mentioned above (FIG. 32). The cylinder is assumed to be centered on each edge, so that the length of the ends of the cylinders to the vertices of the octahedron are all equal (FIG. 31). Ignoring a constant scale factor in absolute intensity, the DNA octahedron is then defined solely by four free param- eters: the height of each cylinder hpy,,, their radius tpy,, and the edge length of the DNA octahedron L,,,, they combine into (FIG, 31). The parameters used for these models involv- ing DNA octahedra were rp,,4=3.0 nm, Npy.,=31.6 nm and Lypy1=39.0 nm, with the best fit shown in FIG. 33. Polydis- persity was then added by varying the DNA octahedron edge length Ly, by 0, 20% Ly. leading to the green curve in FIG. 33. Finally, the model is rescaled by a constant to account for the overall scale factor. When combined in a superlattice, the DNA octahedra align in a cubic structure. The only defining parameter for such a lattice is the length of one of the basis vectors, apy,4 The peak widths from the crystalline peak are modeled by Lorentzian curves, defined by their full width half maximum. (FWHM), o,. Finally, disorder from positional disorder (and/or dynamic motion of objects) extinguishes higher- order peaks through the Debye-Waller factor o,,,. Ignoring scaling factors, the scattering of the superlattice is thus defined by the free parameters: hpy.4, fpy4 and Lyx, for the octahedra, as well as apyy, Op and Gpzy for the lattice. The structure factor S(q) and its model are found in the and FIG. 30. The structure factor here is taken by normal- izing the data or simulated intensity by estimated form factor for freely dispersed octahedra. As this form factor is not measured confidently, it is estimated by an empirically 5 determined power law fit of q'° (FIG. 33). Arange of possible models was assessed; the experimen- tal data can only be explained using the proposed DNA octahedron model. FIG. 34 shows the structure factor of a model where the octahedra are rotated 45 degrees around one of the unit cell basis vectors. The same estimated form factor for freely dispersed DNA octahedra. There is a strong disagreement between the data and this model, demonstrat- ing that octahedron orientation is important. The strong disagreement from a change in orientation can be under- stood by viewing a 2D cross section of the modeled scattered intensity in of the DNA octahedra in both cases (FIG. 35). When the DNA octahedra are assembled in a simple cubic lattice, this form factor is modulated by sharp peaks denoted by the dots in the figure. Changing the DNA octahedra orientation changes the modulation of the peaks and thus the modulation of the peaks in the resultant 1D curve. a w 8 w 8 40 4 2 s 30 Finally, the shape of the octahedron is also crucial. This is demonstrated by comparing the measured structure factor to a case where the cylinders in the DNA octahedra (FIG. 31) are replaced by spheres of radius 3 nm. There is clear disagreement with the experimental data (FIG. 36). In this case, the same estimated form factor for freely dispersed DNA octahedra is used for ease of comparison. Other models including large spheres, spherical shells, filled octa- hedra, and hollow octahedra likewise did not yield agree- ment. DNA octahedra with quantum dots: A sample of DNA octahedra with quantum dots (QDs) in a superlattice was measured using SAXS. The same model of DNA octahedra was used as the previous section. A quantum dot was then added to the model, of radius tgp=2.5 nm and surrounded by proteins of radius r,,,.,=2.5 nm (FIG. 37). The density of the quantum dot is approximately 5.8 g/cm? ''. The streptavidin protein is assumed to have roughly the same density as DNA, near 1.7 g/cm? '?. All samples were modeled as immersed in water of density 1 g/cm*. Three models were computed: 1. DNA octahedra only, 2. QD and proteins only, 3. DNA octahedra with the QD and 6 proteins. The com- puted model structure factors are compared with the data and shown in FIG. 37. For the structure factors presented in FIG. 3 of the main text, the same power law as mentioned in section 3m was used for the model of freely dispersed DNA octahedra and its variants. Models 1 and 2 are the extreme cases, when either the scattering from the octahedra or the quantum dots dominate the scattering, respectively. It is found that neither of these scenarios can fit the data. However, when combining the two together, and adjusting the sizes of the protein and QD such that their scattering is comparable to the DNA octahedra, one obtains relatively good agreement between the modeled curve and the mea- sured data. Since small changes in particle size or density changes the overall scattered intensity of the samples in question, an accurate measurement of the density is unnec- essary in this case. Since the densities and particle sizes are all approximate, the conclusion of this result is not to confirm the particle sizes themselves, but that the presence both the DNA octahedra and QD with protein objects are necessary for the model to agree with the data. Adding proteins: the results for DNA octahedra contain- ing 6 internally-coordinated proteins, located near the ver- tices, at d,,,,.=14.3 nm from the center of the octahedron are shown (FIG. 38). For the structure factors presented in FIG. 4, the same power law as mentioned in section 3m was used for the model of the freely dispersed DNA octahedra and its variants. The difference in the scattering curves is extremely subtle, owing to the small overall change in electron density that the introduction of the proteins engenders. Nevertheless, the computed model curve suggests differences in the scat- tering curves at the same q-values, supporting the hypothesis that the observed differences are a direct result of the protein inclusion. (FIG. 39). In the model, the mass of the protein was fixed to be roughly equal to the mass of one cylinder of DNA. The density was assumed to be the same as that of the DNA octahedra, and the radius was set to be 2.5 nm. At this radius, the form factor contribution of the protein is rela- tively flat, and variations in radii do not affect the results of the model. Since the density is rescaled such that the total mass of the protein remains the same, any variation in this parameter does not lead to an appreciable change. Model for DNA tetrahedron: A sample constructed of DNA tetrahedra was also measured using SAXS. The DNA tetrahedra model is shown in FIG. 40, and described using: Tpna> Dpya and Lyx. The scattering of free tetrahedra was
Page 96
US 12,548,243 B2
31
experimentally unmeasurably weak. However, when com-
bined into a superlattice, the DNA tetrahedra give rise to
distinct structural peaks (FIG. 41). Since it is measured
directly, the form factor for the tetrahedra is estimated
empirically using a q” power law, as can be seen in FIG. 41.
This estimates the overall trend of the expected form factor
(the theoretical model for the form factor is shown in FIG.
41).
Four candidate superlattice models were computed and
compared to the data. The first model was a cubic arrange-
ment of tetrahedra, which is seen in FIG. 42. The tetrahedra
are rotated such that in this cubic arrangement, each tetra-
hedron vertex touches only one other tetrahedron. This is the
expected configuration given the steric packing constraints
for DNA tetrahedra vertices. The second model was a
hexagonal diamond arrangement of tetrahedra. Each tetra-
hedron is oriented such that each vertex connects to exactly
one vertex of another tetrahedron. The third model is a cubic
close-packed arrangement of tetrahedra. In this lattice, the
vertex of each tetrahedron connects to the three vertices of
three other tetrahedra. Finally, the fourth model is this same
model, but where the tetrahedra are placed further apart, so
as to account for the observed scattering peak position. The
models are compared to the data in FIG. 43.
The ‘close-packed’ cubic lattices configurations do not
correctly match the experimental data, and are also unlikely
on physical grounds. The cubic diamond and hexagonal
diamond arrangement of tetrahedra appear to agree with the
data, with cubic diamond more closely matching the experi-
mental data. Given the high degree of structural similarity
between cubic and hexagonal diamond arrangements, it is
possible that the experimental system is a principally cubic
diamond lattice with occasional hexagonal diamond defects.
Tn any case, the scattering curve suggests that tetrahedra are
arranging into a diamond-like configuration, where each
tetrahedron is connected to four neighbors along its vertices.
SC, FCC, BCC and diamond lattices: The SC, FCC, BCC,
and diamond of gold spheres in a DNA lattice are shown in
FIG. 44, FIG. 45, FIG. 46, and FIG. 47, respectively. In each
fit, a model of gold spheres was used. The DNA octahedra
were not modeled due to the fact that the origami effective
electron density is approximately one order-of-magnitude
smaller than the effective electron density of gold. In each
model, candidate fits for each BCC, FCC and simple cubic
are plotted. For the case of diamond, the second case of
cubic diamond is also plotted. The form factor used in the
structure factor S(q) was obtained from fitting the high q
term of the intensity to the known sphere form factor as in
FIG. 48.
Theoretical Considerations.
Wertheim’s theory: In order to provide a starting point, a
description of Wertheim’s theory for associating liquids is
provided. Briefly, an associating particle was defined as one
that possess both short-range and highly directional inter-
actions (FIGS. 49A-49B). By construction, the particles are
only attractive within the patchy regions and repulsive
everywhere else. Thus, the potential was decomposed into
their attractive and repulsive components, 0,(r) and 0,(r),
respectively.
ONO AW). (22)
orsd 3)
eavr={ Or>d’
OsZacrEperGapt") (24)
5
20
25
30
35
40
45
50
60
65
32
where d is the particle diameter, I is the set containing all
N patchy sites in the system, and o and B define the subset
of patchy sites associated with the particles whose potential
is being evaluated. As a example, if an interacting particle
has n patchy sites, then o is the subset of I that identifies
those patchy sites G={Py_p.4, Vyggos + + Uycoen} with b
acting as a dummy indexing variable pointing to where
subset o exists in P.
Through the introduction of Mayer-f functions, f,,(r)=exp
[6(@7/kT]-1, density expansions, graph theory, and cluster
reductions, Wertheim showed that the exact Helmholtz free
energy for an associating fluid can be written as,
A () 25)
Fe fle] -e +3, orate
where p(1) is the single molecule density integrated over all
coordinates (1), p,(1) is the subset of p(1) that consist of
only monomers (“unbonded” particles), A is the standard de
Broglie wavelength, G,_,, is a density operator representing
the density sums of subsets of a sites, and c is an graph set
that describe a sum from singlet to n-body interactions
within the system.
GAB, Pull) 26)
Q7)
Car)
ed =) G
=o
the subscript k indicates the cluster size associated with
k-particle interaction. The value of c cannot be rigorously
determined. However, in the limit of only one patch per
particle (shown in FIG. 1), a single bonding condition was
imposed. This results in the cancellation of all k>2 terms,
resulting in;
ol 08)
62.45 [nstnfica exten 09de0)
Oa
where g,(12) is the two body hard-sphere radial distribution
function. Excess free energy of association of the form is
defined:
A-ay 1 1 @9)
7 = focfinss— Frcs s}m
with X,(1) as the equilibrium monomer fraction
Ke poll) _ -1+¥1+48 G0)
arial 28
Tn the context of two free patchy sites coming together to
form a pair of associated particles, the process can be a
“reaction” where two “unbonded” sites come together to
form a “bond.” Within this framework, A represents the
equilibrium constant of the system:
KeAa2lf(Agstnd?
The above result for a singlet patch particle can be gener-
alize to particles with multiple patches. Briefly, the densities
GDPage 97
US 12,548,243 B2 33 P,(1) and p,(2) in the single patch bonding approximation can be substituted out for the density operators G,_,, and O,_g and interactions are summed over all sites associated with particles o and B. This approximation gives a gener- alized excess Helmholtz free energy for a homogeneous multipatch particle system of the form: 4- =e (G2) =v fe ‘ifinsy- 2 feu ca ere 3) aris 2npd Therefore, for a particle with n patches, the equilibrium constant becomes, Kpnlf(ngoind? co) Eg. 31-33 represent the major results determined by Wertheim for a homogeneous associating fluid with multiple bonding sites per particle. Crystal Reference Derivation. While the results provided by Wertheim provide an accu- rate description for associating fluids, utilizing the hard- sphere fluid as the reference state for a perturbation analysis of crystalline structure will fail for lattice predictions as the crystalline morphology deviates too far from that of a bulk fluid to be properly captured by a first order approximation. Here, the reference state to that of any crystalline structure of interest was casted. Similar to the two approximations made for singlet and multiplet patchy particles, an approximate form for c that accurately reflects the crystalline state was defined. In the limit of a perfect crystalline structure, all particles are connected into a single cluster, thus k=1. This singlet cluster sum incorporates an infinite number of interactions as all particles feel the effect of other particles fo) (0) fy Eh, Feld Petty ey * 85) co = where the subscripts I, II, III etc indicate single-body, two-body, three-body etc interactions, respectively. While this reduces the analysis to only systems containing a single cluster, the infinite sum over all possible n-body interactions was not performed. To address this summation problem, the short attraction constraint for the system was imposed. Within this limit, only adjacent lattice points have a non- vanishing contribution. As a result, the term that remains corresponds to an s-body interaction where s represents the crystal coordination number associated with a given lattice. ce now reduces to a reference term and the corresponding s-body interaction term: (36) = or SD Ler Tm Deeg 9+ Ld dQ)... d+ 1) if a homogeneous system, c'” simplifies to wo 0), Pet 67) 6 =p BL rings 10 20 35 40 3 55 2 3 34 where the subscipt Ii indicate the density of particle i. This equation can employ a small perturbation about a reference crystal lattice (defined by the radial distribution function £.,(t)). While subtle, it is worth noting the key difference between the two approximations. The free energy change due to local perturbations about a homogeneous solution that results from patchy associations is shown in certain ques- tions. On the other hand, certain equations probe how turning on association between particles already occupying lattice sites further stabilizes the reference crystal structure. It can be shown that the resulting equilibrium constant takes the form: rl 68) Ks ae Se gate) where 0(m, n) is a coordination saturation parameter that depends on n, the number of patches, and m, the crystal coordination number associated with the reference structure. o(m, n) acting on f,(r) accounts for association of a refer- ence particle to its neighbors within the crystal lattice. In the limit where m2n, the reference particle possesses an excess number of patches than required by the crystal coordination number. Here, all attractions that stabilize the lattice are satisfied and 6(m, n)=n, indicating that there are n bonds formed per particle within the lattice. In the opposite limit where m<n, the reference crystal has a coordination number greater than that of the number of patchy sites available on the particle. In those situations, the energetic contribution from association is limit to the num- ber of patches on the particle, thus o(m, n)=m. The pre- factor p,”"'/a! accounts for both the indistinguishably and probability of finding n—1 particles within the cutoff inter- action distance. The free energy of lattice formation can then be calculated via the relation AG=-kT In K.. Free energy decomposition: From standard thermody- namics, the free energy of lattice formation can be decom- posed into an enthalpic and entropic component AG=AH— TAS. To assign a functional form to both AH and AS, the expression for K. was simplified by introducing a binomial expansion of the Mayer-f function to give: pr Ink, = In} | G9) n! U(r) va . af f Xe (Hoole |- vgotea] +o] fips car The first and last terms has no energetic term and thus correspond to the entropic contribution and whereas the middle term containing the Boltzmann weight of the inter- particle potential represents the enthalpic contribution to AG, giving: (40) an of e-uf [OE (3 Jex[- 2] ‘sora fe a | H f (1 entra a As a simplifying assumption, this can be a limiting case where the only dominating interaction exist at the the position related to the first coordination shell in the crystal
Page 98
US 12,548,243 B2 35 lattice—r,. Doing so allows us to define a approximation for the crystal pair correlation function as g,,(r)=m8(r-r,). where is indicates the crystal coordination number. Plugging this approximation into the relations for AH and AS, assum- ing that the highest ordered term in the free energy expan- sion dominates, and normalizing by the number of patches per patchy particle result in, m 42) lm nyo mp! 43) ica where U(r,)=u,. These results indicate that the enthalpic contribution stems from patchy association between par- ticles occupying each lattice site whereas the entropic com- ponent comes from counting the number of ways the refer- ence particles can find an associating particle within the lattice. By inspection, AH dominates over AS, emphasizing that patch-patch association drives lattice stabilization and governs the morphological transition between the various structures. 10 20 36 Design of patchy particle model: the cubic, octahedral and tetrahedral nanocages have a degree of truncation at their vertices to provide room for DNA linker placement and facilitate hybridization interactions. To properly model the patchy interactions, a relationship between the degree of truncation and design parameters were derived (FIGS. 50A- 50C). For the both cases, a parameter 6 is defined as the ratio between the shortened vertex length h and the full length L. A degree of truncation term 1 is defined as the ratio of the center to truncated vertex to center to full vertex lengths. By simple geometrical arguments, the degree of truncation 1 is defined as a function of the ratio 6. 1-6 44) Octahedron, Tetrahedron “ Cube 214646 Experimentally, 6 is set as 0.75 and 0.84 for the octahe- dron and cube, respectively. This gives in =0.06 for the cube and )=0.12 for the octahedron. Octahedral DNA staple strands (SEQ ID NOs: 1-120 respectively top to bottom): OC-staple-1 OC-staple-2 oC-staple-3 oc-staple-4 OC-staple-5 OC-staple-6 oC-staple-7 OC~staple-8 oC-staple-9 oC-staple-10 OC-staple-11 OC-staple-12 OC-staple-13 oC~staple-14 oc-staple-15 OC-staple-16 oC-staple-17 OC-staple-18 oc-staple-19 oc-staple-20 oc-staple-21 OC-staple-22 oc-staple-23 oc-staple-24 TCAAAGCGAACCAGACCGITTTATATAGTC GCTTTGAGGACTAAAGAGCAACGGGGAGIT GTAAATCGTCGCTATIGAA TAACTCAAGAA AAGCCTTAAATCAAGACTTGCGGAGCAAAT ATTTTAAGAACTGGCTTGAATTATCAGTGA CTTAAAATTCGCATTATAAACCTAAACTAG AGCACCATTACCATTACAGCAAATGACGGA ATIGCGTAGATTTTCAAAACAGATIGITIG TAACCTGTTTAGCTATITICGCATTCATTC GTCAGAGGGTAATTGAGAACACCAAAATAG CTCCAGCCAGCTTTCCOCTCACGACGTTCS CTCCACTATTAAACAACCACTTTTGGTTCC TAAAGGTGGCAACATAGTAGAAAATAATAA GATAAGTCCTGAACAACTGTTTAAAGAGAA CCTAATACTAAAATGTAAGTTTTACACTAT TCAGAACCGCCACCCICTCAGAGTATTAGC AAGGGAACCGAACTGAGCAGACGGTATCAT CTAAAGATTCAAAACCOCTCACTTGACCCT AGGCGTTAAATAAGAAGACCGTGTCGCAAG CAGGTCGACTCTAGAGCAAGCTICAAGGCG CAGAGCCACCACCCTCTCAGAACTCGAGAG TTCACGTTGAAAATCTIGCCAATGGGATIT AAGTTTTAACGGCGTCGCAGTGTAGAATGG TTGCGTATTGGcecccoccccceTecec TC
Page 99
US 12,548,243 B2 37 -continued 38 Octahedral DNA staple strands (SEQ ID NOs: 1-120 respectively top to bottom} oc-staple-25 oc-staple-26 oc-staple-27 0C-staple-28 0C-staple-29 oc-staple-30 oc-staple-31 oc-staple-32 oc-staple-33 0c-staple-34 OC-staple-35 oc-staple-36 oc-staple-37 oc-staple-38 0c-staple-39 0c-staple-40 o¢-staple-41 oc-staple-42 0C-staple-43 oc-staple-44 oc-staple-45 oc-staple-46 oc-staple-47 oc-staple-48 Oc-staple-49 0c-staple-50 oc-staple-51 oc-staple-52 oc-staple-53 oc-staple-54 Oc-staple-55 0c-staple-56 0c-staple-57 0c-staple-58 oc-staple-59 oc-staple-60 oc-staple-61 0C-staple-62 0C-staple-63 GTCACCAGAGCCATGGTGAATTATCACCAATCAGAAAAGCCT GGACAGAGTTACTTTGTCGARATCCGCGTGTATCACCGTACG CAACATGATTTACGAGCATGGAATAAGTAAGACGACAATARA AACCAGACGCTACGTTAATAAAACGAACATACCACATTCAGG TGACCTACTAGAAAAAGCCCCAGGCAAAGCAATTTCATCTTC TGCCGGAAGGGGACTCGTAACCGTGCATIATATTTTAGTICT AGAACCCCAAATCACCATCTGCGGAATCGAATAAABATTTTT GCTCCATTGTGTACCGTAACACTGAGTTAGTTAGCGTAACCT AGTACCGAATAGGAACCCAAACGGTGTAACCTCAGGAGGTTT CAGTTTGAATGTTTAGTATCATATGCGTAGAATCGCCATAGC AAGATTGTTTTTTAACCAAGAAACCATCGACCCAAAAACAGG: TCAGAGCGCCACCACATAATCAAAATCAGAACGAGTAGTATG GATGGTTGGGAAGAAAAATCCACCAGAAATAATTGGGCTTGA CTCCTTAACGTAGAAACCAAT CAATAATICATCGAGAACAGA AGACACCTTACGCAGAACTGGCATGATTTTCTGTCCAGACAA, GCCAGCTAGGCGATAGCTTAGATTAAGACCTTTTTAACCTGT CCGACTTATTAGGAACGCCATCAAAAATGAGTAACAACCCCA GTCCAATAGCGAGAACCAGACGACGATATTCAACGCAAGGGA CCAAAATACAATATGATATTCAACCGTTAGGCTATCAGGTAA AACAGTACTTGAAAACATATGAGACGGGTCTITTTTAATGGA, TTTCACCGCATTAAAGTCGGGAAACCTGATTTGAATTACCCA GAGAATAGAGCCTTACCGTCTATCAAATGGAGCGGAATTAGA ATAATTARATTTAAAAAACTTTTTCAAACTTTTAACAACGCC GCACCCAGCGTTTTTTATCCGGTATTCTAGGCGAATTATTCA GGAAGCGCCCACAAACAGTTAATGCCCCGACTCCTCAAGATA GTTTGCCTATTCACAGGCAGGTCAGACGCCACCACACCACCC CGCGAGCTTAGTITTTCCCAATTCTGCGCAAGTGTAAAGCCT AGAAGCAACCAAGCCAAAAGAATACACTAATGCCAAAACTCC ATTAAGTATARRGCGGCAAGGCAAAGAAACTAATAGGGTACC CAGTGCCTACATGGGAATTTACCGTTCCACAAGTAAGCAGAT ATAAGGCGCCAAAAGTTGAGATTTAGGATAACGGACCAGTCA, TGCTAAACAGATGAAGAAACCACCAGAATTTAAAAAAAGGCT CAGCCTTGGTTTTGTATTAAGAGGCTGACTGCCTATATCAGA CGGAATAATTCAACCCAGCGCCAAAGACTTATTTTAACGCAA CGCCTGAATTACCCTAATCTTGACAAGACAGACCATGAAAGA ACGCGAGGCTACAACAGTACCTTTTACAAATCGCGCAGAGAA, CAGCGAACAT TAAAAGAGAGTACCTTTACTGAATATAATGAA GGACGTTTAATT TCGACGAGAAACACCACCACTAATGCAGAT AAAGCGCCAAAGTTTATCTTACCGAAGCCCAATAATGAGTAA,
Page 100
US 12,548,243 B2 39 -continued 40 Octahedral DNA staple strands (SEQ ID NOs: 1-120 respectively top to bottom} oc-staple-64 oc-staple-65 0C-staple-66 Oc-staple-67 oc-staple-68 oc-staple-69 0C-staple-70 0C-staple-71 0C-staple-72 0C-staple-73 0C-staple-74 0C-staple-75 OC-staple-76 0C-staple-77 oc-staple-78 0c-staple-79 oc-staple-80 oc-staple-81 0c-staple-82 0C-staple-83 oc-staple-84 0c-staple-85 oc-staple-86 0C-staple-87 oc-staple-88 0c-staple-89 0c-staple-90 oc-staple-91 oc-staple-92 0C-staple-93 oc-staple-94 0c-staple-95 oc-staple-96 0C-staple-97 oc-staple-98 0c-staple-99 0c-staple-100 0c-staple-101 GAGCTCGTTGTARACGCCAGGGTTTTCCARAGCAATAAAGCC AATTATTGTTTTCATGCCTTTAGCGTCAGATAGCACGGARAC AAGTTTCAGACAGCCGGGATCGTCACCCTTCTGTAGCTCAAC ACAAAGAAATTTAGGTAGGGCTTAATTGTATACAACGGAATC AACAAAAATAACTAGGTCTGAGAGACTACGCTGAGTTTCCCT CATAACCTAAAT CAACAGTTCAGAAAACGTCATAAGGATAGC CACGACGAATTCGTGTGGCATCAATTCTITAGCAAAATTACG CCTACCARCAGTAATTTTATCCTGAATCARACAGCCATATGA GATTATAAAGAAACGCCAGTTACAAAATTTACCAACGTCAGA AGTAGATTGAAAAGAATCATGGTCATAGCCGGAAGCATAAGT TAGAATCCATAAATCATTTAACAATTTCTCCCGGCTTAGGTT AAAGGCCAAATATGTTAGAGCTTAATTGAT TGCTCCATGAGG CCAABRAGGAAAGGACAACAGTTTCAGCGAATCATCATATTCC GARATCGATAACCGGATACCGATAGTTGTATCAGCTCCAACG TGAATATTATCAAAATAATGGAAGGGTTAATATTTATCCCAA GAGGAAGCAGGATTCGGGTAAAATACGTARAACACCCCCCAG GGTTGATTTTCCAGCAGACAGCCCTCATTCGTCACGGGATAG CAAGCCCCCACCCTTAGCCCGGAATAGGACGATCTAAAGTTT. TGTAGATATTACGCGGCGATCGGTGCGGGCGCCATCTTCTGG CATCCTATTCAGCTAAAAGGTAAAGTAAAAAGCAAGCCGTTT CAGCTCATATAAGCGTACCCCGGTIGATGTGTCGGATTCTCC CATGTCACAAACGGCATTAAATGTGAGCAATTCGCGTTAAAT AGCGTCACGTATAAGAATTGAGTTAAGCCCTTTTTAAGAAAG. TATAAAGCATCGTAACCAAGTACCGCACCGGCTCTAATATCC ATAGCCCGCGARAATAATTGTATCGGTTCGCCGACAATGAGT AGACAGTTCATATAGGAGAAGCCTTTATAACATTGCCTGAGA, AACAGGTCCCGAAATTGCATCAAAAAGATCTTTGATCATCAG ACTGCCCTTGCCCCGTTGCAGCAAGCGGCAACAGCTTTTTCT TCRAAGGGAGATAGCCCTTATAAATCARGACAACAACCATCG GTAATACGCAAACATGAGAGATCTACAACTAGCTGAGGCCGG GAGATAACAT TAGAAGAATAACATAAAAAGGAAGGATTAGGA CAGATATTACCTGAATACCAAGTTACAATCGGGAGCTATITT CATATAACTAATGAACACAACATACGAGCTGTTTCTTTGGGG ATGTTTTGCTTTTGATCGGAACGAGGGTACTITTTCTITTGATAAGAGGTCATT GGGGTGCCAGTTGAGACCATTAGATACAATTTTCACTGTGTGARATTGTTATCC CTTCGCTGGGCGCAGACGACAGTATCGGGGCACCGTCGCCATTCAGGCTGCGCA TCAGAGCTGGGTAAACGACGGCCAGTGCGATCCCCGTAGTAGCATTAACATCCA TTAGCGGTACAGAGCGGGAGAAT TAACTGCGCTAATTTCGGAACCTATTATTCT
Page 101
US 12,548,243 B2 41 -continued 42 Octahedral DNA staple strands (SEQ ID NOs: 1-120 respectively top to bottom}: 0C-staple-102 0C-staple-103 0C-staple-104 0C-staple-105 0C-staple-106 0¢-staple-107 0c-staple-108 0C-staple-109 0c-staple-110 oc-staple-111 0C-staple-112 0c-staple-113 0C-staple-114 0c-staple-115 0c-staple-116 0c-staple-117 0c-staple-118 0c-staple-119 0C-staple-120 GATATTCTAAAT TGAGCCGGAACGAGGCCCAACTTGGCGCATAGGCTGGCTGAC TGTCGTCATAAGTACAGAACCGCCACCCATTTTCACAGTACARACTACRACGCC CGATTATAAGCGGAGACTTCRAATATCGCGGAAGCCTACGAAGGCACCAACCTA AACATGTACGCGAGTGGTTTGAAATACCTAAACACATTCTTACCAGTATAAAGC GICTGGATTTTGCGTTTTAAA TGCAATGGTGAGAAA TAAATTAATGCCGGAGAG GCCTTGAATCTT TTCCGGAACCGCCTCCCAGAGCCCAGAGCCGCCGCCAGCATT. CGECTGSTGCTTTCCTGAATCGGCCAACGAGGGTGGTGATIGCCCTTCACCGCCT TGATTATCAACTTTACAACTARAGGAATCCAAAAAGTTTGAGTAACATTATCAT ACATAACTTGCCCTAACTITAATCATTGCATTATAACAACATTATTACAGGTAG GTAGCGCCATTAAATTGGGAATTAGAGCGCAAGGCGCACCGTAATCAGTAGCGA TTIATTTTTACCGACAATGCAGAACGCGCGAAAAATCTTTCCTTATCATTCCAAG TTTCAATAGAAGGCAGCGAACCTCCCGATTAGTTGAAACAATAACGGATTCGCC GGGCGACCCCAARAGTATGTTAGCAAACTARAAGAGTCACAATCAATAGAAAAT AGCCGAAAGTCTCTCTTTIGATGATACAAGTGCCTTAAGAGCAAGAAACAATGA, GIGGGAAATCATATAAATATT TAAATTGAATTTTTGTCTGGCCTTCCTGTAGCC CCCACGCGCAAAATGGTTGAGTGTTGTTCGTGGACTTGCTTTCGAGGTGAATTT. ATGACCACTCGTTTGGCTTTTGCAAAAGTTAGACTATATTCATTGAATCCCCCT TCCAAATCTTCTGAATTATTTGCACGTAGGTTTAACGCTAACGAGCGTCTTTCC GGGTTATTTAATTACAATATA TGTGAGTAATTAATAAGAGTCAATAGTGAATTT: In order to attach one gold nanoparticle or quantum dot (525, 605) inside the octahedral DNA frame, add (SEQ ID NO: 121) ‘ ATCCATCACTTCATACTCTACGTIGTIGITGTTGTTGTT’ in front of certain sequences (e.g., OC-staple-98, 100, 101, 109, 111, 115, 116, and 117). In order to attach quantum dot (705) inside the octahedral DNA frame, add (SEQ ID NO: 122) ‘ CTACCATCATACCTACTCTACGTTGTIGTTGTTGTTGTT’ in front of certain sequences (e.g., OC-staple-98, 100, 101, 5 109, 111, 115, 116, and 117). In order to attach 6 strepta- vidins inside the cage, add (SEQ ID NO: 123) ‘ATCCATCACTTCTTT’ in front of certain sequences (e.g., OC-staple-50, 52, 53, 55, 56, 57, 58, 59, 63, 65, 67, 68, 69, 72, 75, 77, 80, 82, 85, 87, 91, 93, 95, and 96). In order to attach 1 internal GOx enzyme, add (SEQ ID NO: 124) \TTCTTCTTCATCCATACTACCATCTAC! 45 in back of certain sequences (OC-staple-78); In order to attach | internal HRP enzyme, add (SEQ ID NO: 125) 50 \CTICTTCTTATACTTCACTACCTAATC! in back of certain sequences (OC-staple-78). These staple strands, when used for interior binding in structural charac- terization were ordered as PAGE-purified, while those used in functional systems (QD and enzyme) were ordered as standard desalted. Both showed high levels of material incorporation. DNA sticky ends for octahedron (OR, m = 22, 1 = 8, n = 22) (SEQ ID NOs: 126-149 respectively, top to bottom): TTTGCGGATGGCCAACTARAGTACGGGCTTGCAGCTACAGAGTTTTTTTTTTTTITTTTTTTTTATCCGTTA, CTTCATCAAGAGAAATCAACGTAACAGAGATTTGTCAATCATTTTTTTITTTTTTITTTTTTTTTATCCGTTA, AAAGATTCATCAGGAATTACGAGGCATGCTCATCCTIATGCGTTTTTITITTTTTTTTITITTTATCCGTTA
Page 102
US 12,548,243 B2 4B -continued. 44 ATAAATCATACATAAATCGGT IGTACTGTGCTGGCATGCCTGTTTTITTTTTTTTTTTTITTTTIATCCGTTA GGTAGCTATT TTAGAGAATCGATGAAAACATTAAATGTGTAGTTTTTITTTTITITTTTTTTTIATCCGTTA CARATGCTTTAAAABATCAGGTCTTTAAGAGCAGCCAGAGGGTTTTTTTTTTTTITTTITTTTTATCCGTTA, TCATATGGTTTACGATTGAGGGAGGGARACGCAATACATACATITITTTITTTTITTTITTTTTATCCGTTA, CAACGCTCAACAGCAGAGGCATTTTCAATCCAATGATAAATATTTTTTPITTTTITITTTTTTTATCCGTTA, AATAGCAATAGCACCAGAAGGAAACCTAAAGCCACTGGTAATTTTTTTTTTTITTTTTTTTTTTATCCGTTA GACAGGAGGT TGAAACAAATAAATCCGCCCCCTCCGCCACCCTITITITITTTTTTTTITTTTTATCCGTTA AGCTTTCATCAACGGATTGACCGTAAAATCGTATAATATITTTITITTTITTITTTTTITTTTTATCCGTTA AGAGCCTAATTTGATTTTTTGTT TAAATCCTGAAATAAAGAATTTTTTTTTTTTTTTTTTTTTTATCCGTTA GCTCACAATTCCGTGAGCTAACTCACTGGAAGTAATGGTCAATTTTTTTTTTTTTTTTTTTTTTATCCGTTA CTTAAACAGCTTATATATTCGGTCGCTTGATGGGGAACAAGATTTTTTTITTTTITITTTTTTTATCCGTTA, AAACGAAAGAGGGCGAAACAAAGTACTGACTATATT CGAGCTTTTTTTTTTTITTTTTTTTTTTATCCGTTA ACTGTTGGGAAGCAGCTGGCGAAAGGATAGGT CAAGATCGCATTT PTTTTTTITTTTTTTTTTTATCCGTTA, GGCCCTGAGAGAAGCAGGCGAAAATCATTGCGTAGAGGCGGTTTTTTTTTTTITTTTTTTTTTTATCCGTTA AACGGGTATTAAGGAATCATTACCGCCAGTAATTCAACAATATTITTTTTTTITTTTTTTTTTTATCCGTTA CAGAATCAAGTTTCGGCATTTTCGGTTAAATATATCACCAGTTTITITITITITIITITITTTITATCCGTTA, GAAACATGAAAGCT CAGTACCAGGCGAAAAATGCTGAACAAATTTTTTTTTTTTTTTTTTTTTTATCCGTTA ATCAAAATCATATATGTAAATGCTGAACAAACACTTGCTICTITITTTTTTTTTITTTTTTTTTATCCGTTA, TGATTGCTTTGAGCAAAAGAAGATGAAATAGCAGAGGTTTTGTTTTTTPTTTTITTTTTTTTTTATCCGTTA TTTGCGGAACAATGGCAATTCATCAATCTGTATAATAATTTTTITT[TTTTTTTITTTTTTTTTTATCCGTTA TGTAGCATTCCAACGTTAGTAAATGAAGTGCCGCGCCACCCTTITITITITTTTITITTTTTITATCCGTTA, DNA sticky end for octahedron (OB, m= 22, 1=8, n respectively, top to bottom) : 22) (SEQ ID NOs: 150-173, TTTGCGGATGGCCAACTAAAGTACGGGCTTGCAGCTACAGAGTTTTTTTTITTTIITTTITTTITTAACGGAT CTTCATCAAGAGAAATCAACCTAACAGAGATTTGTCAATCATTTTTTTTTTTTITTTTTTTTTTTAACGGAT AAAGATTCATCAGGAATTACGAGGCATGCTCATCCTTATGCGTTTTTTTTTTTTITTTTTTTTTTAACGGAT ATAAATCATACATAAATCGGTIGTACTGTGCTGGCATGCCTGTTTTTTTTTTTTTTTTTTTITTTAACGGAT GGTAGCTATT TTAGAGAATCGATGAAAACATTAAATGTGTAGTTTITTTITTTTITTTTTTTTTTAACGGAT CABATGCTTTAAAAAATCAGGTCTTTAAGAGCAGCCAGAGGGTTTITTTITTTTITITITTTITTAACGGAT TCATATGGTTTACGATTGAGGGAGGGARACGCAATACATACATITITTTITTTIITTTITTTTTTARCGGAT CRACGCTCAACAGCAGAGGCATTTTCAATCCRATGATABATATITITTTTTTTIITTTTTTTTTTARCGGAT AATAGCAATAGCACCAGAAGGAAACCTAAAGCCACTGGTAATTTTTTTTTTITITTTTTTTITTTAACGGAT GACAGGAGGT TGAAACAAA TAAATCCGCCCCCTCCGCCACCCTTTITTTTTITTTTTTTTTITTTAACGGAT AGCTTTCATCAACGGATTGACCGTAAAATCGTATAATATITTTTTITTTITITTTTTTTTTTTTTAACGGAT AGAGCCTAATTTGATTTTTTGTT TABATCCTGAAATABAGAATTTTTTTTTTTTTTTTTTTTTTTARCGGAT GCTCACAATTCCGTGAGCTAACTCACTGGAAGTAATGGTCAATTTTTTTTTTTTITTTTTTTTTTARCGGAT CTTAAACAGCTTATATATTCGGTCGCTTGATGGGGRACAAGATTTTTTTTTTTTTTTTTTTTTTTAACGGAT AAACGAAAGAGGGCGAAACAAAGTACTGACTATATTCGAGCTITITTTTTTTTTITTTTTTITTTAACGGAT ACTGTTGGGAAGCAGCTGGCGAAAGGATAGGT CAAGATCGCATTTTTTTTTTTTTTTTTTTTTTTAACGGAT GGCCCTGAGAGAAGCAGGCGAAAATCATTGCGTAGAGGCGGTTTTTTTTTTTTTTTTTTTTTTTTAACGGAT
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US 12,548,243 B2 45 -continued. 46 AACGGGTATTAAGGAATCATTACCGCCAGTAATTCAACAATATTTTTTTITITTITITTTTTITTAACGGAT CAGAATCAAGTTTCGGCATTTTICGGTTAAATATATCACCAGTTITITITITITIITITITTTITTAACGGAT GAAACATGAAAGCTCAGTACCAGGCGAABAATGCTGAACAAATTTTTTTTTTTTTTTTTTTTTTTARCGGAT ATCARAATCATATATGTARATGCTGAACBAACACTTGCTICTTTITTTTTTITTTTTTTTTTTTTARCGGAT TGATTGCTTTGAGCAAAAGAAGATGAAATAGCAGAGGTTTTGITTTTTTITTTTITTTTTTTITTAACGGAT TTTGCGGAACAATGGCAATTCATCAATCTGTATAATAATTTITITITITITTTTITTTTTTTITTAACGGAT TGTAGCATTCCAACGTTAGTAAATGAAGTGCCGCGCCACCCTTTTTTITITTTTITTTITTTTTTAACGGAT Cubic staple sequence (SEQ ID NOs: 174-293, respectively, top to bottom): Cub-staple-1 TARATATTGACGGAAAATTGAGGTTGTCAC Cub-staple-2 CCGRACARAGTTACCAABAAGTATAAGCCC Cub-staple-3 TGAATCGGCCAACGCGGTGCCAGAATGAGT Cub-staple-4 AATCGTAAAACTAGCAAGAATCGGGGTAGC Cub-staple-5 AGAAGCCTTTATTTCAGTAATACGCAAAAT Cub-staple-6 ATTGTGAATTACCTTAAATTTCATCAGTGA Cub-staple-7 ATAGAAAGGAACAACTTTTCAGCTAGCGTA Cub-staple-8 CATGTAATTTAGGCAGTATTTAATGCGTTA, Cub-staple-9 ACGTTGTAAAACGACGGGTTTTCAAGGGCG Cub-staple-10 GACTTCARATATCGCGAAGAGGAAATCAAA Cub-staple-11 CAAATAAGAAACGATTATTATTTGAATCTT Cub-staple-12 GAGTGAATAACCTTGCATAAATCTCAAGAA, Cub-staple-13 ATTGCGTAGATTTTCAAAACAGATTGTTTG Cub-staple-14 GTICCACTATTAAAGAACCAGTTTIGGTTCC Cub-staple-15 CCCAATTCTGCGAACGCATATARAATATAA, Cub-staple-16 ATTTACCGTTCCAGTARBAGCGCTTGAGGC Cub-staple-17 GGCTTTTGCAAAAGAAAACCAAACAAAAGG Cub-staple-18 AACTTTTTCAAATATAACARAGATTTTAAC Cub-staple-19 CGTACTCAGGAGGTTTGGAATAGTCCTCAA, Cub-staple-20 CCGTAATGGGATAGGTACAAACGAAAATAA Cub-staple-21 TAATCAAAATCACCGGGTTIGCCGTTTGCC Cub-staple-22 AGCCGTTTTTATTTTCTCATCGACCTAATT: Cub-staple-23 AGCCGGAACGAGGCGCCTGCTCCCAAGCGC Cub-staple-24 ATCGGAACGAGGGTAGAGCAGCGAACCGAT Cub-staple-25 ARAAATGAGTTACAGCGTCTTTCCAGAGAATCATCATATTCC Cub-staple-26 GAATTACATTCTAGAGGATCCCCGGGTAATCCGCTCACAATT Cub-staple-27 AAGCAAAGACATCTGCCAGTTTGAGGGGCCGCTTCTGGTGAG Cub-staple-28 ACCCCGGTGAGAGTCTACAAAGGCTATCTCGCAAGCGGTCCA, Cub-staple-29 GAGAAGGGCCTGTACCATGTACCGTAACCCACCCTCCACCCT Cub-staple-30 TGAATATTATCAAAATAATGGAAGGGTTGCGCCTGTTTATCA, Cub-staple-31 CCTACCAACAGTAATATAAAGTACCGACAATGCAGAACGCTC Cub-staple-32 GATTATAATAAGTCCAACATGTTCAGCTAAAAGGTCGTCAGA,
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US 12,548,243 B2 47 -continued. 48 Cub-staple-33 Cub-staple-34 Cub-staple-35 Cub-staple-36 Cub-staple-37 Cub-staple-38 Cub-staple-39 Cub-staple-40 Cub-staple-41 Cub-staple-42 Cub-staple-43 Cub-staple-44 Cub-staple-45 Cub-staple-46 Cub-staple-47 Cub-staple-48 Cub-staple-49 Cub-staple-50 Cub-staple-51 Cub-staple-52 Cub-staple-53 Cub-staple-54 Cub-staple-55 Cub-staple-56 Cub-staple-57 Cub-staple-58 Cub-staple-59 Cub-staple-60 Cub-staple-61 Cub-staple-62 Cub-staple-63 Cub-staple-64 Cub-staple-é65 Cub-staple-66 Cub-staple-67 Cub-staple-68 Cub-staple-69 Cub-staple-70 Cub-staple-71 CCTGATACCGAACTCACCGACTTGAGCCGGCCGGAAACGTCG ARACGCACTTACCGGARACARTGARATATACACCATCAATAT TACCGCGGTATTARAAACCAATCAATAATTCGCCTTABATCAA, TAATAATTTGCTAATGTCGICTTTCCAGATGCTTGATACCGA, GAAACAAAGCAGCAATTACCATTAGCAAATTTGGGCAATCAT AGGTCAGAAACACTTACGAAGGCACCAAGGAAGT TTACATGG CATTGCCTTGATAACCAGGGTGGTTTTTGAGAGAGTTGCAAC TAAGCAAGAAACGCTAGCAARCGTAGAAGAACTGGGATARAA, ATATTCGTCTGRAACCGTATARACAGTTATRAGT TTACAGAG TAAAGTAAGATACACAGT CAGGACGTTGGTAGAAAGATTCAC AGAACGGCCCAATAGCAAGCCTCCCTCACACTTATCATTCCA TGCTGTATACCACACAACATTATTACAGGGAAGAATTAGTTT. CAGAACCGGGTTGATTAGCGGGGTTTTGTACACCAGTACAAA GARATCGAATATCAAATTAACTGAACACAGAATAATCCAACG CAGTAATGGGCT TAAGTATARAGCCAACAGGCGRATTATTCA, ATCTTCTTGATGCAGGGTTATATAACTACTCAGTACCAGGCC ATAAGGCCCAATAACTGAAAAGGTGGCAAATAACCTTAAGAA AAGGGAAAATTGTGCGGAGATTTGTATCAGCACCAATGAAAC TGGGATTTTTTTCACGTTGAAAATGTTTCCGAATTTTCTGTA GAGTAGTATTATACTTTCGCAAATGGTCTCAATTCTACTAAC TCAAAGCGCGGATTCCTGACTATTATAGTTCATCAACATTTA, GAGCTAAAGCTCATAACGTTAATATTTTAAAACAGAGGCGGT TICGCGTCCATTCGCCAGCTTTCCGGCAACGACGAGTGTAGA, AACAGTAAAGAGAACAGTACCTTTTACAAATCGCGCAGAGAA, CCGGAACGGTCATAGTAGCGCGTTTTCACGGCTGTCTTTCCC GCTATTACTTTITTCATTTAACAATTTCCAGCTGGCGAAAAA, TAACCCTGTAAAATCTCCAAAAAAAAGGATTTCTTAAACACA, CTGGCTCAAATTGGGACGAGAAACACCACAATAGTAGTAGCA, TCGGTTGGAACCCTCGGAATACCCAABAAATACATACATAAA, CTTTTGATCCTCATGCCTTGATATTCACTTGAGGCABAAGAA TAGCTATATAATAACATATATTTTAAATAGACAGTCAAATAA, TAAATGCGAACCGCCACCCTCAGAGCCAACTGAGTTTCGTTG ACCAACGCAGATGAAGAAACCACCAGAATTTAAAATAACGTC CCRAAGATCACCGTGACCAACTT TGAAACAAGAGTAATCTCG TACGAGCTGCTATTCCTCCCGACTIGCGTTATCCGGAATCAT CATTTTCCGCARATCAGATATAGAAGGCGGAGGT TT TGAAGG ATCGGTGCGGAAGCTGTGTGAAATTGTTCCGAGCTCCAAGCT GGTAATACGT TTACGTAAGAGCAACACTACGTTAGTAAATCT ATTTGCCAAAATAGACCGTCTATCAAATGGAGCGGAATTAGA
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US 12,548,243 B2 49 -continued. 50 Cub-staple-72 Cub-staple-73 Cub-staple-74 Cub-staple-75 Cub-staple-76 Cub-staple-77 Cub-staple-78 Cub-staple-79 Cub-staple-80 Cub-staple-81 Cub-staple-82 Cub-staple-83 Cub-staple-84 Cub-staple-85 Cub-staple-86 Cub-staple-87 Cub-staple-88 Cub-staple-89 Cub-staple-90 Cub-staple-91 Cub-staple-92 Cub-staple-93 Cub-staple-94 Cub-staple-95 Cub-staple-96 Cub-staple-97 Cub-staple-98 Cub-staple-99 Cub-staple-100 Cub-staple-101 Cub-staple-102 Cub-staple-103 Cub-staple-104 Cub-staple-105 Cub-staple-106 Cub-staple-107 Cub-staple-108 Cub-staple-109 Cub-staple-110 Cub-staple-111 TTTAGCGCCACCAGACCCTCAGAGCCGCGAGCCGCGCCACCA, TATTTTTTTGCCCCCACCGCCTGGCCCTCTTTICACATATGT ATTTTTATACCAAATCAGAGCATARAGCGCRAGGTGGCAACA ACGATCTGCCGACATGCTTTCGAGGTGACTCCARATTGCGAA AATCAATATTACCCTGGCTGACCTTCATGAGGACATTAAAGG AATAATAAACCGTTGTGAGAAAGGCCGGGCAATGCACCGAGG TGGECECCCCETCETCCTGTAGCCAGCTICCCGGAAACCAGG AACAAAATTTATCAGACGCTGAGAAGAGCTTAGAAAATCGTC TCAAAGGGAGATAGCCCTTATAAATCAACCCAGAGGGTAATT TACAAATTACCTGAATACCARGTTACAATCGGGAGTTCGAGC AGGCCGCGGACTAAGGAGTGTACTGGTAAATGCCCCCTGCAC AATTACGTTTAAACTATTCATTGAATCCAGACTGGCAGAGGG GCAAGGCCTGCAGGTCGACTAATTTTCCTCGGGGGATGTGCT GCTTTGATTTTGCGAGGCTTIGCAGGGAGAACTATTTCGGAAC GTCGAGAGCCACCCTCAGACCTAAATTTCACGGATAAGTGCC TIGCGTATTTCCAGTAATTGCGTTGCGCAGATTAAATTTTTG GTAACAAATCGTAACCGTGACCAGACCGGAAAATGTGAGCGA, AATCAGGATTTTTGTAATTGCTCCTTTTGAAGCAATCGAGCT TGAATTACAAAAGGTCATATGGT TTACCATTGACAAGAACCG CTCCGGCTAATTACTAAATAAGAATAAAAATGGTTTAATTTC GACCATTCGGTGTCATGTTTTAAATATGGAATCAGTTGAGAT AAATAAATGATACAAGACTITTTCATGACCTARAACGAAAAA ATAGCCCGCGAAAACAGCCTTTACAGAGCCTGAACAAAGTTA TGCCCGCTTGGGCGTCAGAARAGCCCCAGTTAARATTCGCGT TGCATGCGATTAAGCTTCGCTATTACGCATTTCCACACAACA ACAATAGCTTCTGAATTATTTGCACGTAGGT TTAAAAAGTAATTCTGTCCAGAC TAGTGAATTAAT TAAATGGAAACAGTACTTCTGTATCCTTGAARACATAGCGAT AGAACCATCAGACTGCCCCCTTATTAGCAACCAGACACCCTCAGAACCGCCACC AGAGGTCTCTTTACGCATCAAAAAGATTTTTTAATACTCCAACAGGTCAGGATT TAGTTGCAAAGTTTACAACTT TCAACAGAAAGGAAAGGAGCCTTTAATTGTATC GATATTCAGAGCRAAAGCCCTTTTTAAGGAAGGRACTGAGTAATGTGTAGGTAA TGATTATCTAACGAAAATAAACAGCCATITTTGITGTTTGAGTAACATTATCAT CAAATGCAGGCATACAGACGACGATAAAGTTTTGCATAGCGTCCAATACTGCGG TIAAATCCTCACATTCGGGAAACCTGTCCGGGGAGGAAGATIGTATAAGCAAAT TIAGGAAGCTCAACTGGAAGTTTCATTCAGTAGA TAAATCTACGTTAATAAAAC CTATTATGTCGCTGGGATCGTCACCCTCCAACGGCTTAACGGGGTCAGTGCCTT. CATCGATAGTACAATCGAAAT CCGCGACAGACGGTAATTAGAGCCAGCAAAATC GATATTCAGAAAATGCGACAT TCAACCGTTATTCAGATGAACGGTGTACAGACC TIAACATTTGCCCTGCTTGAGATGGTTTIGCGATTTGTTTAGCTATATTTTICAT TTTCAATTCTTACCATTGAGAAT CGCCAAGGCAT TAABACAATAACGGATTCGCC
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US 12,548,243 B2 51 32 -continued. Cub-staple-112 CARAGCGCTGGCCTGATTCTCCGTGGGACACGTTGCAGTATCGGCCTCAGGAAG Cub-staple-113 GAGCGCTGCAAAATGGTTGAGTGTTGTTCGTGGACCATARRAACAGGGRAGCGC Cub-staple-114 GGRATCATTAGGTTAATCCARTCGCAAGTTT TAGTTGAAATACCGACCGTGTGA Cub-staple-115 TATAAAATAAAGCCAACATTATGACCCTACGCAAGCATGATTAAGACTCCTTAT Cub-staple-116 CTACAACATTAGGATATAAGTATAGCCCAGTACCGCATTTTCAGGGATAGCAAG Cub-staple-117 GATTAGTATGTAGAACCAAGTACCGCACATCGTAGGTATT CTAAGAACGCGAGG Cub-staple-118 TACACTAACGAT TGTAAAGCCAGAATGGAGCGTCATCCATTAAACGGGTAARAT Cub-staple-119 TACGAGCCGGGCCTTTGGGTAACGCCAGGCCAGTGCGAATTCGTAATCATGGTC Cub-staple-120 CGCTGGTGAGAGATCTGGAGCAAACAAGTGT CAR TCCAGTGAGACGGGCAACAG In order to attach one gold nanoparticle inside the cubic 49 DNA frame, add (SEQ ID NO: 294) \ TCACTTCATACTCTACGTTGTTGTTGTTGTTGTTGTTGTTGTTGTT ' w in front of the certain sequences (e.g., Cub-staple-97, 105, 106, 107, 110, 111, 118, and 120) with PAGE purification. DNA sticky end for cube-1 (CR, m= 12, 1 = 8, n= 12) (SEQ ID NOs: 295-318, respectively, top to bottom) : ACCAGTAGCACCCCGTAATCAGTAGCATTATACATGTTACTITTTTTTTTTTTTATCCGTTA, ATTAGACGGGAGGAGAGATAACCCACTTGATGGGGAACAAGATTTTTTTTTTTTATCCGTTA, AGGCGCATAGGCAAATCAACGTAACAGTTTATTGAGGGAAGGTTTTTTTTTTTTATCCGTTA, CCCAATAGGAACGCATTCCACAGACACTGAGACGTGTATCACTTTTTT?ITTTTATCCGTTA, AGCTTAGATTAAAAATCATAGGT CTGACAAACAAATATATGTTTTTTTTTTTTIATCCGTTA, ACGTAATGCCACCATCTTTGACCCCCCAGGAGGAGTCTCTGATTITTTTTTTTTATCCGTTA, CTCAGAGCCACCAGCCGCCGCCAGCAGAATCARATCTTTTCATTTITITITITIATCCGTTA ATAGCTGTTTCCATAAAGTGTAAAGCTGTTGGGCCAGTCACGTTTTTTTTTTTTATCCGTTA, GAGTAACAGTGCCATGAAAGTAT TAACACGCATAAAGACAGCTTTTTTTTTTTTATCCGTTA, AGATTCAAAAGGCTAGCTGATAAATTATTGAGTAGCAGATAGTTTTTTTTTTTTATCCGTTA GAACTAACGGAATTCAACTAATGCAGATTGCTGCAGTTGATTTTTTTTTTTTTTATCCGTTA TTTGCGGAACAATGGCAATTCATCAATTATCCTATCCCAATCTITITITITITIATCCGTTA TACGCAGTATGTAAAGACACCACGGAAGAATTATTTIGCGGGTTTTTTTITTTTATCCGTTA TGATTGCTTTGAGCAAAAGAAGATGATATCATACAACGCCAATTTTTTTTTTTIATCCGTTA TIGGGGCGCGAGAT CATACAGGCAAGTGCTCATACTTTAATCTTITITITITTTIATCCGTTA CTIGATTGCCCTTAGCAGGCGAARATCCCGGAGAATGAACGGTTTTTTTTTTTTIATCCGTTA ATCGCACTCCAGCCATTCAGGCTGCGGCCATCAGCGGATIGATTTTTTTTTTTTATCCGTTA, AATCGTCATAAAAGTTCAGAAAACGAATAACGCATAGCGAGATTT TT TTTTTTTATCCGTTA CGTTTTAGCGAATTGCACCCAGCTACATCCCATGAACAAGCATTTTTTTTTITIATCCGTTA GACGACAATAAACTGAACAAGAAAAAATCCTGAAATAAAGAATTTTTTTTTTTTIATCCGTTA, TARATAAGGCGTTAGAAAAAGCCTGTACTACCTACGCGAGAATTTTTTTITTTTATCCGTTA GGTTTATCAGCTATGACAACAACCATTCATAGTGGAGTGAGATTTTTTTTTTTTATCCGTTA,
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US 12,548,243 B2 53 34 -cont inued ATTTAAATTGTATTTTTAACCAATAGGGTGCCTCTGCATTAATTTTTTTITITIATCCGTTA, AGAGAGTACCTTCGGATGGCT TAGAGGACCATAAGCCCGAAATTTTTTTTTTTTATCCGTTA, DNA sticky end for cube-2 (CB, m= 12, 1 = 8, n = 12) (SEQ ID NOs: 319-342, respectively, top to bottom): ACCAGTAGCACCCCGTAATCAGTAGCATTATACATGTTACTTITTTITTTTTTTTTAACGGAT ATTAGACGGGAGGAGAGATAACCCACTTGATGGGGAACAAGATTTTTTTTTTTTTAACGGAT AGGCGCATAGGCAAATCAACGTAACAGTTTAT TGAGGGAAGGTTT TTTTTTTTTTAACGGAT CCCAATAGGAACGCATTCCACAGACACTGAGACGTGTATCACTITITTTITTTTTAACGGAT AGCTTAGATTAAAAATCATAGGTCTGACAAACRAATATATGTTTTTTTTTTTTTTAACGGAT ACGTRATGCCACCATCTTTGACCCCCCAGGAGGAGTCTCTGATTTTTTTTTTTTTAACGGAT CTCAGAGCCACCAGCCGCCGCCAGCAGAATCAAATCTTTTCATITTTTTITTTTTAACGGAT ATAGCTGTTTCCATAAAGTGTAAAGCTGTTGGGCCAGTCACGTTTTTTTTTTTTTAACGGAT GAGTAACAGTGCCATGAAAGTAT TAACACGCATAAAGACAGCTTT TTTTTTTTTTAACGGAT AGATTCAAAAGGCTAGCTGATARATTATTGAGTAGCAGATAGTTTTTTTTTTTTTAACGGAT GAACTAACGGAATT CAACTAATGCAGATTGCTGCAGTTGATITTTITTTITTTITAACGGAT TTTGCGGAACAATGGCAATTCATCAATTATCCTATCCCAATCTITITITITTTITAACGGAT TACGCAGTATGTAAAGACACCACGGAAGAATTATTTTGCGGGTTTTTTTTTTTTTAACGGAT TGATTGCTTTGAGCAAAAGAAGATGATATCATACAACGCCAATTTTTTTTTTTTTAACGGAT TTGGGGCGCGAGAT CATACAGGCAAGTGCTCATACTTTAATCTTTTTTTTTTTTTAACGGAT CTIGATTGCCCTTAGCAGGCGAARATCCCGGAGAATGAACGGTTTITTTTTITTTTTAACGGAT ATCGCACTCCAGCCATTCAGGCTGCGGCCATCAGCGGATIGATTTTTTTTTTTTTAACGGAT AATCGTCATAAAAGTTCAGAAAACGAATAACGCATAGCGAGATTTTTTTTTTTITAACGGAT CGTTTTAGCGAATTGCACCCAGCTACATCCCATGAACAAGCATTTTTTTTTTTTTAACGGAT GACGACAATAAACTGAACAAGAAAAAATCCTGRAATABAGAATTT TTTTTTTTTTAACGGAT TARATAAGGCGTTAGAAAAAGCCTGTACTACCTACGCGAGAATTTTTTTTTTTTTAACGGAT GGTTTATCAGCTATGACAACAACCATTCATAGTGGAGTGAGATTTTTTTTTTTTTAACGGAT ATTTAAATTGTATTTTTAACCAATAGGGTGCCTCTGCATTAATTITTTTTTTTITAACGGAT AGAGAGTACCTTCGGATGGCT TAGAGGACCATAAGCCCGAAATTTTTTTITTTTTAACGGAT Tetrahedral DNA staple strands (SEQ ID NOs: 343-502, respectively, top to bottom) tet-staple-1 ATTGTGTAGCCGTCCCCGAACATACCGRACGAACCCAGC tet-staple-2 ATTTGTATTGAGGACAACTCGGAAGATA tet-staple-3 AGGACAGATCTTGAGAATAACATAAAAAAACACCCGAAT tet-staple-4 ACGTAGCCGGAACGAGGCCCACAATGAACAATTTAATG tet-staple-5 CAGGCGCATACCGCGACCTGCTCCACCAATAAGGGAGAACCTAAAA, tet-staple-6 | CTCATCTAAAATACAAAGARACCACCAGAAGGAGCGGAATT tet-staple-7 GCTGACGCGCATTAGAAAAGTTTGAGTCAACCTAARAGGCTG tet-staple-8 AGGAAGTTTCCATTAAACGGGTTTGACCCAACGGAG tet-staple-9 | AAAATGARAATAGCAGCCTTTGGGTAATTGAGC tet-staple-10 GGAACTTCATCAAGAGTAATGA,
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US 12,548,243 B2 55 56 -cont inued tet-staple-11 CATTTTTICCTTTGAATAGATTAAAATATCTTTAGTGAACCTTGAAAAA tet-staple-12 AACGTAATGCCACTACGAATACACCTGATAA, tet-staple-13 TTTACARACAATTCGATTTAGAAGTATTAGAC tet-staple-14 GCTAATATCAGACGAAGCCCTTTTTAAGAAA, tet-staple-15 TGAAAATAGCAATAGCTCCAGAAGAAGACTC tet-staple-16 GATTAGCGAAATCGAAAGAGGCAAAAGAAGGCACAACATTAT tet-staple-17 ATGGTTAAGCTGTTACTGTGTACAGAC tet-staple-18 ATAACGTCACCTTGCGAGCACTAACAAAGAGCAAGAGCAATA tet-staple-19 TCAACGCTGAGAGCCAGACCAGCATATTARAGCGG tet-staple-20 CTTATTARTTAGTCAGTCAGRACAGAGACAAGAACCGGATATTCATTACCCAA tet-staple-21 TCTAAAGATTAAAAGTTATTAATTTTACTACTAATA tet-staple-22 AAACAAATATCAAACCCGGTTATCAATACATTCATCGCTAAAACA, tet-staple-23 GACAATATTTTTGAATGGCTCGCAGTATGTTAGCAAACG tet-staple-24 CGCGAACCATGATTGARACCGAGGAAACAACA, tet-staple-25 AAACAGACAGTGCCATATCTGGTCAGTTGGCAA tet-staple-26 TAATTTGATTTACGACTCATCCGCCGCCAGCATTGGAAA tet-staple-27 AACGAGCAATCAATGGGTATTGTTGAGG tet-staple-28 TCAACGTAACAAAGCTGCACGATTTTTTGTTTAACGT tet-staple-29 GTGAATAGGTTTAATTATACCAGTCAGGGAACTAACCAC tet-staple-30 TTGTTATCCCAATCCARAGGRATACGGAACAAGCCACC tet-staple-31 TTAGTTGTCTAAGAGCGCCCAATAGCAAGCAAATCAGATA tet-staple-32 GCACCCAGCTACAATTTTTTTCGCTGAGGCTTGCAGGGAGTCATCGCCCACGCATAACCGATTTTTTTATCCTGAATCTTAC tet-staple-33 TAGTAAGAAGAAAAAGCCGTTTTTATTCTCCCGACTAACGAG tet-staple-34 TTGGATTGGGCTTGAGATAGGC tet-staple-35 AGGAATGTACCGCAGCATGTAGATAAGTCCTGAAAAGCGTCCAGTCTC tet-staple-36 ACCACGCGAGGCGTTTTGCCTTAACAGAGCC tet-staple-37 TTATCATTCCAAGAACAATCGGCTGTCTTTCC tet-staple-38 GATTCATCAGTTACACTATCATAACCCTCGTT tet-staple-39 ATTTTACGAGGCATAGTATAGCCCACCACCG tet-staple-40 ATCCTACCAGTTACGGGAGGTTTTGARAGCGAACTTCATCGT tet-staple-41 TGTTCAGCTAATGCAGAACGCGCCTGTTTATATGA, tet-staple-42 GCGTTTTCATCGGCATTTTCGGTCAAGAGCAGAGATTTATAAGAATCATTCA, tet-staple-43 TTTTCACGTTCCAGTCAAGAAAAATAAATAACGCCAGCCATC tet-staple-44 TACTTAARGCCAGAATGACAGGAGAAACCAACATT tet-staple-45 GAACCGCCCCTCAGACATTATTGGCTCATTTCAACTTTAATCATTGTGAATTAC tet-staple-46 TGAATTTCCAGAGCGAGAACAAGCAATCACTATCCC tet-staple-47 GCGATACATGGCTTTTGCAACAATAGAAACCGTCTTTCATCAAGA, tet-staple-48 ACCCTCACAGAGCCCCTTATTAGCGTTTAAAG tet-staple-49 ACCACCAACTAATCAARATCACCGGAACGAGCCGCATAARACACG tet-staple-50 CAGGTCAATCCTCAAGGAGTGTACTGGTAATAAGTTT
Page 109
US 12,548,243 B2 57 -cont inued 58 tet-staple-51 tet-staple-52 tet-staple-53 tet-staple-54 tet-staple-55 tet-staple-56 tet-staple-57 tet-staple-538 tet-staple-59 tet-staple-60 tet-staple-61 tet-staple-62 tet-staple-63 tet-staple-64 tet-staple-65 tet-staple-66 tet-staple-67 tet-staple-68 tet-staple-69 tet-staple-70 tet-staple-71 tet-staple-72 tet-staple-73 tet-staple-74 tet-staple-75 tet-staple-76 tet-staple-77 tet-staple-78 tet-staple-79 tet-staple-80 tet-staple-81 tet-staple-82 tet-staple-83 tet-staple-34 tet-staple-85 tet-staple-86 tet-staple-87 tet-staple-838 tet-staple-89 GAATGACATCACCGAACGTCATCTGGCCAACAGAGAAAC TTTAAACCATTTGGATTACCACCTICTG ACTTCAARACAGGTGGATGGCTTAGAGCTGTTTTATTCC CGCGACTATTATAGTCACAGT TGAAATATGCTGGCAGA, GAGCTTCAAGGCAAAAATCAGGTCTAACGAGTGCTGTAGGACCAGT GCGTCCAATAGCGATCAAGTTTGCCTTTAGCGTCAGACTGT AGAGTACCTTTAATTGCTTTTGAATCGCCATATTTAACAACGGCCAA, CCAGACCTGAATATATCGATAGCAGCATTGCCAGAGAGCGAA CAGACGACGATARBAACCARRATACTGCCAAATGC ATTGCGGAAGCAAACTCCATAT AGTAGCGGCCGGATCACCGAGGTAAATATTGACGTGTCACAAAGACAC GAAGAGGCTTTTGCAAAGTTTAGAAAAACGA, AATCACCAGTAGCACCGAATTAGAGCCAGCAA GAAGTTTCATTCAATAACCTGTTTAGCTATA CAAAGATACATTTCGCAGGTGGCAGCAAAGA, TGAATTCATAAATGGTAATAGTAAAATAGAAGTTCCGTAATC ATTTTCTGCGTTACCCTGTTTTAATTC AGCATTAGTTTATTTGAAATTATTCATATT TAGTTTAGTAGT TGGGCAACATATAAAAGATAGAACTTAGCAAGACA ATTAGCATTTACATAACTAAATTTTTGCCAGGATTAG CACGGAAGGGACATCCAATGAAACCAATAGTAAAGG GCAATCAATAGAAAATTGAGGGAACTTGAGCAGTTCAGCTGGATA AGAAAATACATACATAGCGTAAGAATACGTGGCA, CAATCGTCTGAAATGGATTARAATTAAGCAATAAAGCCT TTCACCAAGGCAAGTCAATTCTACTAATGACC ACCTGAARAAGGTGTTTACCAGCGCCAAAGACA CACGACGTTACATCTTTCAATCGGAACGAGGGTAGCAAT TAACGCCCGGATTCCGCGCAGTACAGAG GTCATAGCCGGAAGGCCCTCATAGTTAGACGTTAGGGAG TICGCATGCCTGCAGGTTTTCAGCTARATGACGCTITT TIGTTATCCTTACGACGGCCAGTGCAAGGAACTGTCGTCCTCAGCA, GCTATTAAGGCAAAATTAATTACATTTAACAATTTCATTTGAATT AATTCCATCTAAAGTAGAAGATGATGAGCGCAACTGGCTCAC ACCGCTTCTGGTGCCGGRAACCCGCCAGCAGTTGGG ACTACRACGCCTGTAGCATTCCGTATGGGATTTT ACGACACRACATACGAGCTGT TCAAGATTATTCAGGGAGAAAGGTT TAACGTCAGTGAATAAATCCTGA AAAGCGCCATTCGCCATGTGCGGGTCCCAGT TACCAAGTTACAAAATGCCTGATTGCTTTGAA
Page 110
US 12,548,243 B2 59 60 -cont inued tet-staple-90 tet-staple-91 tet-staple-92 tet-staple-93 tet-staple-94 tet-staple-95 tet-staple-96 tet-staple-97 tet-staple-98 tet-staple-99 tet-staple-100 tet-staple-101 tet-staple-102 tet-staple-103 tet-staple-104 tet-staple-105 tet-staple-106 tet-staple-107 tet-staple-108 tet-staple-109 tet-staple-110 tet-staple-111 tet-staple-112 tet-staple-113 tet-staple-114 tet-staple-115 tet-staple-116 tet-staple-117 tet-staple-118 tet-staple-119 tet-staple-120 tet-staple-121 tet-staple-122 tet-staple-123 tet-staple-124 tet-staple-125 tet-staple-126 tet-staple-127 tet-staple-128 tet-staple-129 GCTAAACAACTTAATCTCCAAABAAAAGGCT TGATAATAATTTTTTCATTTATCAACAATGA, TACCTTTTGTAAAGGGAAGGGCGATCGTCAGGCTARCAAACA GAAGAATAGACAAGCTTCTGTGTGAAA TCTTAATTATACTTCATGAATATACAGCTAAAGGAATGAATT, CCATGATGGCAATTCATCAACGGCAGGCGAAAAAC CAACAACTAAAGGCATTTT CTACAGACACATAAAGTGTARAGCCTGGGGTGCC TIGTTTGACAGCATTACCTGAGCAAATTAATAACAG ATATGGAAGGGTTAGAAGATTTTCACAATABAGGGTTTCCTCTTC ATCATATTCCTGATGGACTARAGACTITTTC GCGGGATTGCGCCGGCTIGCTTTCGAGGTTGC GCTTTGATATCAGATATCAAAATTATTTGCACG ACAGCTGATAGCCCCTCAGAGATAAAGTACCGACAAACA TTTTCACACCGTACGCCACCCAAGTAAT GTTCCGAGTTGTTCTCARAATCATAGGTGTTGGGTCGAG GGCAGTTGCAGCAAGCGAAGAACGTATATAAGTAATTT ATAAATCAAAATTCACCGCCTGGCCCARATATAACCTCCTCGAGCC GGCCAACRATTGCGGTACCGTAACACTGAGTTTCGTCACCAGTAC AGCCCGACTACCTTTTTTTCAGGGATACAGTCGGGAAAGAAT AATGAGTGAGCTAACTCACATTGCGCGGGGTTTTTC GACGCTGAGAAGAGTCAATAGTTAAATGCTGATG GAGAGATAGGGTTGAGTAATC AATAGGCGCCACCGGAATAGCCAGGCGGATAAGTTATTATTAATGCCC CATTTGCGCTCACTGCCTGCATTAACGGGCA CGCCACCCTCAGAACCTCAGGAGGTTTAGTAC CABATCCAATCGTTGAAATACCGACCGTGTG AAATTCTGACCTAAATTACACCGGCTTACCA ATAAGTATTGCCCCCTGTCGTGCCAGCCGCTTTCGCAAGCCC ATCACTTTTTCTGAGAGAAAATCCCTT GCCTGTTTCGGAACCGCCGTCGAGAGGTTTAGTTAAGAARAA, AAGACAGTGCCCGTATARAAGGTATCAGAACAACC CGCTCAACAGTAGGGCAATTTCCTTTTGATAAGAGGTCAGTACGGTGTCTG GTATAAACCAACATCTATATGGAATTTACAGTTTGGAACAAGAGTCCACTATT CCTGCCTAGAGAATCCACCACCCTCATTATGTTGAT GTTCTGARACATGAAAGCTCAGTAGTGTATCCAGTGAGATGAATC AACGGGGTCAGTGCCAGACGACGACAATAAACAA AGGCAGAACAAATTAATCATAATTACTATT TC TCTGTCCTIGAGTAAGGCTGAGACTCCTCAAGA, ACCTTGCGCATTAAGCCAGCTAATCACCATCAATACAGG ATCAATAAGCTCATTTCGCGTCAACCGT
Page 111
US 12,548,243 B2 61 62 -cont inued tet-staple-130 CTTTTTTAATGGAAACAGCAGCCAGCTTTCCGG tet-staple-131 AAGAACGTGGACTCCAACCATAGCGATAGCTTAGATT tet-staple-132 GGCGARAGATTTTACACCGAGTARAAGAAATACTICCTT tet-staple-133 GTCTTCCCTTAGAATCCCTATCGGCTTTGATTAGGTAA tet-staple-134 GGAAGATGGTGTAGCGTGGGAACAAACGGCGGATTGACCG tet-staple-135 AACAGGCATCACGCAARATGTGAGCGAATCTGCCAGGAGCTA tet-staple-136 TGTCAGGCCGATTAAAGGAACC tet-staple-137 CCGTCGTCCTGTAATTTTTGATATTTAAATTGTAGAGAATCTTGCCTG tet-staple-138 CTCATGGGCGCATCGTACAGTATCGTGAATA tet-staple-139 ACGCCATCAAAAATAATTTTTAACCAATAGGA tet-staple-140 CTGAGTAGAAGATGGARATACCTACATTTTGACG tet-staple-141 GCTTTGCAACAGGAARACCAAAAAATAAAAA tet-staple-142 AAATTCTTCTGTATGAGGGGACGACGAACCGTGCGTAACAAC tet-staple-143 AGAGCATARAGCTAAATCGGTTGTAACGCTCAACTCARATTGAARAGTCAAAG tet-staple-144 TTTGCGAGCAAACAAAACGTTAATATTATATTACCGAATACT tet-staple-145 AACTCTACAAAGGCTATTGATATTCTGGCCTGATT tet-staple-146 TCATATGTACCCCGGGGTTARCACCGCCTGCAAGGTGAGGCGGTCAGTGGCAGAAAAGCCCCAAAAACAGGAAGATTGTAGTAA tet-staple-147 TITTTAGTAATGTGTAGTAATGTGAGGCGACAGGAACGGTACGCCAGAATCCTG tet-staple-148 AGAGTCTACAGTCATTCATCAACATTAACATTGTTA tet-staple-149 TCAGATGAACGGTAATCTAAGCAATTAAATCTATGTGAGGCCTCA tet-staple-150 AGATTCACGCAAGGCATTATGACCCTGTCCAG tet-staple-151 GCCGGAGGGGGAGAAGCCTTTATTTCARAAAGGGTTTGTAGCGTC tet-staple-152 TCTAGCTTGAGAGATAGCATGTCAA tet-staple-153 AAACACCAGTGCAAAATAAACAGCCTGCAGATTACGTTACACCAGA tet-staple-154 GAACAACTAAATATTATCCCTGACGAG tet-staple-155 AGAGCGGTTAATCGTCGCTATTATCCAGAATTAACCGGAGAAAG tet-staple-156 CCAGGTAATAATTAATTTATCATAGAATC tet-staple-157 CGTAATGAACTCAATATTTTCATCGCCCAGAATACCCAAAAGAACTGGTGATAGCTTAACTGCAG tet-staple-158 CGTAATGAACTCAATATTT TAATAAARTARACATCCAATAAATCATACGTCACACCTCAACATTA tet-staple-159 CGTAATGAACTCAATATTTTGCGAAAGGARCAGCTTGATACCGATAGTCGTCACCTTTCCAGCGTA tet-staple-160 CGTAATGAACTCAATATTTTAGTAATAATT TAGTATCATATGCGTTATGGCATTTGGCTTAGCTGA Staple sequences from 157 to 160 (PAGE purified) are the ones to bind with 10 nm AuNP inside. DNA sticky ends for tetrahedron (TR, m= 22, 1 = 8, n = 22, PAGE purified) (SEQ ID NOs: 503-526, respectively, top to bottom): AGGAAGAGTTTTTTTTTTTTITTTTTTTTTGCGCGAAACAAAGTACCCAGCGATTATACCAA, AGGAAGAGTTTITTTTTTTTTTTTTTTTTT TT TACCGAACTGACCAACTGGTCAATCATAAGGGA, AGGAAGAGTTTITTTTTTTTTTTITTTTTTTTTATCAACAGTTGAAAGGAATTGAGGAATCAA. AGGAAGAGTTTITITTTTTTTTTTTTTTTTAGTAAGCAGATAGCCGAACAAAGTTAATCT TACGAGATAACGCAGACTTGAAAG AGGAAGAGTTTTTTTTTTITTTTITTTTTTTTTTAGAAGGCTTATCCGGTATCTATTTTCAACGCT AGGAAGAGTTTITTTTTTITITTTTTTTTTCTIATGCGATTTTAAGAACTACAGGTAGABA
Page 112
US 12,548,243 B2 63 64 -cont inued AGGAAGAGTTTTTTTTTTTTTITTTTTTTTTATTCACAAACAAATAAGACGATTGGCCTIGAT AGGAAGAGTTTTTTTTTTITITTTTTTTTTTTTACCCTCAGAACCGCCACTCCCTCAGAGCCGCC AGGAAGAGTTTTTTTTTTITITTTTTTTTTTTCATTGAATCCCCCTGGAATCGTCATARATA, AGGAAGAGTTTTTTTTTTTTITTTTTTTTTTT TAAGAT TAAGAGGAAGCAGCGGATTGCATCAAA AGGAAGAGTTTTTTTTTTTTTTTTTTTTTTTTTAAAGGGCGACAT TCAACCGATTGAGGCATA AGGAAGAGTTTITTTTTTTTTTTITTTTTTTTTT CATT TGGGGCGCGAGCTGAAAAAATGGT CCATATAAGAAGCAACCGAAAG. AGGAAGAGTTTITTTTTTTTTTTTTTTTTTTGCTGCAAGGCGATTATGGCGARAGGGGGATG AGGAAGAGTTTITTTTTTTTITTTTTTTTTTTTCCGAGCTCGAAT TCGTAGAGGATCCCCGGGTA, AGGAAGAGTTTTTTTTTTTTTTTTTTTTTTTTTTARAACAGAAATAAAGARATTGCGTACCTA. AGGAAGAGTTTTITTTTTTTTTTTTTTTTTCCAAAAGGAGCCT TTAATTGTAT CGGCGTTGAAT CAACAGCGACTCTAATCATG, AGGAAGAGTTTTTTTTTTTTITTTTTTTTTAT TGGGCGCCAGGGTGGAGAGGCGGTTTGCGT AGGAAGAGTTTITTTTTTTTTTTTTTTTTT TT TGGCGAAAATCCTGTTTCTGGTTTGCCCCAGCA, AGGAAGAGTTTITTTTTTTTTTTTTTTTTT TT TGAAGGATTAGGA TTAGCGGGGTTTTGTATT AGGAAGAGTTTITTTTTTTTTTTTTTTTTTATAAATAAGGCGT TAAATAAGAA TAATAATGGTCAAGACAGTCCACGGATGGTG AGGAAGAGTTTTTTTTTTTTTTTITTTTTTTTTTAATGGGATAGGTCACGTTCGCACTCTACATAA AGGAAGAGTTTTTTTTTTTTTTTTTTTTTTAGAAGTGTTTTTATAATCAAACATCACTTGC AGGAAGAGTTTTTTTTTTTTTTTTTTTTTTAGAGGGTAGCTATTT TGATAAAT TAATGCCGG AGGAAGAGTTTITTTTTTTTTTTITTTTTTTTTAAATGCAATGCCTGAGAACCCTCATATATITT, DNA sticky end for tetrahedron (TB, m= 22, 1 = 8, n= 22, PAGE purified) (SEQ ID NOs: 527-550, respectively, top to bottom) : CICTTCCTITTTTITTTTTTTITTTTTTTTGCGCGAAACAAAGTACCCAGCGATTATACCAA CICTTCCTITTTTITTTTTTTITITTTTTITTTACCGAACTGACCAACTGGTCAATCATAAGGGA, CICTICCTITTTTITTTTTTTITTTTTTTITTTATCAACAGTTGAAAGGAATTGAGGAATCAA, CTCTTCCTITTTTITTTTTTTITTTTTTTTAGTAAGCAGATAGCCGAACAAAGTTAATCT TACGAGATAACGCAGACTTGARAG CTCTTCCTITTTTITTTTTTTITTTTTTTTTTTTAGAAGGCTTATCCGGTATCTATTTTCAACGCT CICTTCCTITTTTITTTTTTTITTTTTTTTCTTATGCGATTTTAAGAACTACAGGTAGABA, CICTTCCTITTTTTTTTTTTTITTTTTTTTATTCACAAACAAATAAGACGATTGGCCTTGAT CICTTCCTITTTTITTTTTITITITTTTTITTTACCCTCAGAACCGCCACTCCCTCAGAGCCGCC CICTTCCTITTITITTTTTTTITTTTTTTITTCATTGAATCCCCCTGGAATCGTCATAAATA CTCTTCCTITTTTITTTTTTTITTTTTTTTTTTAAGATTAAGAGGAAGCAGCGGATTGCATCARA, CTCTTCCTITTTTITTTTTTTITTTTTTTTTT TABAGGGCGACAT TCAACCGATTGAGGCATA, CICTTCCTITTTTITTTTTTTITTTTTTTITTTTCATTTGGGGCGCGAGCTGAAAAAATGGT CCATATAAGAAGCAACCGAAAG CICTTCCTITTTTTTTTTTTTITTTTTTTTIGCTGCAAGGCGATTATGGCGAAAGGGGGATG CICTTCCTITTTTITTTTTTTITTTTTTTTTTTCCGAGCTCGAATTCGTAGAGGATCCCCGGGTA, CICTTCCTITTTTITTTTTTTITTTTTTTTTTTTARAACAGARATAAAGAAATTGCGTACCTA, CTCTTCCPITTTTILTTTTTTITTTTTTTTCCAAAAGGAGCCT TTAATTGTAT CGGCGTTGAATCAACAGCGACTCTAATCATG CICTTCCTITTTTITTTTTTTITITTTTTTATTGGGCECCAGGGTGGAGAGGCGGTTTGCGT CICTICCTITTTTITTTTTTTITITTTTTITTIGGCGAAAATCCTGTTTCTGGTITGCCCCAGCA, CICTTCCTITTTTTTTTTTTTITTTTTTTTTTIGAAGGATTAGGATTAGCGGGGTTTTGTATT CICTTCCTITTTTITITTTTTITTTTTTTTATAAATAAGGCGT TAAATAAGAATAATAATGGTCAAGACAGTCCACGGATGGTG
Page 113
US 12,548,243 B2
65
66
-cont inued
CICTTCCTITTITITTTITITITITTTTTITTTTAATGGGATAGGTCACGTTCGCACTCTACATAA,
CICTTCCTITTTTITTTTTTTITTTTTTTTAGAAGTGTTTTTATAATCAAACATCACTTGC
CTICTTCCTITTTTITITTTTTITTTTTTTTAGAGGGTAGCTATTTTGATARATTAATGCCGG
CICTTCCTITTTTITTTTTTTITTTTTTTITTTAAATGCAATGCCTGAGAACCCTCATATATITT
Modified DNA sequence attached on QDs (525, 605) and streptavidin for octahedral system
{HPLC purified} (SEQ ID NO: 551}:
TATGAAGTGATGGATGAT-Biot in
Modified DNA sequence attached on QDs (705)
ID NO: 552):
TAGGTATGATGGTAGTAG- Biotin
for octahedral system (HPLC purified) (SEQ
Modified DNA sequence attached on gold nanoparticle for octahedral system (HPLC
purified) (SEQ ID NO: 553):
TATGRAGTGATGGATGAT- SH
Modified DNA sequence attached on gold nanoparticle for cubic system (HPLC purified)
(SEQ ID NO: 554)
GTAGAGTATGAAGTGATGGATGATGATGATGAT- SH
Modified DNA sequence attached on gold nanoparticle for tetrahedral system (HPLC
purified) (SEQ ID NO: 555):
TATTGAGTTCATTACGTTITTTITITTITTTTTTTTT-SH
Modified DNA sequence attached on glucose oxidase for enzyme lattice (standard desalting)
SEQ ID NO: 556);
SH-TTTTTGTAGATGGTAGTATGGAT
Modified DNA sequence attached on horseradish peroxidase for enzyme lattice (standard
desalting) (SEQ ID NO: 557):
SH-TTTTTGATTAGGTAGTGAAGTAT
Example 3: Valence-Programmable Nanoparticle
Architectures 3
The disclosed subject matter provides a sphere-like DNA
mesh structure (FIG. 51A) as symmetric frames, which can
be programmed to exhibit different prescribed valence
modes, and consequently, used to coordinate nanoparticles
into pre-defined architectures. Moreover, specific positions
and types of bonds can be fully prescribed. This approach
can offer designability over different valence modes using
the same underlying high-symmetry frame, including vari-
ous subset symmetries, arbitrarily prescribed helix-like
valence, and valence with different types of affinities.
Accordingly, a variety of cluster architectures with single-
type and multi-type nanoparticles can be rationally formed.
Furthermore, using planar and spatial imaging and in situ s
scattering methods, such programmable frames can coordi-
nate nanoparticles into a variety of corresponding cluster
architectures possessing symmetric, helical, and site-specific
nanoparticle organizations.
Self-assembly of DNA meshframe: The sequences of 5:
staple strands were designed by vHelix28. Staple strands
(Integrated DNA Technologies) and M13mp18 scaffold
(Bayou Biolabs) were mixed in 0.5xTE buffer (5 mM Tris,
1 mM EDTA, pH 8.0, supplemented with 10 mM MgCl,).
The solution was annealed from 80° C. to 60° C. ata cooling
rate of 1 min/° C. and from 60° C. to 20° C. at a rate of 23
min’? C.
DNA modification of gold nanoparticles: The thiolated
DNA strands (HPLC, Integrated DNA Technologies) were
first reduced by tris(2-carboxyethyl) phosphine (TCEP)
solution (Sigma-Aldrich) with a molar ratio of 1:100 in the
water at 20° C. After the incubation for 1.5 h, the thiolated
&
s
DNA strands were purified by removing small molecules
with MicroSpin G-25 columns (GE Healthcare). Then the
purified thiolated DNA strands were mixed with aqueous
spherical gold nanoparticle (AUNP) solution (Ted Pella)
with a ratio of 300:1 for 10 nm AuNPs, 700:1 for 15 nm
AuNPs, 1000:1 for 20 nm AuNPs, and 2100:1 for 30 nm
AuNPs. After 2 h of incubation at 20° C., 10x phosphate-
buffered saline (PBS) (100 mM, pH 7.4) was added to bring
the final solution to be 1xPBS (10 mM, pH 7.4). For 15 nm,
20 nm, and 30 nm AuNPs, 10% SDS was added to bring the
final concentration to 0.01% SDS. After another 2 h of
incubation, stepwise addition of salting buffer (1xPBS buf-
fer with 2
M sodium chloride) increased the concentration of the
sodium chloride to 0.3 M. The solution was aged for 12 h.
To remove excess thiolated DNA strands, DNA-AuNP con-
jugates were washed four times by centrifuge. The super-
natant was removed, and the fresh washing buffer (1xPBS
buffer with 100 mM sodium chloride) was used to rinse and
disperse the DNA-AuNP conjugates. The purified DNA-
AuNP conjugates were quantified by measuring the absor-
bance at 520 nm on PerkinElmer Lambda 25 spectropho-
tometer.
Assembly and purification of AuNP clusters: DNA
meshframe was mixed with AuNPs with the ratio of 3 N:1
(N is the valency number) in 0.5xTE (supplemented with 10
mM MgCl.) and annealed from 50° C. to 20° C. for 12 h.
The annealed samples and 1 kb DNA molecular weight
marker (New England Biolabs) were loaded to a native 1.5%
agarose gel with 0.5xSYBR Gold (running buffer: 0.5xTBE
buffer, containing 44.5 mM Tris, 44.5 mM boric acid, and 1
mM EDTA, supplemented with 11 mM MgCl.) and gel
electrophoresis was performed at 60 volts for 3 h in an icePage 114
US 12,548,243 B2 67 bath. Target bands were excised and cut into small pieces. The gel pieces were placed into Freeze *N Squeeze columns (Bio-Rad Laboratories) and centrifuged at 3000xg for 5 min to obtain purified AuNP clusters. For spherical helix clusters, DNA mesh frame was first purified with agarose gel elec- trophoresis as described above and quantified by measuring the absorbance at 260 nm on PerkinElmer Lambda 35 spectrophotometer. Then, the purified DNA mesh-frame was mixed with AuNPs with a ratio of 3 N:1 and annealed from 37°C. to 20° C. for 12 h. Finally, the annealed sample was purified with agarose gel electrophoresis. Negative-stained TEM: Three microliters of samples were loaded on the glow-discharged, carbon-coated grid (300 mesh, Ted Pella) for 1 min, and the excess sample was removed by a piece of filter paper. Next, the grid was incubated in 2% uranyl acetate aqueous solution for 30 s, followed by using a piece of filter paper to dry it. TEM imaging was performed on a JEOL 1400 at 120 kV. Cryogenic electron tomography: Copper mesh grids (Car- bon Film 300 mesh, Copper, Ted Pella) were held for 10s 2 and glow discharged for 20 s. The cryogenic sample was then prepared using the FEI Vitrobot with typical parameters of3 uL sample, the temperature at 4° C., force at 0, humidity at 100%, wait time 4 s, and blot time 5 s. The as-prepared sample was transferred to a liquid nitrogen tank to be stored for later use. A single tilt cryogenic tomography holder (Gatan 626) was cooled down to below 90 K under liquid nitrogen before the sample was loaded. The holder was then inserted into the JEOL 1400 microscope to collect tomog- raphy image series under 120 keV from around -60 degrees to 60 degrees at 10 degree intervals. The original images were first converted into a stack image using the ImageJ software. Contrast inversion, image alignment, and tilt axis refinement were carried out in Tomviz manually. The refined image stack was further reconstructed into 3D volumes using the Simultaneous Iterative Reconstructive Technique (SIRT) algorithm embedded in Tomviz. The reconstructed 3D volumes were then filtered and segmented in Avizo software to get the 3D center positions. Small-angle X-ray scattering (SAXS): SAXS measure- ments were performed at BNL National Synchrotron Light Source I] (NSLS-II) Complex Material Scattering (CMS) beamline. The purified samples were injected into glass capillary tubes for X-ray scattering experiments, which were performed under room temperature. Structure factors S(q), where q is the wavevector, were obtained by the radial integration of 2D patterns and were divided by a nanopar- ticle form factor obtained from the scattering of solution- dispersed nanoparticles. Measurements of circular dichroism and optical absorp- 5 tion for spherical helix clusters: Purified spherical helix cluster was mixed with the Gold Enhancement reagent (Nanoprobes) with a volume ratio of 1:0.3 and incubated at room temperature for ~30 min-43 The circular dichroism and optical absorption of helical clusters were measured by a Jasco J-815 CD spectrometer. Design of valence-programmable nanoparticle clusters: The disclosed subject matter provides methods for program- ing the desired nanoparticle (NP) cluster architecture using, a DNA scaffold. It is advantageous to program the under- lying scaffold to be capable of exhibiting the desired valence. Moreover, such valence does not have to be limited to symmetry modes found in atomic systems, but it can address a wider space due to the ability to fully prescribe bond locations and types. Although, in general, DNA- binding sites can be placed in desired locations on the DNA scaffold, the specific design and geometrical limitations of S a 8 w 40 68 the scaffold become restricting factors quickly. Indeed, the overall shape of the DNA construct can have a major impact on the nanoparticle-cluster architecture. In order to mini- mize any effect arising from DNA-scaffold shape anisotropy, such a scaffold can possess the symmetry. In other words, it can be designed to be as close as possible to a sphere. At the same time, it can provide binding sites for nanoparticles around this topology. Such objects can have the capability for bond programming in order to afford different types of valence modes through the 3D placement of specific binding sites on this object. Unlike atomic systems, different sites can be distinguished through orthogonal DNA-encoding, and this so-called polychro- matic valence offers distinctive bonds for DNA-encoded NP bindings. Such a desired programmable DNA object can address the challenge of creating a designed cluster (FIG. 51A). DNA origami was used to fabricate a sphere-like mesh construct 28, which has a pentakis icosidodecahedron shape (FIG. 51B). Icosahedral symmetry (Ih) is a common sym- metry for polyhedrons approximating a sphere. This struc- ture can contain vertices, with each vertex being a junction of either five or six edges. 90% of the edges (108 of 120) contain one DNA double helix and the other 10% contain two DNA double helices, with an average length of -15.7 nm (-47 base pairs (bp), assuming 3.5 nm/10.5 bp for DNA double helix). For this highly symmetrical object, the desired vertex, surrounded either by six or eight helices, can be used as a binding site for NPs by encoding six identical single-stranded DNA (ssDNA) around it, i.e., incorporating so-called “sticky ends” for NP binding. Such sticky ends were designed with a 2-base inner spacer region and 11-base outer recognition region. By choosing and encoding desired subgroups of vertices on a sphere-like meshframe, a desired valence mode was designed while maintaining the shape of the underlying DNA scaffold. Subsequently, DNA-capped NPs can bind to those designated sites and form designed cluster architectures. Furthermore, polychromatic valence can be generated by encoding the meshframe with different types, or sequence specificities, of sticky ends to recognize different correspondingly encoded NPs. Using this method, NP assemblies with symmetric valence, such as a five-fold cluster, nanoclusters with arbitrary valence, such as a spheri- cal helix cluster, and multitype nanoclusters, such as a three-component chiral cluster, were achieved as demon- strated in FIG. 51B. FIGS. 51A and 51B show a nanoparticle-cluster self- assembly directed by the valence-programmable DNA meshframe. FIG. 51A: Conceptual illustration of a nanopar- ticle (NP) cluster coordinated by sphere-like frame structure with arbitrarily prescribed valence modes and different types of binding affinities (shown as colors). Designated vertices provide binding affinities to corresponding DNA-encoded NPs (shown with matched colors). FIG. 51B: Designed DNA meshframe origami, pentakis icosidodecahedron (gray skeleton), for programming designed NP cluster architec- tures. Zoomed-in vertex shows that it is formed by six edges, with each edge consisting of one double helix. Dark gray lines indicate staple strands and light gray lines indicate a templating DNA. The DNA meshframe can be encoded by introducing ssDNA around the vertex. An encoded vertex is zoomed-in to show that six identical sticky ends (green curves) protrude from a designated vertex. Sticky ends anchored on designated vertices form valence modes of triangular bipyramid and helix (top and middle). Distinctive sets of sticky ends (strands with different colors) can be anchored at designated vertices (middle and bottom). NPs
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US 12,548,243 B2 69 (red balls), capped with complementary DNA shells, are assembled into designed clusters through their coordination around the meshframe corresponding with the programmed vertices, for example (from top to bottom): symmetric nanocluster, arbitrarily prescribed nanocluster with chiral helical valence mode, and multitype NP cluster. Symmetric architectures: First, to explore the valence- programmability of the DNA sphere-like meshframe, a class of frames with various symmetric valence modes was designed, from two to six, corresponding to the geometries of dumbbell, triangle, square, triangular bipyramid (TBP), and octahedra, respectively (FIG. 52A). NPs functionalized with DNA can bind through hybridization to complemen- tary-encoded sites, resulting in the assembly of matching clusters with designed architectures (FIG. 52B). To fabricate the programmable meshframes, M13mp18 scaffold was annealed with a specific set of staple strands. Then, the preassembled frames were mixed and annealed with gold NPs (AuNPs, 10 nm core diameter) to construct NP clusters, followed by purification with agarose gel elec- trophoresis. Representative TEM images demonstrate the projections of five types of nanoclusters, consistent with corresponding designs (FIG. 52C). In comparison with AuNPs, stained meshframe edges formed by only one or two duplexes, have a weak contrast. Different projections of these clusters were observed, with some clusters being deformed on a TEM grid due to the limited rigidity of meshframe that did not withstand drying process during a TEM sample preparation. Statistical analysis shows a high yield for all assembled types of the cluster architectures: —90% of dumbbell, triangular and square nanoclusters show the correct number and positions of NPs (of —500 for each type of cluster), and >80% for TBP and octahedral nano- clusters exhibit expected morphology (of —400 for each type of cluster) (FIG. 52D). TEM statistics also shows that -99% of binding sites on the meshframe are occupied by NPs. A high NP attachment yield per binding site owes to the rational design of sticky-end distribution and binding strength. Among the occupied sites, 98% are specifically bound by individual particles as designed, and 2% are shared by two particles. The double occupancy phenomenon results from non-equilibrium hybridization, where a second NP binds before all sticky ends at a given site can connect with the first NP. This effect of double occupancy is reduced for larger NPs. For example, clusters assembled with 20 nm AuNPs show 99% meshframe site occupancy by individual NPs and that only 1% of sites are shared by two NPs. To reveal the spatial arrangement of NPs in the assembled 3D nanoclusters, such as TBP and octahedra arrangement, cryo-EM based tomography was employed. FIG. 52E shows that the five NPs of the reconstructed TBP cluster are arranged in a designed manner. To quantify TBP cluster structural parameters, distances between NPs on the diago- nal (one d1) were measured, on the horizonal triangular plane (three d2) and on the lateral plane (six d3). The distances agree well with designed values, indicating the precise, successful arrangement of NPs on the sphere as shown in FIG. 52E, bottom. Similarly, tomographic mea- surement, and reconstruction were carried out for an octa- hedral nanocluster, and the results also demonstrate (FIG. 52F) an excellent correspondence between the targeted and realized architectures of the interparticle distances (three dl, twelve d3). FIG. 52 shows structures of NP cluster architectures assembled by programmed meshframe and revealed by TEM and cryo-TEM tomography. FIG. 52A (From top to bottom) shows designed DNA meshframes with different 45 60 70 valence numbers: two, three, four, five, and six, correspond- ing to the geometry of dumbbell, triangle, square, TBP and octahedra, respectively. FIG. 52B shows NPs decorated with DNA are assembled into clusters, whose architecture is determined by the meshframe valence mode. FIG. 52C shows representative negative-stained TEM images of assembled NP clusters based on meshframes with different valence modes; scale bar, 100 nm (insets: zoomed-in images; side length, 70 nm). FIG. 52D shows NP cluster population histograms. FIG. 52E shows a reconstructed 3D structure of TBP cluster from cryo-TEM based tomography (top). Designed center-to-center distances between NPs (bottom, shadow columns): d1=64.8 nm, d2=56.1 nm, and d3=45.8 nm. Measured distances between NPs of one recon- structed cluster (bottom, solid columns): d1=64.6 nm, d2=55.842.7 nm, and d3=46.1+2.1 nm. FIG. 52 F shows reconstructed 3D structure of octahedral cluster by cryo- TEM tomography (top). Designed NP distances (bottom, shadow columns): d1=64.8 nm and d3=45.8 nm, and mea- sured cryo-TEM tomography distances averaged over two independently reconstructed clusters (bottom, solid col- umns): d1=66.3+2.0 nm and d3=46.943.8 nm. Error bars indicate standard deviation of experimental data. To probe the structures of formed clusters in the actual buffer conditions in which the clusters were assembled, in situ synchrotron-based small-angle X-ray scattering (SAXS) was employed. Purified nanoclusters, dispersed in solution, were placed in a quartz capillary, and then probed at room temperature by a collimated X-ray beam (1=0.92 A) at the Complex Materials Scattering (CMS) beamline of National Synchrotron Light Source If (NSLS-ID at Brookhaven National Laboratory. The scattering pattern was collected with a Dectris Pilatus3x2 M pixel-array detector and con- verted to 1D scattering intensity versus wavevector transfer, q. Structure factor, S(q), for each nanocluster was extracted from the 1D scattering intensity by subtracting background and dividing the intensity by the form factor of AuNPs41,42 (FIG. 53A), which was measured for dispersed AuNPs. Interparticle center-to-center distances are derived by fitting S(g) with the function below: wy sinfgd y) 5) i= 1 rd is, a 2 Siy=1+5 N is the NP number in a specific designed cluster, and d,, is the center-to-center distance for each pair of NPs in a cluster. For dumbbell and triangular nanoclusters, only one type of interparticle distance exists, which is d1 between diagonal nanoparticles in the dumbbell nanocluster and d2 between nanoparticles on the triangular plane in the triangular nano- cluster. For the square nanocluster, two types of interparticle distances, dl between diagonal nanoparticles and d3 between adjacent nanoparticles, exist. The average interpar- ticle distances for five-cluster architectures are d1=68.1+0.3 nm, d2=58.1+0.1 nm, and d3=47.2+0.3 nm, respectively, which are close to the expected values based on the design: d1=64.8 nm, d2=56.1 nm, and d3=45.8 nm. FIG. 54 shows In-liquid structures of NP cluster archi- tectures revealed by in situ SAXS. FIG. 54A shows an extracted structure factor S(q) from in situ SAXS for designed clusters with the following meshframe valence numbers (from bottom to top): two, three, four, five, and six, respectively. Fitted S(q) are shown. FIG. 54B shows inter- particle distances derived from SAXS (squares) for NP
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US 12,548,243 B2 71 clusters: d1=68.4 nm for dumbbell cluster, d2=58.0 nm for triangular cluster, dl=68.3 nm and d3=47.4 nm for square cluster, d1=67.9 nm, d2=58.2 nm and d3=46.8 nm for TBP cluster, d1=67.6 nm and d3=47.3 nm for octahedral cluster. Interparticle distances computed for the designed architec- tures (dash lines): d1=64.8 nm, d2=56.1 nm, and d3=45.8 nm. Arbitrarily designed cluster: spherical helix architecture: To demonstrate the versatility and flexibility of this assem- bly strategy for creating complex and arbitrarily designed clusters, an architecture in which NPs are located in a helical pattern on the surface of sphere-like DNA object (FIG. 54) was implemented. DNA meshframe was programed to have 13 binding sites at designated vertices, encoding a left- handed spherical helix cluster formed by 13 NPs (FIG. 54A). Two different sets of sticky ends, placed in alternating order between the sites, with five identical sequences per site are used. By alternating these two distinctive sets of sticky ends, site competition was reduced between neighboring NPs and thus increase the accuracy of NP localization on the designated vertices. Accordingly, two types of 10 nm AuNPs decorated with different DNA shells are used to bind with the helically positioned binding sites. For this high valence DNA scaffold, an excess of DNA staple strands used in scaffold formation can be removed before mixing with NPs, in order to minimize inter-cluster crosslinking. The mixtures of purified meshframes and AuNPs were annealed from 37° C. to 20° C. for 12 h, followed by purification with agarose gel electrophoresis and analysis with negative-stained TEM. Representative TEM images show that the NP cluster matches the designed helical morphology (FIG. 54B left), with 88.4% of binding sites occupied by NPs (FIG. 54B, right), as measured from 611 clusters. This yield is lower than that of symmetric clusters discussed above (-99%), due to more complex NP arrangements in a helical pattern and stronger steric interactions between adjacent DNA-capped AuNPs To unravel information regarding particle organization for the spherical helix valence mode on the meshframe, cryo- TEM-based tomography was used to characterize the 3D structure of individual clusters. To establish a comparison with a prescribed helical valence arrangement, the particle sites on the sphere-like meshframe were numbered from 1 to 13 (FIG. 54C, left). Seven particles (Particle 4-10) are located on the equatorial plane of the sphere. Particle 1 and Particle 13 are located on the summits of Upper (U) and Lower (L) hemispheres, respectively. The structure was projected from both the U and L summits. For visual guidance, in the center of the cluster, a gray sphere was added, whose radius is equal to the distance between the 5 meshframe center and NP surface. Both projections show a uniform NP arrangement on the equatorial plane and NP positions on the upper and lower hemispheres (FIG. 54C, right). The reconstructed nanocluster is presented in both 3D view and 2D projections in FIG. 54D, where a gray sphere provides visual guidance as FIG. 54C. The coordinate of sphere center and sphere radius are fitted using the obtained 3D coordinates of all NPs (numbered 1-13) in the recon- structed cluster. The tomography results (FIG. 54D, left) show that seven NPs (Particles 4-10) are on the equatorial plane, three NPs (Particle 1-3) on the upper sphere, and three (Particles 11-13) on the lower sphere. The visualized 3D spherical helix pattern of NPs matches well with the designed helical cluster architecture. The U and L projec- tions (FIG. 54D, right) demonstrate that Particles 4-10 are arranged relatively uniformly on the equatorial plane. The S a w 8 w 8 40 4 2 s B 2 locations of Particles 1-3 and Particles 11-13 indicate that their placement pattern corresponds to a left-handed chiral- ity, which agrees with the prescribed valence mode. Small NP shifts (-5 nm) from expected positions are observed in the recon-structed structure, which can result from interpar- ticle repulsion and sample preparation for imaging. Since each binding site has multiple sticky ends, NPs can hybrid- ize at slightly different configurations at the same site, which affects the precision of a NP placement. Although there are twelve values of interparticle center-to-center distances (D) for clusters with thirteen NPs (FIG. 54E), there are only two different D in our designed locations of NPs due to two types of edge lengths of the meshframe: ten D values are of 20.2 nm and the other two of 22.8 nm. Since the diameter of 10 nm AuNPs with DNA shells is -20 nm (21 nucleotide (nt) for the DNA shell), which is close to the designed D value, steric repulsion can result in some displacement of NPs. Tomograph measurements show a small increase of D (24.024.9 nm) over its two design values, as averaged from three independently reconstructed clusters. Such an increase of D is consistent with the SAXS measurements, which show a corresponding shift of the primary scattering peak that arises from nearest neighbor interparticle distances. Another obtained value is the distance between the center of meshframe and the center of individual AuNP in the nano- cluster (FIG, 54E), R=34.5+3.8 nm, which matched well the expected value (d1/2=32.4 nm) and SAXS measurements (68.1/2=34.0 nm). The SAXS measurements for spherical helical clusters provide coarse information about internal cluster organization due to the sensitivity of a structure factor to an interparticle distance distribution of interparticle distances in comparison to the symmetrical cluster. In solution, the optical absorption spectrum of spherical helix clusters, with characteristic absorption peak at 520 nm, closely resembled the surface plasmon resonance (SPR) mode of individual AuNPs, which indicates a weak coupling between NPs. Due to the chiral nature of the helical cluster of plasmonic NPs, a different absorption for incident light of left and right circular polarization can lead to a circular dichroism (CD) signature near the SPR. To enhance the plasmonic resonance through interparticle coupling, NP size increases to -13 nm using gold enhancement reagent; this also decreases interparticle distances accordingly. A corre- sponding plasmonic redshift from 520 nm to 532 nm (FIG. 54F) was observed. A positive CD signal was detected at wavelength shorter than 500 nm, with a negative dip around the SPR peak. This observation is consistent with our numerical simulation of the absorption and CD spectra of AuNP spherical helix clusters (FIG. 54G), using discrete dipole approximation (DDA). This type of cluster combines a helical arrangement of NPs with overall spherical shape, and that compact and symmetric design is distinct from previously reported bar-like clusters with a spiral NP arrangement. FIG. 54 shows design and structural and optical charac- terization of the spherical helix NP cluster. FIG, 54.4 shows a schematic of the spherical helix NP cluster. The spherical helix valence mode is realized for the meshframe by pro- gramming two alternating sets of sticky ends (green and purple, with five strands) at desired vertices. Two types of AuNPs with identical 10 nm core size but distinct DNA shells are directed through the hybridization to binding sites and assembled into a spherical helix cluster. FIG. 54B shows (Left) representative negative-stained TEM image of spheri- cal helix clusters; scale bar, 200 nm (inset: zoomed-in image ofa spherical helix cluster; side length, 150 nm) and (Right) population analysis of assembled spherical helix clusters
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US 12,548,243 B2 73 (from 611 measured clusters). FIG. 54C shows designed NP positions of the spherical helix cluster (numbered from 1 to 13) on a sphere-like meshframe (left). Seven particles (Par- ticles 4-10) are on the equatorial plane of the sphere-like structure. Particle 1 and Particle 13 denote the summits of Upper and Lower hemispheres, respectively. Projections of designed spherical helix cluster from the Upper (upper right) and Lower (lower right) summits. The grey sphere provides visual guidance. The line with arrow defines particles on the equatorial plane (Particles 4-10). FIG. 54D shows a spheri- cal helix cluster reconstructed from cryo-TEM tomography. 3D view of the reconstructed structure (left) and projections from the Upper (upper right) and Lower (lower right) summits. FIG. 54E shows a center-to-center distance between adjacent NPs, D, and center-to-center distance between sphere-like meshframe and NPs, R, were measured. Designed distances (bottom histogram, shadow columns): D=20.2 nm and 22.8 nm, and R=32.4 nm. Average distances obtained from three independently reconstructed clusters (bottom histogram, solid columns): D=24.0+4.9 nm and 2 R=34.523.8 nm. Error bars indicate standard deviation of experimental data. FIG. 54 F shows a computed CD spec- trum and absorption spectrum for spherical helix clusters. FIG. 54G shows an experimental CD spectrum, Lorentzian fit, and absorption spectrum for spherical helix clusters. Multitype nanoparticles clusters: The addressability of the sphere-like DNA meshframe not only allows prescribing directional valence through the location of binding sites, but also encoding each site independently such that it provides aflinity only to the NP with matching encoding, as deter- mined by the complementarity between sticky ends at the vertex and ssDNA in a given NP shell. This encoded, so-called polychromatic valence can open opportunities for assembly of multitype NP cluster architectures (FIG. 55). As a demonstration of this capability, a meshframe was pro- gramed with two- (FIG. 55A, I-II) and three-color valence modes (FIG. 55A, III-V) for assembly of different multitype NP clusters based on the same five-fold directional valence (FIG. 55B) discussed previously. Specifically, the binding sites of a TBP linking frame (labelled 1-5, FIG. 55A) are encoded independently with two and three sets of sticky ends. AuNPs with diameters of 10 nm (P1), 15 nm (P2), 20 nm (P3), and 30 nm (P4) were coated with specific DNA shells that are complementary to respective sticky ends on the meshframe. Guided by this polychromatic valence, dif- ferent types of AuNPs were anchored to prescribed sites. FIG. 56 (1) shows a two-component nanocluster with three-fold symmetry, with two P3 particles located at sites 1 and 2 and three P1 particles at sites 3, 4, and 5, providing a label of P1(345)P3(12). Similarly, a nonsymmetric two- 5 component nanocluster P1(245)P4(13) was designed and assembled (FIG. 55B (II)). Then, a three-color valence meshframe was used to form symmetric and nonsymmetric nanoclusters: P1(345)P2(2)P3(1) with three-fold symmetry (FIG. 55B (III)), nonsymmetric achiral P1(245)P2()P3(1) (FIG. 55B (IV)) and nonsymmetric chiral P1(24)P2(3)P3 (15) (FIG. 55B (V)). Representative TEM images show that the NP arrangements for all five hetero-clusters based on the sphere-like meshframe with polychromatic valence agree well with our design (FIG. 55C). The statistical analysis indicates that this hetero-cluster assembly process can be realized with relatively high yield, with -80% of correct NP composition (out of -420 clusters for each cluster type) (FIG. 55D). To obtain information about the spatial arrangement of NPs in multitype NP clusters, nonsymmetric chiral cluster (P1(24)P2(3)P3(15)) was evaluated using cryo-TEM tomog- S a w 8 40 4 74 raphy. As shown in FIG, 55E, the reconstruction confirms that three types of AuNPs are arranged in the designed manner, with two P3 particles at vertices 1 and 5, one P2 particle at vertex 3 and two P1 particles at vertices 2 and 4. The interparticle distances were obtained from eight recon- structed nanoclusters. The center-to-center distances between diagonal NPs (d12) and between NPs on the horizonal triangular plane (d34, d45, and d35) are in excel- lent agreement with the designed values (FIG. 55E, right). FIG. 55 shows the design and characterization of multi- type NPs clusters assembled with a meshframe of directional and polychromatic valence modes. FIG. 55A shows meshframe programmed with a valence of TBP geometry and encoded sites at selected vertices, labelled from 1 to 5, results in polychromatic valence. (I-II) two-color valence. (III-V) three-color valence. FIG. 55B shows multitype NP clusters. (I) P1(345)P3(12) with three P1 (10 nm AuNP) at sites 3-4-5 and two P3 (20 nm AuNP) on sites 1-2; (II) P1(245)P4(13) with three P1 at sites 2-4-5 and two P4 (30 nm AuNP) at sites 1-3; (III) P1(345) P2(2)P3(1) with three PI at sites 3-4-5, one P2 (15 nm AuNP) at site 2 and one P3 at site 1; (TV) P1(245) P2(3)P3(1) with three P1 at sites 2-4-5, one P2 at site 3 and one P3 at site 1; (V) P1(24) P2(3)P3(15) with two P1 at sites 2-4, one P2 at site 3 and two P3 at sites 1-5. FIG. 55C shows representative negative- stained TEM images of NP hetero-clusters; scale bar, 200 nm (insets: zoomed-in images; side length, 100 nm). FIG. 55D shows population histograms for corresponding multi- type NP clusters. FIG. 55E shows a cryo-TEM tomography reconstructed P1(24)P2(3)P3(15) cluster (left). Designed interparticle distances (right, shadow columns): ,,,=69.8 nm, 734—58.3 nm, d45=60.5 nm, and ,3;=62.6 nm. Measured distances, as averaged over eight independently recon- structed P1(24)P2(3)P3(15) clusters (right, solid columns): d12=66.824.3 nm, d34=61.424.6 nm, d45=63.824.4 nm, and d35=67.8+7.2 nm. Error bars indicate standard devia- tion of experimental data. The disclosed subject matter provides methods for assem- bly of designed nanoparticles clusters using valence-pro- grammable DNA mesh-frame with 3D control of NP posi- tions and incorporation of different types of NPs. Spatially and type-defined (polychromatic) valence modes can be rationally designed and programmed using a highly sym- metric, sphere-like frame that serves as a universal 3D scaffold for coordinating NPs in designed 3D patterns. Nanoclusters with symmetric and arbitrary valence modes can be created with high yield and high structural fidelity, including different prescribed symmetries and helical orga- nizations. The polychromatic valence can permit assembly of multitype NP clusters. The quantitative agreement between the designed and assembled structures was dem- onstrated using a combination of TEM, tomography and X-ray scattering methods. A demonstrated, broadly appli- cable valence-programmable assembly strategy opens new routes for the rational fabrication of NP architectures via self-assembly, with customized architectures, compositions, and function. Example 4: Engineering Organization of DNA Nano-Chambers Through Dimensionally Controlled and Multi-Sequence Encoded Differentiated Bonds The disclosed subject matter provides a nanoscale system that possesses the following features (FIGS. 56 and 57): (i) a rigid 3D semiclosed cuboid geometry that can carry nanocargo; (ii) differentiated bonds, through DNA-encod- ing, for prescribing interobject connectivity in three
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US 12,548,243 B2 15 orthogonal directions and their local alignments; (iii) mul- tisequence encoding of bonds (polychromatic bonds) within each axis that have a robust prescription of interactions along with control over a relative orientation of objects. As the fundamental building block, a DNA nano-chamber (DNC), a hollow DNA cuboid, was used. Such an object has an intrinsically relatively low symmetry, which well sup- ports the designability of complex architectures, has well- defined internal arrangement of duplexes, and possesses an internal compartment that allows it to host a nanoparticle or a molecule or a biomolecular complex. Two openings of the DNC also permit the ease of inserting and release of nanocargo. Thus, the DNC combines a simple yet relevant geometrical shape with a potential utility for exploring assembly using differentiated bonds. A facile approach to regulate the programmable assembly of these blocks into multidimensional ordered arrays with different degrees of complexity through control of differentiated polychromatic bonds (FIG. 57B). Using electron microscopy and in situ 2 X-ray scattering methods, engaging bonds along one (Z), two (X,Y) and three (X, Y, Z) axes that results in the formation of 1D, 2D, and 3D ordered arrays, respectively were observed, while the strength of the bonds and their differences have effect on the assembled structures. The disclosed subject matter also provides the resultant arrays that can serve as versatile platforms to coordinate the organization of gold nanoparticles (AuNPs). Design of DNA Nanochamber and Establishment of Dif- ferentiated Polychromatic Bonds: The disclosed subject matter provides building blocks (DNCs) with bonds that are fully differentiated in the X, Y, and Z directions and within each axis (FIG. 57A). DNC binding properties can be determined by 6 unique types of bonds: X-, X+, Y-, Y+, Z-, and Z+. They are referred as X, Y, and Z bonds. Moreover, each bond was encoded via multisequence strands, so-called “sticky ends”, that offer a high degree of uniqueness for encoding, orthogonality of interactions, orientation, and positioning control. Since each sticky end is encoded with a distinct DNA sequence, it is referred to as color, and this differentiation allow us to establish polychromatic X, Y, and Z bonds. The bonds are located at the well-defined positions along the surface of the DNC. The assembly of DNC monomers into a larger-scale organization is dictated by these polychromatic bonds, while the specific details of bonds can influence the assembly process. The disclosed subject matter offers the designability of anisotropic and differentiated bonds for engineering ordered arrays using the same basis nanoobject, the DNA nanochamber. DNC can include a total of 80 DNA duplex cylinders was 5 designed by caDNAno. The DNC has an open cavity of size ~25x25x28 nm?, which is fenced by a double layer of DNA. duplexes. Binding strands for inter-chamber connections can be independently tuned and are located in the middle of the DNC walls for XY directions and at the edges of the DNC opening for Z direction. Each binding strand consists of an 8-base recognition sequence (sticky ends) and a 22-base poly-T DNA spacer. The poly-T spacers endow the flexibil- ity of linkages and mediate steric effects, both of which are beneficial for DNC-DNC recognition during assembly. In addition to differentiated bonds along orthogonal X, X*, YY, Y*, Z, and Z* axes, all of the 8-base sticky ends within each axis are entirely distinct as denoted by different num- bers (e. g., 1-16 in XY). Not only do these polychromatic bonds enable the connected DNCs to align correctly and prevent uncontrolled shifting, but, more importantly, they also endow orientational control of monomers within S 40 4 716 ordered arrays. These design principles of programmable polychromatic bonds are applied to 1D, 2D, and 3D assem- blies. DNA Nanochamber Construction and Nanoparticles Encapsulation: DNC was formed by folding an M13mp18 single-stranded (ss) scaffold with 224 short staple oligonu- cleo-tides (FIG. 58A), through slowly cooling from 80° C. to room temperature. The successful formation of DNC was first analyzed by agarose gel electrophoresis (FIG. 58B). The gel-purified products were then examined by negative- staining transmission electron microscopy (TEM). Hollow DNA frames with a height of 28 nm were observed (FIG. 58C), which unambiguously confirmed the formation of the designed construct. Cryo-TEM was utilized to further verify the DNC structure. As shown in FIG. 58D, hollow cuboid architectures with a central cavity of 25 nm and wall thickness of -5 nm can be visualized directly, in good agreement with double layer packing of DNA helix. The binding strands for inter chamber ligation can be readily incorporated through introducing selected staple strands containing extension sequences of sticky ends during the DNA origami folding protocol (see DNC with bonds in FIG. 58A). DNCs can be encoded with internal and external anchor- ing strands for carrying nanocargo, in this case AuNPs, within the cavity or outside of the sidewall (FIG. 58E), respectively. The integration of DNCs and AuNPs was achieved by annealing the DNCs encoded with anchoring strands and AuNPs functionalized with complementary ssDNA strands. The AuNPs and DNCs (e.g., 60 WL of 10 nM) were mixed in a stoichiometric ratio of 2.5:1, and subjected to an overnight annealing procedure. As illustra- tive examples, AuNPs with core diameters of 10 nm (18- base DNA shell) and 20 nm (18-base DNA shell) were employed to coordinate with the DNCs, respectively. TEM imaging after gel purification confirms a successful encap- sulation of 10 nm AuNPs in the DNCs with a yield of 93% (FIG. 58F), and a binding of 20 nm AuNPs onto the sidewall with a yield of 81% (FIG. 58G). It is worth noting that each cavity (25 nm) and sidewall (28 nm) can accommodate only one DNA-coated AuNP, which is essential for controlling the arrangement of AuNPs within ordered arrays. 1D Assembly of DNA Nanochambers: By utilizing DNCs with fully prescribed binding characteristics as underlying building blocks, the disclosed subject matter provides the assembly of 1D linear arrays, which can represent a nanoscale polymer analog. The use of differentiated bonds and type-encoded DNCs allows constructing of a variety of nanoparticle architectures including homopolymers, sequence-prescribed heteropolymers, and helical polymers. To assemble the chain-like 1D arrays, sticky ends are placed as extensions of selected staples at the end of the DNA duplex along the Z-axis. Two types of DNCs (e.g., Z and Z') containing sticky ends complementary to each other were designed, giving rise to two kinds of bifunctional monomers. For each monomer, up to 32 binding strands can be attached at the predefined locations of DNC edges (FIG. 57B), where their 8-base sticky end sequences are completely different from each other. This multisequence encoding along the Z-axis allows for a high degree of specificity of DNC-DNC bindings and for controlling their relative orientation along Z direction. 8-base sticky ends were selected because their melting temperatures are within a favorable experimental annealing temperature window (20-50° C.). 6-base or 10-base sticky ends were tested using identical annealing protocol. Unconnected or aggregated structures were observed. Therefore, 8-base sticky ends design was used.
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US 12,548,243 B2 77 Analogous to classic step-growth polymerization, the liga- tion of DNCs was induced by slowly cooling mixtures containing equal amount of two monomers (typically, 100 uL of 10 nM). To enable a proper antiparallel directionality for the hybridization of sticky ends, Z' monomer was spe- cifically designed to rotate 900 clockwise relative to Z monomer in the direction of polymerization. The disclosed DNC system with Z bonds can have a similarity with a classic step-growth polymerization, for which the prerequisite of synthesizing long polymer chains is highly reactive monomers. In order to identify the high “reactivity” for 1D growth, the chain formation was evalu- ated by varying the number of sticky ends on each side of DNC (from 8, 16, 24 to 32). When 8 sticky ends were used, oligomers with an average of 4 monomers long were observed. By increasing the number of sticky ends to 16, both oligomers and polymers were yielded with an average of 8 monomers per chain. Further increase of sticky end number (24 and 32) results in longer nanopolymer chains. Notably, using 32 sticky ends, discrete chains with microm- eter lengths containing on average 30 monomers were obtained (FIG. 59A). The formation of chains without which disordered aggregates was observed. Annealing involves a slow cooling of the sample from 50 to 20° C. During this process, there is a temperature window within which quasi- equilibrium assembly occurs with reversible bindings of individual sticky ends. For an assembly regime within this favorable temperature window (FTW), the growth rate can depend on the inter-DNC interaction strength and binding probabilities, which for the given sticky end motifs are determined by the number of sticky ends. Since all systems follow the same annealing protocol, the same amount of time was spent in the FTW. This results in the assembly of the longest chains for the 32 sticky ends design, which has the largest attraction interaction among the discussed sys- tems. On the basis of the design of 32 sticky ends, different types of linear AuNPs chains were fabricated via a “one-pot” assembly process, where DNA-coated AuNPs, Z, and Z' DNCs encoded with internal anchoring strands were simply mixed and annealed to grow 1D nanoparticles arrays. This process is referred as a “one-pot” because AuNPs loading and DNC polymerization occur concurrently in one anneal- ing protocol. To ensure satisfactory loading efficiency of AuNPs, the following features were implemented: (i) stron- ger bonds, based on 12-base internal anchoring strands, for AuNPs encapsulation, in comparison with 8-base sticky ends for polymerization and (ii) the excess of AuNPs (molar ratio AuNPs/DNC=2/1). This one-pot approach is also applied to the 2D and 3D assembly. The alternating/homo- 5 chains that are alternatively/fully filled with 10 nm AuNPs were produced by encoding single (Z or Z'V/both (Z and Z') DNC monomers with internal anchoring strands. The desired nanostructures were directly visualized from TEM images in FIGS. 59B and 59C, with a success rate of 83% and 89% for filling alternating chains and homochains with AuNPs, respectively. Given a full prescription of bonds and DNCs encoding for both external and internal connections, the presented approach for constructing polymer-like chains is both robust and versatile. It can be adapted to build chains with increas- ing complexity and arbitrary order of different monomers. To demonstrate this versatility, four different kinds of DNCs were specifically designed (e. g., A, B, C, and D), whose complementary sticky ends allow for connecting with each other sequentially (FIG. 59D). Upon annealing in a rigorous equal molar ratio, the desired nanoscale heteropolymer with S a w 8 w 40 45 2 s B 78 a definitive sequence of ABCD was produced. Notably, the sequence-controlled chains offer a platform for controlled nanoparticle arrangement in a complex linear array. For example, the disclosed subject matter provides a nanoarchi- tecture in which two filled and two empty DNCs are alternatively arranged (FIG. 59E). Within this nano-archi- tecture, monomers A and B are designed with internal anchoring strands to capture 10 nm AuNPs, while monomers C and D do not have these strands. To stress the significance of monomer orientation control through multisequence bonds, a helical organization was created within the 1D array by placing 20 nm AuNPs on the specifically designated sidewall of four types (A, B, C, and D) of DNCs. This assembly requires the specific orientation of monomers relative to their adjacent neighbors, and the discussed multisequence scheme permits for such a realiza- tion (FIG. 59D). The defined 1D arrays with helical nan- oparticle organization were successfully fabricated and veri- fied by TEM imaging (FIG. 59F). While certain studies used a single DNA origami construct as a template for the fabrication of AuNPs helices, the disclosed helical organi- zation is derived from the assembly of multiple types of DNC monomers. As each monomer can be independently programmed, this can provide new opportunities to tailor chiral organizations in terms of their length, composition, and architecture, and potentially allow for dynamic regula- tion of the formed structures. 2D Assembly of DNA Nanochambers: Four-fold symme- try of DNC in the lateral direction (XY, FIG. 57B) permits for the manipulation of 2D square arrays by encoding bonds on four sidewalls of adjacent, complementary DNCs (e. g., XY and X'Y’). Up to four distinct, sticky ends with a defined order were used to prescribe a recognition zone on each DNC face along the center-line in the XY plane of each sidewall. This encoding provides positioning restrictions ensuring the ligation of two neighboring DNCs in the same plane, whereas the implementation of distinct sequences for face-to-face bonds prevents any undesired lateral shifting. To induce 2D array formation, equal amounts of two complementary DNC mixtures were subjected to a 48 h annealing process (cooling from 50 to 20° C.). FIGS. 60A-60D demonstrate the successful assembly of 2D arrays for the different number of sticky ends in the XY-plane (total per DNC: 4, 8, 12, and 16, or per DNC’s face: 1, 2, 3, and 4). For 4 sticky ends, the binding strength is too weak to bridge neighboring DNCs (FIG. 60A). As the number of sticky ends increases from 8 to 12, and finally to 16, the average domain size of 2D arrays increases dramatically from 22 to 45 to 156 DNCs per array (FIGS. 60B-60E), respectively. This positive correlation between the number of sticky ends and the size of the 2D array to the increased inter-DNC hybridization energy and binding probability was observed. The process of lattice formation occurs during annealing in the FTW, similar to the 1D case. The same mechanism (as discussed for 1D case) is realized for 2D system: the growth rate within FTW influences the size of the crystalline domain, and that rate is higher for the larger number of the sticky ends. With 16 sticky ends, the forma- tion of the prescribed large-domains of DNC 2D lattice was observed (FIG. 60D). Small angle X-ray scattering (SAXS) characterization reveals a scattering profile for 2D square lattices. The scattering peaks corresponding to Bragg reflec- tion planes and their positions agree well with the modeled scattering. The center-to-center distance of two adjacent DNCs is 44.2 nm based on the first-order peak and is consistent with that measured from TEM observations.
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US 12,548,243 B2 79 These results validate that the formation of 2D ordered arrangements of DNCs within the XY-plane occurs in bulk. To further understand the results on the assembly of the 2D DNC arrays, molecular dynamics simulations were per- formed. A cubic hard object was used as a proxy for the designed DNC and the Kern-Frenkel potential to mimic inter-DNC bonds. In order to reflect the specificity of bonds in the experimental system, different types (“colors”) of bonds were designed for each sticky end strand on the cube, wherein only the same color of bonds can bind to each other. Using Monte Carlo simulation of hard polyhedral (HOOMD-blue package), the systems were cooled with 4, 8, 12, and 16 bonds down from high temperature and caleu- lated the average assembled cluster sizes (FIGS. 60E-60H). Similar to the experimental findings, the average cluster size becomes larger as the number of bonds increase from 4 to 16. This effect is due to a higher growth rate of ordered domains for a larger number of sticky ends. The simulation indicates that the crystallization rate can significantly affect the final size of the clusters, and the binding probability enhances as the number of bonds increases, leading to larger sizes of clusters within the same simulation time scale. Next, DNCs that were encoded with internal anchoring strands to form 2D lattices of AuNPs were used. With the design of 16 bonds per DNC, 2D lattices of AUNPs were produced via a one-pot protocol where AuNPs were annealed with DNCs (molar ratio: AUNPs/DNC=2/1). TEM observation and well-defined SAXS pattern validate the formation of the intended 2D AuNP lattices (FIGS. 601 and 60J). Compared with the SAXS pattern of vacant DNC arrays, the SAXS scattering profile in FIG. 60J is much sharper and fully reflecting the AuNPs arrangement due to high X-ray contrast of AuNPs relative to DNA. The nor- malized peak positions in the SAXS pattern show a qn/q1 ratio of 1:V2:2:V5 . . . (q1=0.143 nm-1), unambiguously identifying a conventional 2D-square lattice. The close correspondence with the model peak positions (FIG. 60J) indicates a formation of well-defined square lattice. More- over, the center-to-center distance of two neighboring AuNPs obtaining from SAXS (d1=43.9 nm) is in good agreement with that from TEM image (~44 nm), further suggesting a high degree of order for assembled AuNP lattices. To emphasize the versatility of this approach for engi- neering particle arrangements, a chessboard-like 2D lattice was designed with alternative loading of AuNP in DNCs (FIG. 60K). The chess-board-like 2D lattices were also characterized by SAXS, where the experimental scattering peaks are consistent with the model (FIG. 60L). The result- ing lattice with well-defined order has a V2-fold increase in 5 the center-to-center nanoparticle distance (d1=62.8 nm) in comparison with the previous design for a flat square lattice shown in FIG. 60J. 3D Assembly of DNA Nanochambers: The presented techniques were used to create DNCs with differentiated bonds in all three orthogonal directions and to explore their assembly behaviors. The complementary sticky ends were anchored along the three axes of DNC to construct the 3D assembly, combining approaches for the formation of the aforementioned 1D and 2D arrays. This seemingly simple addition of Z and XY interactions induces a quite nontrivial assembly behavior in 3D due to the competition between this longitudinal (Z) and lateral (XY) binding modes. Com- pared with 1D or 2D arrays, the growth of 3D structures within the XY-plane versus along the Z-axis resembles a tug-of-war, and this interplay has significant impacts on the assembly kinetics and the structures formed. In an effort to S a w 8 w 8 40 2 s 80 control the assembly process for DNCs with XYZ bonds, the 3D design was used with 16 sticky ends within the XY-plane that can afford well-defined 2D arrays, and varied the number of sticky ends along the Z-axis from 8, 16, 24, to 32 (FIG. 61A). For simplicity, they are referred as XYm-Zn, where m and n denote the number of sticky ends prescribed in the XY-plane and along the Z-axis, respectively. Sequences of these (m+n) sticky ends attached to a single DNC are entirely distinct from each other, and bonds for each axis (X, Y, and Z) are differentiated from each other. Following 3D assembly of these DNCs, SAXS measure- ments were performed to explore the structures of the resultant assemblies. FIG. 61B illustrates both experimental and modeled SAXS patterns of assembled structures for the relevant designs (m=16 and n=8, 16, 24-32). To enhance the X-ray scattering signal, the DNCs for all designs were filled with 10 nm AuNPs. Two additional experimental scattering patterns are shown as controls for the 3D assembly. The first control is the XY0-Z32 (interparticle distance c=37.8 nm) that assembles into 1D chains, due to the absence of a lateral binding mode, and the other one is the XY16-Z0 (lattice constant a=b=43.9 nm) that assembles into 2D arrays (adapted from FIG. 60J), due to the absence of the longitu- dinal binding mode. On the basis of the symmetry of DNC and these controls, one can expect that assembled 3D DNC arrays can correspond to that of a lattice with a primitive tetragonal unit cell whose lattice parameters are a=b=43.9 nm, c=37.8 nm, a=B=y=90°. A modeled scattering pattern for this tetragonal lattice (TL) was thus generated and was displayed on the same plot. However, the comparison of the generated scattering pattern ofa tetragonal lattice to the experimentally obtained patterns for the XY16-Z8 and XY16-Z16 systems revealed signifi- cant differences. The structure factors (S(q)) of XY16-Z8 and XY16-Z16 instead resemble that of XY16-Z0 (2D arrays), implying that the lateral growth dominates the assembly process. Similar to 1D and 2D systems discussed above, the growth of the 3D system within FTW of anneal- ing can depend on the relative interaction strength of XYZ bonds. This can result in anisotropic growth or limited growth along the XY and Z direction. Since the growth rate in the XY and Z direction can be different for bond designs, the assembled structure is likely to be anisotropic, reflecting differences in growth rates. To further elucidate the structure 5 of the assemblies, detailed electron microscopy was con- ducted. In FIGS. 61D and 61E, the TEM images of XY16-Z16 with and without encaging AuNPs, were shown, respec- tively. Large domains consisting of 2D AuNP lattices can be clearly visualized in FIG. 61D. Considering the 2D projec- tion feature of TEM imaging, it is challenging to directly identify 3D architectures from TEM observation. However, the disclosed subject matter provides methods to capture two different kinds of DNC packing patterns within a single domain, which are parallel alignment of short 1D chains along the Z-axis and a 2D array pattern within the XY-plane (FIG. 61E), respectively. These two observed scenarios result from two projection views that are perpendicular to and parallel with Z direction of the 3D structures (FIG. 61E). Taken together, these results support that the growth of structures dominates within the XY-plane, leading to the formation of 3D structures with a limited number of layers along the Z-axis for XY16-Z8 and XY16-Z16. The scattering profiles evolve with increasing bond strength (the number of sticky ends) along the Z-axis. For XY16-Z24 and XY16-Z32, the structure factors exhibit significant deviation from that of the 2D control (XY16-Z0),
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US 12,548,243 B2 81 but these patterns still do not resemble that of the expected tetragonal lattice. In the case of a strong longitudinal binding mode, XY16-Z32, the scattering profile exhibits a peak at q=0.166 nm-1 (guided by the dash line) that corresponds to the center-to-center distance of two adjacent DNCs in 1D chains, as seen in the SAXS data of the 1D control sample (XY0-Z32). Note that this peak is not present in XY16-Z8 and XY16-Z16 scattering patterns. This suggests that the packing of DNCs along the Z-axis becomes a key structural feature of this system. Such a feature is directly confirmed by TEM imaging of XY16-Z32 (FIG. 61C) in which a high aspect ratio bundle of DNCs is observed. To further analyze the above observation for the XY16- 732 system, SAXS analysis was performed to understand both an internal order of assembly and the morphology of assembled structure. A finite 3D array was formed contain- ing 3 (X)x3 (Y)x10 (Z) AuNPs-filled DNCs within which DNCs are arranged in a tetragonal unit cell, as an example of a Z-elongated assembled structure. This finite model captures the feature of fiber-like 3D structures, as observed in FIG. 61C. The scattering profile of this 3x3x10 model demonstrates an excellent match with the structure factor of the XY16-Z32 system, which further verify the feature of stacking along the Z-axis. This scattering model does not uniquely identify that the structure formed can be 3x3x10, but it rather indicates that the 3D assemblies formed have tetragonal internal organization and a high aspect ratio, which signifies a different growth rate along the XY and Z directions. Taken together, the XY16-Z8 and XY16-Z16 systems exhibit 2D-dominated morphology due to the rela- tively weak longitudinal binding. In contrast, for XY 16-224 and XY16-Z32 systems with a strong longitudinal binding, a growth of 3D structure along Z-axis becomes dominant, leading to the formation of 3D structures with aspect ratio emphasized in the Z-direction. Within these both layer-like and fiber-like mesoscale 3D assemblies, a tetragonal orga- nization of DNCs is still maintained due to their well- defined structure and prescribed binding modes. To understand the effects of relative binding energies along the Z-axis versus in the XY-plane on the crystalliza- tion process, molecular simulations of assembled 3D struc- tures were performed. The design principles and multi- sequence features of the bonds are similar to the 2D case, but one bond was added on each corner of the cube along the Z-axis. The simulations was performed by keeping the number of bonds constant in the XY-plane, and the binding energy of each bond fixed (€/K,T=3.0). The binding energy of each bond in Z direction can be varied (3.0<BE/ K,T<S5.0). The shape of clusters changes from the pancake- like 2D-dominated structures for €©/K,T=3.0 (FIG. 61H) to 5 cubic-like structures for €,/K,,T=4.0 (FIG. 61G), and fiber- like 1D-dominated structures for €/K,T=5.0 (FIG. 61F). The relative shape anisotropy («2=(3/2(A; *+A) “+A; *))/ ((hy?-+h3 24h, *)?)-14) was used to quantify the shape of these resultant crystallites, where i°2 are the eigenvalues of the gyration tensor of the clusters. The K2 for the largest clusters in the system was calculated, and obtained «2=0.24, 0.06, and 0.75, for pancake-like, cubic-like, and fiber-like crystallites, respectively. These values correspond to ideal disk-like (k2=0.25), cubic-like (k2=0), and rod-like (k2=1) geometry, respectively, which further confirm the formation of 2D-dominated, cubic, and 1D-dominated crystallites in the molecular simulation. The 2D-layer dominated (FIG. 61H) and 1D-fiber domi- nated 3D structures (FIG. 61F) predicted by simulation are qualitatively consistent with the disclosed observations. Simulations predict the formation of assemblies with cubic S a w 8 w 8 40 3: 2 s 82 morphology (FIG. 61G). The cubic-like morphologies can occur in certain ranges of relative binding energy (ez/exy) along the Z-axis versus in the XY-plane. In simulation, the disclosed techniques allow to systematically tune the value of ez/exy to catch a cubic 3D lattice. The changes of ez/exy can be discrete in certain designs. As illustrated in FIG. 614, the binding energy along the Z-axis changes in a step of 8 bonds (8, 16, 24, and 32). A location of sticky ends for the XY (along the center of face) and Z (at edges) bonds can play a significant role due to the entropic effects and connectivity properties of individual sticky ends. The disclosed subject matter provides a versatile strategy for creating nanoscale objects, DNA nanochambers (DNCs), with differentiated and dimensionally controlled bonds. Such objects offer polychromatic bonds, where each bond provides individual encoding with orientation and position control of inter-DNC binding, and the ability to carry nanocargoes. The ability to prescribe binding modes allows for the programmable assembly of DNCs into 1D, 2D, and 3D arrays. These structural variations are rationally achieved through fully prescribed bonds encoded along the X, Y, and Z orthogonal axes of DNCs. The disclosed subject matter explores the engineering of different architectures using this novel object. The disclosed subject matter can provide the followings: (i) Z encoded DNCs create homopo- lymers and heteropolymers with the ability to control the sequences of monomers and relative orientation, thus, gen- erating helical polymers; (ii) XY encoded DNCs form 2D ordered square arrays with regulated chamber-filling pat- terns wherein the sizes of the 2D ordered arrays depend on the bonds strength; and (iii) three axes encoded DNCs assemble into mesoscale-sized 3D tetragonal arrays whose morphology is a result of competing Z- and XY-bonds. Both experimental and computational studies reveal that the design of binding modes is a crucial factor in the formation of the desired structures. These results provide a new insight into the governing principles of programmable assembly and 5 offer a versatile approach, using bond encoding, for building complex organizations from functional nano-objects that can be readily integrated with nanochambers. All patents, patent applications, publications, product descriptions, and protocols, cited in this specification are hereby incorporated by reference in their entireties. In case of a conflict in terminology, the present disclosure controls. While it will become apparent that the subject matter herein described is well calculated to achieve the benefits and advantages set forth above, the presently disclosed subject matter is not to be limited in scope by the specific embodiments described herein. It will be appreciated that the disclosed subject matter is susceptible to modification, variation, and change without departing from the spirit thereof. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.
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US 12,548,243 B2 83 84 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 557 <210> SEQ ID NO 1 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-1 <400> SEQUENCE: 1 tcaaagegaa ccagaccgtt ttatatagtc 30 <210> SEQ ID NO 2 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-2 <400> SEQUENCE: 2 getttgagga ctaaagagea acggggagtt 30 <210> SEQ ID NO 3 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-3 <400> SEQUENCE: 3 gtaaategte gctattgaat aactcaagaa 30 <210> SEQ ID NO 4 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-4 <400> SEQUENCE: 4 aagecttaaa teaagacttg cggageaaat 30 <210> SEQ ID NO 5 <21l> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-5 <400> SEQUENCE: 5 attttaagaa ctggcttgaa ttatcagtga 30 <210> SEQ ID NO 6 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-6 <400> SEQUENCE: 6 gttaaaatte geattataaa cgtaaactag 30
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85 US 12,548,243 B2 -cont inued 86 <210> SEQ ID NO 7 <21l> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description name: OC-staple-7 <400> SEQUENCE: 7 agcaccatta ccattacage aaatgacgga <210> SEQ ID NO 8 <211l> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description name: OC-staple-8 <400> SEQUENCE: 8 attgegtaga ttttcaaaac agattgtttg <210> SEQ ID NO 9 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description name: OC-staple-9 <400> SEQUENCE: 9 taacctgttt agetatttte geatteatte <210> SEQ ID NO 10 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description name: 0C-staple-10 <400> SEQUENCE: 10 gtcagagggt aattgagaac accaaaatag <210> SEQ ID NO 11 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: name: 0C-staple-11 <400> SEQUENCE: 11 etecagecag ctttececte aggacgttgg <210> SEQ ID NO 12 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: name: OC-staple-12 <400> SEQUENCE: 12 gtccactatt aaagaaccag ttttggttce Octahedral DNA staple strand, Octahedral DNA staple strand, Octahedral DNA staple strand, Octahedral DNA staple strand, Octahedral DNA staple strand, Octahedral DNA staple strand, 30 30 30 30 30 30
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US 12,548,243 B2 87 88 -cont inued <210> SEQ ID NO 13 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-13 <400> SEQUENCE: 13 taaaggtgge aacatagtag aaaataataa 30 <210> SEQ ID NO 14 <21l> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-14 <400> SEQUENCE: 14 gataagtect gaacaactgt ttaaagagaa 30 <210> SEQ ID NO 15 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-15 <400> SEQUENCE: 15 ggtaatagta aaatgtaagt tttacactat 30 <210> SEQ ID NO 16 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-16 <400> SEQUENCE: 16 teagaacege cacectctca gagtattage 30 <210> SEQ ID NO 17 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-17 <400> SEQUENCE: 17 aagggaaceg aactgageag acggtatcat 30 <210> SEQ ID NO 18 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-18 <400> SEQUENCE: 18 gtaaagatte aaaaggectg agttgacect 30
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US 12,548,243 B2 89 90 -cont inued <210> SEQ ID No 19 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-19 <400> SEQUENCE: 19 aggegttaaa taagaagace gtgtegeaag 30 <210> SEQ ID NO 20 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-20 <400> SEQUENCE: 20 caggtegact ctagagcaag cttcaaggeg 30 <210> SEQ ID NO 21 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-21 <400> SEQUENCE: 21 cagagecace accctctcag aactcgagag 30 <210> SEQ ID NO 22 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-22 <400> SEQUENCE: 22 tteacgttga aaatettgeg aatgggattt 30 <210> SEQ ID NO 23 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-23 <400> SEQUENCE: 23 aagttttaac ggggteggag tgtagaatgg 30 <210> SEQ ID NO 24 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-24 <400> SEQUENCE: 24 ttgegtattg ggegecegeg gggtgegete 30 <210> SEQ ID NO 25
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US 12,548,243 B2 91 -cont inued <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-25 <400> SEQUENCE: 25 gtcaccagag ccatggytgaa ttatcaccaa teagaaaage ct <210> SEQ ID NO 26 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-26 <400> SEQUENCE: 26 ggacagagtt actttgtega aatccgegtg tatcacegta cg <210> SEQ ID NO 27 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-27 <400> SEQUENCE: 27 caacatgatt tacgagcatg gaataagtaa gacgacaata aa <210> SEQ ID NO 28 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-28 <400> SEQUENCE: 28 aaccagacge tacgttaata aaacgaacat accacattca gg <210> SEQ ID NO 29 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-29 <400> SEQUENCE: 29 tgacctacta gaaaaagece caggcaaage aatttcatct te <210> SEQ ID NO 30 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-30 <400> SEQUENCE: 30 tgecggaagg ggactcgtaa ccgtgcatta tattttagtt ct «210> SEQ ID NO 31 <211> LENGTH: 42
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US 12,548,243 B2 93 -cont inued 94 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-31 <400> SEQUENCE: 31 agaaccccaa atcaccatct geggaatcga ataaaaattt tt <210> SEQ ID NO 32 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-32 <400> SEQUENCE: 32 getecattgt gtacegtaac actgagttag ttagegtaac ct <210> SEQ ID NO 33 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-33 <400> SEQUENCE: 33 agtaccgaat aggaacccaa acggtgtaac ctcaggaggt tt <210> SEQ ID NO 34 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-34 <400> SEQUENCE: 34 cagtttgaat gtttagtate atatgegtag aategecata ge <210> SEQ ID NO 35 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-35 <400> SEQUENCE: 35 aagattgttt tttaaccaag aaaccatcga cccaaaaaca gg <210> SEQ ID NO 36 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-36 <400> SEQUENCE: 36 tcagagegcc accacataat caaaatcaga acgagtagta tg <210> SEQ ID NO 37 <211> LENGTH: 42 <212> TYPE: DNA 42 42 42 42 42 42
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US 12,548,243 B2 95 -cont inued 96 <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-37 <400> SEQUENCE: 37 gatggttggyg aagaaaaatc caccagaaat aattgggctt ga <210> SEQ ID NO 38 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-38 <400> SEQUENCE: 38 ctecttaacg tagaaaccaa tcaataattc atcgagaaca ga <210> SEQ ID NO 39 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-39 <400> SEQUENCE: 39 agacacctta cgcagaactg geatgatttt ctgtccagac aa <210> SEQ ID NO 40 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-40 <400> SEQUENCE: 40 gecagetagg cgatagctta gattaagace tttttaacct gt <210> SEQ ID NO 41 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-41 <400> SEQUENCE: 41 cogacttatt aggaacgcca tcaaaaatga gtaacaacce ca <210> SEQ ID NO 42 <211l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-42 <400> SEQUENCE: 42 gtccaatage gagaaccaga cgacgatatt caacgcaagy ga <210> SEQ ID NO 43 <211> LENGTH: 42 «212> TYPE: DNA <213> ORGANISM: Artificial sequence 42 42 42 42 42 42
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US 12,548,243 B2 97 -cont inued 98 <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-43 <400> SEQUENCE: 43 coaaaataca atatgatatt caaccgttag gctatcaggt aa <210> SEQ ID NO 44 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-44 <400> SEQUENCE: 44 aacagtactt gaaaacatat gagacgggtc ttttttaatg ga <210> SEQ ID NO 45 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-45 <400> SEQUENCE: 45 tttcacegca ttaaagtcgg gaaacctgat ttgaattace ca <210> SEQ ID NO 46 <211l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-46 <400> SEQUENCE: 46 gagaatagag ccttacegtc tatcaaatgg ageggaatta ga <210> SEQ ID NO 47 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-47 <400> SEQUENCE: 47 ataattaaat ttaaaaaact ttttcaaact tttaacaacg cc <210> SEQ ID NO 48 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-48 <400> SEQUENCE: 48 geacccageg ttttttatce ggtattctag gcgaattatt ca <210> SEQ ID NO 49 <211> LENGTH: 42 <212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE: 42 42 42 42 42 42
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US 12,548,243 B2 99 100 -cont inued <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-49 <400> SEQUENCE: 49 ggaagegece acaaacagtt aatgececga ctcctcaaga ta 42 <210> SEQ ID NO 50 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-50 <400> SEQUENCE: 50 gtttgectat tcacaggeag gtcagacgee accacaceac ce 42 <210> SEQ ID NO 51 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-51 <400> SEQUENCE: 51 egegagetta gtttttccca attctgegca agtgtaaage ct 42 <210> SEQ ID NO 52 <211l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-52 <400> SEQUENCE: 52 agaagcaace aagccaaaag aatacactaa tgccaaaact cc 42 <210> SEQ ID NO 53 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-53 <400> SEQUENCE: 53 attaagtata aagcggcaag gcaaagaaac taatagggta cc 42 <210> SEQ ID NO 54 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-54 <400> SEQUENCE: 54 cagtgectac atgggaattt accgttccac aagtaagcag at 42 «210> SEQ ID NO 55 <211> LENGTH: 42 «212> TYPE: DNA <213> ORGANISM: Artificial sequence «220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand,
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US 12,548,243 B2 101 102 -cont inued name: OC-staple-55 <400> SEQUENCE: 55 ataaggegcc aaaagttgag atttaggata acggaccagt ca 42 <210> SEQ ID NO 56 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-56 <400> SEQUENCE: 56 tgctaaacag atgaagaaac caccagaatt taaaaaaagg ct 42 <210> SEQ ID NO 57 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-57 <400> SEQUENCE: 57 cagecttggt tttgtattaa gaggctgact gcctatatca ga 42 <210> SEQ ID NO 58 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-58 <400> SEQUENCE: 58 cggaataatt caacccageg ccaaagactt attttaacge aa 42 <210> SEQ ID NO 59 <211l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-59 <400> SEQUENCE: 59 egectgaatt accctaatct tgacaagaca gaccatgaaa ga 42 <210> SEQ ID NO 60 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-60 <400> SEQUENCE: 60 acgegaggct acaacagtac cttttacaaa togegcagag aa 42 <210> SEQ ID NO 61 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-61
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US 12,548,243 B2 103 104 -cont inued <400> SEQUENCE: 61 cagegaacat taaaagagag tacctttact gaatataatg aa 42 <210> SEQ ID NO 62 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-62 <400> SEQUENCE: 62 ggacgtttaa tttegacgag aaacaccace actaatgcag at 42 <210> SEQ ID NO 63 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-63 <400> SEQUENCE: 63 aaagegecaa agtttatett acegaagece aataatgagt aa 42 <210> SEQ ID NO 64 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-64 <400> SEQUENCE: 64 gagetegttg taaacgecag ggttttccaa agcaataaag cc 42 <210> SEQ ID NO 65 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-65 <400> SEQUENCE: 65 aattattgtt ttcatgectt tagegtcaga tagcacggaa ac 42 <210> SEQ ID NO 66 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-66 <400> SEQUENCE: 66 aagtttcaga cageegggat cgtcacectt ctgtagetca ac 42 <210> SEQ ID NO 67 <211l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-67
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US 12,548,243 B2 105 106 -cont inued <400> SEQUENCE: 67 acaaagaaat ttaggtaggg cttaattgta tacaacggaa tc 42 <210> SEQ ID NO 68 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-68 <400> SEQUENCE: 68 aacaaaaata actaggtctg agagactacg ctgagtttec ct 42 <210> SEQ ID NO 69 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-69 <400> SEQUENCE: 69 cataacctaa atcaacagtt cagaaaacgt cataaggata ge 42 <210> SEQ ID NO 70 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-70 <400> SEQUENCE: 70 cacgacgaat tegtgtggea tcaattcttt agcaaaatta og 42 <210> SEQ ID NO 71 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-71 <400> SEQUENCE: 71 cctaccaaca gtaattttat cctgaatcaa acagecatat ga 42 <210> SEQ ID NO 72 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-72 <400> SEQUENCE: 72 gattataaag aaacgccagt tacaaaattt accaacgtca ga 42 <210> SEQ ID NO 73 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-73 <400> SEQUENCE: 73
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US 12,548,243 B2 107 -cont inued 108 agtagattga aaagaatcat ggtcatagec ggaagcataa gt 42 <210> SEQ ID NO 74 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-74 <400> SEQUENCE: 74 tagaatccat aaatcattta acaatttcte ccggettagg tt 42 <210> SEQ ID NO 75 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-75 <400> SEQUENCE: 75 aaaggecaaa tatgttagag cttaattgat tgetecatga gg 42 <210> SEQ ID NO 76 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-76 <400> SEQUENCE: 76 ccaaaaggaa aggacaacag tttcagegaa tcateatatt ce 42 <210> SEQ ID NO 77 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-77 <400> SEQUENCE: 77 gaaatcgata accggatace gatagttgta tcagetecaa cg 42 <210> SEQ ID NO 78 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-78 <400> SEQUENCE: 78 tgaatattat caaaataatg gaagggttaa tatttatece aa 42 <210> SEQ ID NO 79 <211l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-79 <400> SEQUENCE: 79
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US 12,548,243 B2 109 -cont inued 110 gaggaageag gattegggta aaatacgtaa aacaccccce ag <210> SEQ ID NO 80 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-80 <400> SEQUENCE: 80 ggttgatttt ccagcagaca gecctcatte gtcacgggat ag <210> SEQ ID NO 81 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-81 <400> SEQUENCE: 81 caagecceea cecttagece ggaataggac gatctaaagt tt <210> SEQ ID NO 82 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-82 <400> SEQUENCE: 82 tgtagatatt acgeggegat cggtgeggge gecatettct gg <210> SEQ ID NO 83 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-83 <400> SEQUENCE: 83 catectatte agctaaaagg taaagtaaaa agcaageegt tt <210> SEQ ID NO 84 <211l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-84 <400> SEQUENCE: 84 cagetcatat aagegtacce cggttgatgt gtcggattct cc <210> SEQ ID NO 85 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-85 <400> SEQUENCE: 85 catgteacaa acggeattaa atgtgageaa ttegegttaa at 42 42 42 42 42 42 42
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US 12,548,243 B2 111 -cont inued 112 <210> SEQ ID NO 86 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-86 <400> SEQUENCE: 86 agegtcacgt ataagaattg agttaagece tttttaagaa ag <210> SEQ ID NO 87 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-87 <400> SEQUENCE: 87 tataaagcat cgtaaccaag tacegeaceg getgtaatat cc <210> SEQ ID NO 88 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-88 <400> SEQUENCE: 88 atagecegeg aaaataattg tateggttcg ccgacaatga gt <210> SEQ ID NO 89 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-89 <400> SEQUENCE: 89 agacagttca tataggagaa gectttataa cattgectga ga <210> SEQ ID No 90 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-90 <400> SEQUENCE: 90 aacaggtece gaaattgeat caaaaagate tttgatcate ag <210> SEQ ID NO 91 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-91 <400> SEQUENCE: 91 actgecettg cccegttgea geaageggea acagettttt ct 42 42 42 42 42 42
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US 12,548,243 B2 113 -cont inued 114 <210> SEQ ID NO 92 <211l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-92 <400> SEQUENCE: 92 tcaaagggag atagecctta taaatcaaga caacaaccat cg <210> SEQ ID NO 93 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-93 <400> SEQUENCE: 93 gtaatacgea aacatgagag atctacaact agetgaggee gg <210> SEQ ID NO 94 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-94 <400> SEQUENCE: 94 gagataacat tagaagaata acataaaaag gaaggattag ga <210> SEQ ID NO 95 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-95 <400> SEQUENCE: 95 cagatattac ctgaatacca agttacaate gggagetatt tt <210> SEQ ID No 96 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-96 <400> SEQUENCE: 96 catataacta atgaacacaa catacgaget gtttctttgg gg <210> SEQ ID NO 97 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-97 <400> SEQUENCE: 97 atgttttget tttgategga acgagggtac tttttctttt gataagaggt catt 42 42 42 42 42 54
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US 12,548,243 B2 115 -cont inued 116 <210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 98 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Octahedral DNA staple name: 0C-staple-98 SEQUENCE: 98 ggggtgecag ttygagaccat tagatacaat tttcactgtg tgaaattgtt atec <210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 99 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Octahedral DNA staple name: OC-staple-99 SEQUENCE: 99 cttegetggg cgcagacgac agtategggg cacegtcgce attcaggetg cgea <210> <211l> <212> <213> «220> <223> <400> SEQ ID NO 100 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Octahedral DNA staple name: OC-staple-100 SEQUENCE: 100 teagagetgg gtaaacgacg gecagtgega teceegtagt ageattaaca teca <210> <211l> <212> <213> <220> <223> <400> SEQ ID NO 101 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Octahedral DNA staple name: OC-staple-101 SEQUENCE: 101 ttageggtac agagegggag aattaactge gctaatttcg gaacctatta ttet <210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 102 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Octahedral DNA staple name: OC-staple-102 SEQUENCE: 102 gatattctaa attgageegg aacgaggece aacttggege ataggetgge tgac «210> <211l> <212> <213> <220> <223> <400> SEQ ID NO 103 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Octahedral DNA staple name: OC-staple-103 SEQUENCE: 103 tgtegtcata agtacagaac cgecacccat tttcacagta caaactacaa cgee <210> SEQ ID NO 104 strand, strand, strand, strand, strand, strand, 54 54 54 54 54 54
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US 12,548,243 B2 117 -cont inued 118 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-104 <400> SEQUENCE: 104 egattataag cggagacttc aaatatcgeg gaagectacg aaggcaccaa ccta <210> SEQ ID NO 105 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-105 <400> SEQUENCE: 105 aacatgtacg cgagtggttt gaaataccta aacacattct taccagtata aage <210> SEQ ID NO 106 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-106 <400> SEQUENCE: 106 gtetggattt tgcegttttaa atgcaatggt gagaaataaa ttaatgecgg agag <210> SEQ ID NO 107 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-107 <400> SEQUENCE: 107 gecttgaate ttttecggaa cegectecca gagcecagag cegeegecag catt <210> SEQ ID NO 108 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-108 <400> SEQUENCE: 108 egetggtget ttcctgaate ggccaacgag ggtggtgatt geecttcace geet <210> SEQ ID NO 109 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-109 <400> SEQUENCE: 109 tgattatcaa ctttacaact aaaggaatcc aaaaagtttg agtaacatta tcat «210> SEQ ID NO 110 <211> LENGTH: 54 54 54 54 54 54 54
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US 12,548,243 B2 -cont inued <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, <400> name: 0C-staple-110 SEQUENCE: 110 acataacttg ccctaacttt aatcattgca ttataacaac attattacag gtag <210> «21l> <212> <213> <220> <223> <400> SEQ ID NO 111 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Octahedral DNA staple name: 0C-staple-111 SEQUENCE: 111 gtagegeeat taaattygga attagagege aaggegcace gtaatcagta gega <210> <21l> <212> <213> <220> <223> «<400> SEQ ID NO 112 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Octahedral DNA staple name: OC-staple-112 SEQUENCE: 112 ttatttttac cgacaatgca gaacgegega aaaatctttc cttatcattc caag <210> <211l> <212> <213> <220> <223> <400> SEQ ID NO 113 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Octahedral DNA staple name: OC-staple-113 SEQUENCE: 113 tttcaataga aggcagegaa cctccegatt agttgaaaca ataacggatt cgce <210> <211l> <212> <213> <220> <223> <400> SEQ ID NO 114 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Octahedral DNA staple name: OC-staple-114 SEQUENCE: 114 gagegacece aaaagtatgt tagcaaacta aaagagtcac aatcaataga aaat <210> «<21l> «212> <213> <220> <223> <400> SEQ ID NO 115 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Octahedral DNA staple name: 0C-staple-115 SEQUENCE: 115 agecgaaagt ctctcttttg atgatacaag tyccttaaga gcaagaaaca atga <210> «21Ll> <212> SEQ ID NO 116 LENGTH: 54 TYPE: DNA strand, strand, strand, strand, strand, 54 54 54 54 54 54
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US 12,548,243 B2 121 -cont inued 122 <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-116 <400> SEQUENCE: 116 gtgggaaate atataaatat ttaaattgaa tetttgtetg gecttectgt agee <210> SEQ ID NO 117 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-117 <400> SEQUENCE: 117 eccacgegea aaatggttga gtgttgttcg tggacttget ttegaggtga attt <210> SEQ ID NO 118 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: 0C-staple-118 <400> SEQUENCE: 118 atgaccacte gtttggcttt tgcaaaagtt agactatatt cattgaatce cect <210> SEQ ID NO 119 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-119 <400> SEQUENCE: 119 tecaaatctt ctgaattatt tgcacgtagg tttaacgcta acgagegtct ttce <210> SEQ ID NO 120 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Octahedral DNA staple strand, name: OC-staple-120 <400> SEQUENCE: 120 gggttattta attacaatat atgtgagtaa ttaataagag tcaatagtga attt <210> SEQ ID NO 121 <21l> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct element: gold nanoparticle or quantum dot attachment sequence <400> SEQUENCE: 121 atccatcact teatactcta cgttgttgtt gttgttgtt <210> SEQ ID NO 122 <211> LENGTH: 39 «212> TYPE: DNA <213> ORGANISM: Artificial sequence 54 54 54 54 54 39
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US 12,548,243 B2 123 124 -cont inued <220> <223> <400> FEATURE: OTHER INFORMATION: Description: Construct element: gold nanoparticle or quantum dot attachment sequence SEQUENCE: 122 ctaccatcat acctactcta cgttgttgtt gttgttgtt 39 «210> «21Ll> <212> «213> <220> <223> <400> SEQ ID NO 123 LENGTH: 15 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Construct element: streptavidins attachment sequence SEQUENCE: 123 atccateact tettt 15 «210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 124 LENGTH: 27 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Construct element: GOx enzyme attachment sequence SEQUENCE: 124 ttettettca tecatactac catctac 27 <210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 125 LENGTH: 27 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Construct element: HRP enzyme attachment sequence SEQUENCE: 125 cttcttctta tacttcacta cctaatc 27 <210> <211> <212> <213> <220> <223> <400> SEQ ID NO 126 LENGTH: 72 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron SEQUENCE: 126 tttgeggatg gccaactaaa gtacgggett gcagetacag agtttttttt tttttttttt 60 ttttatcegt ta 72 <210> «<21l> «212> <213> <220> <223> SEQ ID NO 127 LENGTH: 72 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 127 cttcatcaag agaaatcaac gtaacagaga tttgtcaate attttttttt tttttttttt 60 ttttatcegt ta 72 <210> SEQ ID NO 128
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US 12,548,243 B2 125 126 -cont inued <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 128 aaagattcat caggaattac gaggeatget catecttatg cgtttttttt trtttttttt 60 ttttatecgt ta 72 <210> SEQ ID NO 129 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 129 ataaatcata cataaategg ttgtactgtg ctggcatgee tgtttttttt tttttttttt 60 ttttateegt ta 72 <210> SEQ ID NO 130 <211> LENGTH: 72 <212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 130 ggtagetatt ttagagaate gatgaaaaca ttaaatgtgt agtttttttt ttttttette 60 ttttatcegt ta 72 «210> SEQ ID NO 131 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 131 caaatgettt aaaaaatcag gtctttaaga gcagecagag ggtttttttt tttttttttt 60 ttttatecgt ta 72 <210> SEQ ID NO 132 <211> LENGTH: 72 «212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 132 teatatggtt tacgattgag ggagggaaac gcaatacata catttttttt tttttttttt 60 ttttateegt ta 72 <210> SEQ ID NO 133 <21l> LENGTH: 72 <212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE:
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US 12,548,243 B2 127 128 -cont inued <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 133 caacgetcaa cagcagagge attttcaatc caatgataaa tatttttttt tttttttttt 60 ttttatcegt ta 72 «210> SEQ ID NO 134 <211> LENGTH: 72 «212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 134 aatageaata gcaccagaag gaaacctaaa gecactggta attttttttt tttttttttt 60 ttttatcegt ta 72 <210> SEQ ID NO 135 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 135 gacaggaggt tgaaacaaat aaatccgece cetcegecac cotttttttt tttttttttt 60 ttttatcegt ta 72 <210> SEQ ID NO 136 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 136 agetttcate aacggattga cegtaaaate gtataatatt tttttttttt ttttttettt 60 ttttatcegt ta 72 <210> SEQ ID NO 137 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 137 agagectaat ttgatttttt gtttaaatcc tgaaataaag aatttttttt tttttttttt 60 ttttatcegt ta 72 <210> SEQ ID NO 138 <211> LENGTH: 72 «212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 138
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US 12,548,243 B2 129 -cont inued 130 getcacaatt cegtgageta actcactgga agtaatggtc aatttttttt tttttttttt 60 ttttatcegt ta 72 <210> SEQ ID NO 139 «<211> LENGTH: 72 «212> TYPE: DNA «213> ORGANISM: Artificial sequence «220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 139 cttaaacage ttatatatte ggtegettga tggggaacaa gatttttttt ttttttettt 60 ttttatcegt ta 72 «210> SEQ ID NO 140 «<211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 140 aaacgaaaga gggcgaaaca aagtactgac tatattcgag cttttttttt tttttttttt 60 ttttatecgt ta 72 <210> SEQ ID NO 141 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 141 actgttggga agcagctgge gaaaggatag gtcaagatcg catttttttt tttttttett 60 ttttateegt ta 72 <210> SEQ ID NO 142 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 142 ggceetgaga gaagcaggey aaaatcatty cgtagaggeg gtttttttet tettttettt 60 ttttatcegt ta 72 <210> SEQ ID NO 143 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 143 aacgggtatt aaggaatcat taccgecagt aattcaacaa tatttttttt ttttttettt 60 ttttatcegt ta 72
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US 12,548,243 B2 131 132 -cont inued <210> SEQ ID NO 144 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 144 cagaatcaag tttcggcatt ttcggttaaa tatatcacca gttttttttt tttttttttt 60 ttttatecgt ta 72 <210> SEQ ID NO 145 <211> LENGTH: 72 «212> TYPE: DNA «213> ORGANISM: Artificial sequence «220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 145 gaaacatgaa agctcagtac caggegaaaa atgetgaaca aatttttttt tttttteett 60 ttttateegt ta 72 «210> SEQ ID NO 146 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 146 atcaaaatca tatatgtaaa tgctgaacaa acacttgctt cttttttttt tttttttttt 60 ttttatecgt ta 72 <210> SEQ ID NO 147 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 147 tgattgettt gagcaaaaga agatgaaata gcagaggttt tgtttttttt tttttttttt 60 ttttatcegt ta 72 <210> SEQ ID NO 148 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 148 tttgeggaac aatggcaatt catcaatctg tataataatt tttttttttt tttttttttt 60 ttttatcegt ta 72 «210> SEQ ID NO 149 <211> LENGTH: 72
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US 12,548,243 B2 133 134 -cont inued <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 149 tgtageatte caacgttagt aaatgaagtg ccgegecace cttttttttt tttttttttt 60 ttttatcegt ta 72 <210> SEQ ID NO 150 <21l> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 150 tttgeggatg gecaactaaa gtacgggott gcagetacag agtttttttt tttttttttt 60 tttttaacgg at 72 <210> SEQ ID NO 151 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 151 cttcatcaag agaaatcaac gtaacagaga tttgtcaate attttttttt ttttttettt 60 tttttaacgg at 72 <210> SEQ ID NO 152 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 152 aaagattcat caggaattac gaggcatgct catccttatg cgtttttttt tttttttttt 60 tttttaacgg at 72 <210> SEQ ID NO 153 <211> LENGTH: 72 <212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 153 ataaatcata cataaategg ttgtactgtg ctggeatgee tgtttttttt ttttttettt 60 tttttaacgg at 72 «210> SEQ ID NO 154 <211> LENGTH: 72 «212> TYPE: DNA <213> ORGANISM: Artificial sequence «220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky
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US 12,548,243 B2 135 136 -cont inued ends for octahedron <400> SEQUENCE: 154 ggtagetatt ttagagaatc gatgaaaaca ttaaatgtgt agtttttttt ttttttrttt 60 tttttaacgg at 72 «210> SEQ ID NO 155 «211> LENGTH: 72 «212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 155 caaatgettt aaaaaatcag gtctttaaga gcagecagag ggtttttttt tttttttttt 60 tttttaacgg at 72 <210> SEQ ID NO 156 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 156 teatatggtt tacgattgag ggagggaaac gcaatacata catttttttt tttttttttt 60 tttttaacgg at 72 <210> SEQ ID NO 157 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 157 caacgetcaa cagcagagge attttcaatc caatgataaa tatttttttt ttttttcttt 60 tttttaacgg at 72 <210> SEQ ID NO 158 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 158 aatageaata geaccagaag gaaacctaaa gecactggta attttttttt ttttteeett 60 tttttaacgg at 72 <210> SEQ ID NO 159 <211> LENGTH: 72 <212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 159
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US 12,548,243 B2 137 -cont inued 138 gacaggaggt tgaaacaaat aaatccgece cctcegecac cetttttttt tttttttttt 60 tttttaacgg at 72 <210> SEQ ID NO 160 <211> LENGTH: 72 «212> TYPE: DNA «213> ORGANISM: Artificial sequence «220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 160 agettteate aacggattga cegtaaaate gtataatatt ttttttttrt tttttteettt 60 tttttaacgg at 72 «210> SEQ ID NO 161 «<211> LENGTH: 72 «212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 161 agagcctaat ttgatttttt gtttaaatcc tgaaataaag aatttttttt tttttttttt 60 tttttaacgg at 72 <210> SEQ ID NO 162 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 162 getcacaatt cegtgageta actcactgga agtaatggte aatttttttt tttttttttt 60 tttttaacgg at 72 <210> SEQ ID NO 163 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 163 cttaaacage ttatatattc ggtegettga tggggaacaa gatttttttt ttttttettt 60 tttttaacgg at 72 <210> SEQ ID NO 164 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 164 aaacgaaaga gggcgaaaca aagtactgac tatattcgag cttttttttt trtttttttt 60 tttttaacgg at 72
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US 12,548,243 B2 139 140 -cont inued <210> SEQ ID NO 165 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 165 actgttggga agcagctgge gaaaggatag gtcaagatcg catttttttt tttttttttt 60 tttttaacgg at 72 <210> SEQ ID NO 166 <211> LENGTH: 72 <212> TYPE: DNA «213> ORGANISM: Artificial sequence «220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 166 ggecctgaga gaagcaggeg aaaatcatty cgtagaggeg gttttttttt tettttettt 60 tttttaacgg at 72 <210> SEQ ID NO 167 <211> LENGTH: 72 «212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 167 aacgggtatt aaggaatcat taccgecagt aattcaacaa tatttttttt ttttttettt 60 tttttaacgg at 72 <210> SEQ ID NO 168 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 168 cagaatcaag tttcggcatt ttcggttaaa tatatcacca gttttttttt tttttttttt 60 tttttaacgg at 72 <210> SEQ ID NO 169 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 169 gaaacatgaa agctcagtac caggcgaaaa atgctgaaca aatttttttt tttttttttt 60 tttttaacgg at 72 <210> SEQ ID NO 170 «<211> LENGTH: 72 <212> TYPE: DNA
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US 12,548,243 B2 141 142 -cont inued <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 170 atcaaaatca tatatgtaaa tgctgaacaa acacttgett cttttttttt tttttttttt 60 tttttaacgg at 72 <210> SEQ ID NO 171 «211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 171 tgattgettt gagcaaaaga agatgaaata gcagaggttt tgtttttttt ttttttettt 60 tttttaacgg at 72 <210> SEQ ID NO 172 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 172 tttgeggaac aatggcaatt catcaatctg tataataatt tttttttttt ttttttettt 60 tttttaacgg at 72 <210> SEQ ID NO 173 <211> LENGTH: 72 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky ends for octahedron <400> SEQUENCE: 173 tgtageatte caacgttagt aaatgaagtg ccgegecace cttttttttt ttttttettt 60 tttttaacgg at 72 <210> SEQ ID NO 174 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic DNA staple sequence,, name Cub-staple-1 <400> SEQUENCE: 174 taaatattga cggaaaattg aggttgtcac 30 <210> SEQ ID NO 175 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic DNA staple sequence,, name Cub-staple-2 <400> SEQUENCE: 175
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143 US 12,548,243 B2 -cont inued 144 ccgaacaaag ttaccaaaaa gtataagecc <210> SEQ ID NO 176 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description Cub-staple-3 <400> SEQUENCE: 176 tgaateggee aacgeggtge cagaatgagt <210> SEQ ID NO 177 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description Cub-staple-4 <400> SEQUENCE: 177 aatcgtaaaa ctagcaagaa toggggtage <210> SEQ ID NO 178 <211> LENGTH: 30 «212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cub-staple-5 <400> SEQUENCE: 178 agaagecttt atttcagtaa tacgeaaaat <210> SEQ ID NO 179 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cub-staple-6 <400> SEQUENCE: 179 attgtgaatt accttaaatt tcatcagtga gtg: gt g: <210> SEQ ID No 180 <21l> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description Cub-staple-7 <400> SEQUENCE: 180 atagaaagga acaactttte agetagegta <210> SEQ ID NO 181 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description Cub-staple-8 <400> SEQUENCE: 181 30 Cubic DNA staple sequence,, name 30 Cubic DNA staple sequence,, name 30 Cubic DNA staple sequence,, name: 30 Cubic DNA staple sequence,, name: 30 Cubic DNA staple sequence,, name 30 Cubic DNA staple sequence,, name
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145 US 12,548,243 B2 -cont inued 146 catgtaattt aggcagtatt taatgegtta <210> SEQ ID NO 182 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description Cub-staple-9 <400> SEQUENCE: 182 acgttgtaaa acgacgggtt ttcaagggeg <210> SEQ ID NO 183 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description Cub-staple-10 <400> SEQUENCE: 183 gacttcaaat atcgegaaga ggaaatcaaa <210> SEQ ID NO 184 <211> LENGTH: 30 <212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cub-staple-11 <400> SEQUENCE: 184 caaataagaa acgattatta tttgaatett <210> SEQ ID NO 185 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cub-staple-12 <400> SEQUENCE: 185 gagtgaataa ccttgeataa atctcaagaa <210> SEQ ID NO 186 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cub-staple-13 <400> SEQUENCE: 186 attgegtaga ttttcaaaac agattgtttg <210> SEQ ID NO 187 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description Cub-staple-14 <400> SEQUENCE: 187 gtccactatt aaagaaceag ttttggttce 30 Cubic DNA staple sequence,, name 30 Cubic DNA staple sequence,, name 30 Cubic DNA staple sequence,, name: 30 Cubic DNA staple sequence,, name: 30 Cubic DNA staple sequence,, name: 30 Cubic DNA staple sequence,, name 30
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US 12,548,243 B2 147 148 -cont inued <210> SEQ ID No 188 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description Cub-staple-15 <400> SEQUENCE: 188 cccaattctg cgaacgcata taaaatataa <210> SEQ ID NO 189 <211l> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description Cub-staple-16 <400> SEQUENCE: 189 atttacegtt ccagtaaaag cgcttgagge <210> SEQ ID NO 190 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description Cub-staple-17 <400> SEQUENCE: 190 ggettttgea aaagaaaace aaacaaaagg <210> SEQ ID NO 191 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description Cub-staple-18 <400> SEQUENCE: 191 aactttttca aatataacaa agattttaac <210> SEQ ID NO 192 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cub-staple-19 <400> SEQUENCE: 192 egtacteagg aggtttggaa tagtectcaa <210> <211l> <212> <213> <220> SEQ ID NO 193 LENGTH: 30 TYPE: DNA ORGANISM: Artificial sequence FEATURE: <223> OTHER INFORMATION: Description: Cub-staple-20 <400> SEQUENCE: 193 cegtaatggg ataggtacaa acgaaaataa Cubic DNA staple sequence,, name 30 Cubic DNA staple sequence,, name 30 Cubic DNA staple sequence,, name 30 Cubic DNA staple sequence,, name 30 Cubic DNA staple sequence,, name: 30 Cubic DNA staple sequence,, name: 30
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US 12,548,243 B2 149 150 -cont inued <210> SEQ ID NO 194 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic DNA staple sequence,, name: Cub-staple-21 <400> SEQUENCE: 194 taatcaaaat cacegggttt geegtttgec 30 <210> SEQ ID NO 195 <21l> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic DNA staple sequence,, name Cub-staple-22 <400> SEQUENCE: 195 agecgttttt attttctcat cgacctaatt 30 <210> SEQ ID NO 196 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic DNA staple sequence,, name Cub-staple-23 <400> SEQUENCE: 196 agecggaacg aggegectge teccaagege 30 <210> SEQ ID NO 197 <211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic DNA staple sequence,, name Cub-staple-24 <400> SEQUENCE: 197 ateggaacga gggtagagea gegaacegat 30 <210> SEQ ID No 198 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic DNA staple sequence,, name: Cub-staple-25 <400> SEQUENCE: 198 aaaaatgagt tacagegtct ttecagagaa tcatcatatt cc 42 <210> SEQ ID NO 199 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic DNA staple sequence,, name Cub-staple-26 <400> SEQUENCE: 199 gaattacatt ctagaggate ceegggtaat ccegetcacaa tt 42
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US 12,548,243 B2 151 152 -cont inued <210> SEQ ID No 200 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic DNA staple sequence,, name: Cub-staple-27 <400> SEQUENCE: 200 aagcaaagac atctgecagt ttgaggggee gettctggtg <210> <21l> <212> <213> <220> <223> SEQ ID NO 201 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-28 <400> SEQUENCE: 201 accceggtga gagtctacaa aggetatctc gcaageggtc <210> <211l> <212> <213> «220> <223> SEQ ID NO 202 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-29 <400> SEQUENCE: 202 gagaagggee tgtaccatgt accgtaacce accctecace <210> <211l> <212> <213> <220> <223> SEQ ID NO 203 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-30 <400> SEQUENCE: 203 tgaatattat caaaataatg gaagggttge gectgtttat <210> <21l> <212> <213> <220> <223> SEQ ID NO 204 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-31 <400> SEQUENCE: 204 ectaccaaca gtaatataaa gtaccgacaa tgcagaacge «210> <211l> <212> <213> <220> <223> SEQ ID NO 205 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-32 <400> SEQUENCE: 205 gattataata agtccaacat gttcagctaa aaggtegtca <210> SEQ ID NO 206 42 DNA staple sequence,, name: 42 DNA staple sequence,, name 42 DNA staple sequence,, name 42 DNA staple sequence,, name: 42 DNA staple sequence,, name: 42
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US 12,548,243 B2 153 154 -cont inued <21l> <212> <213> <220> <223> <400> LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-33 SEQUENCE: 206 ectgataceg aactcacega cttgageegg ccggaaacgt <210> «21l> <212> <213> <220> <223> <400> SEQ ID NO 207 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-34 SEQUENCE: 207 aaacgeactt accggaaaca atgaaatata caccatcaat <210> <211l> <212> <213> <220> <223> «<400> SEQ ID NO 208 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-35 SEQUENCE: 208 taccgeggta ttaaaaccaa tcaataattc gocttaaate <210> <211l> <212> <213> <220> <223> <400> SEQ ID NO 209 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-36 SEQUENCE: 209 taataatttg ctaatgtegt ctttccagat gottgatacc <210> <211l> <212> <213> <220> <223> <400> SEQ ID NO 210 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-37 SEQUENCE: 210 gaaacaaage agcaattace attagcaaat ttgggcaate <210> «211> <212> <213> <220> <223> <400> SEQ ID NO 211 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-38 SEQUENCE: 211 aggtcagaaa cacttacgaa ggcaccaagg aagtttacat «210> <211l> SEQ ID NO 212 LENGTH: 42 DNA staple sequence,, name: DI DNA staple sequence,, name DNA staple sequence,, name ga DNA staple sequence,, name DNA staple sequence,, name: gg 42 NA staple sequence,, name: 42 42 42 42 42
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US 12,548,243 B2 155 156 -cont inued <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic Cub-staple-39 <400> SEQUENCE: 212 cattgecttg ataaccaggg tggtttttga gagagttyca <210> «21l> <212> <213> <220> <223> SEQ ID NO 213 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-40 <400> SEQUENCE: 213 taagcaagaa acgctagcaa acgtagaaga actgggataa <210> <21l> <212> <213> <220> <223> SEQ ID NO 214 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-41 <400> SEQUENCE: 214 atattegtct gaaaccgtat aaacagttat aagtttacag <210> <211l> <212> <213> <220> <223> SEQ ID NO 215 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-42 <400> SEQUENCE: 215 taaagtaaga tacacagtca ggacgttggt agaaagattc <210> <211l> <212> <213> <220> <223> SEQ ID NO 216 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-43 <400> SEQUENCE: 216 agaacggece aatagcaage ctcecteaca cttatcattc <210> «<21l> «212> <213> <220> <223> SEQ ID NO 217 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-44 <400> SEQUENCE: 217 tgctgtatac cacacaacat tattacaggg aagaattagt <210> SEQ ID NO 218 <211> LENGTH: 42 <212> TYPE: DNA DNA staple sequence,, name: 42 DNA staple sequence,, name: 42 DNA staple sequence,, name: 42 DNA staple sequence,, name 42 DNA staple sequence,, name 42 DNA staple sequence,, name 42
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US 12,548,243 B2 157 158 -cont inued <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic Cub-staple-45 <400> SEQUENCE: 218 cagaaceggg ttgattageg gggttttgta caccagtaca «210> «21l> <212> «213> <220> <223> SEQ ID NO 219 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-46 <400> SEQUENCE: 219 gaaategaat atcaaattaa ctgaacacag aataatccaa <210> <21l> <212> <213> <220> <223> SEQ ID NO 220 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-47 <400> SEQUENCE: 220 cagtaatggg cttaagtata aagecaacag gegaattatt <210> <21l> <212> <213> <220> <223> SEQ ID NO 221 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-48 <400> SEQUENCE: 221 atettcttga tgcagggtta tataactact cagtaccagg <210> <211l> <212> <213> <220> <223> SEQ ID NO 222 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-49 <400> SEQUENCE: 222 ataaggecca ataactyaaa aggtygcaaa taaccttaag <210> «<21l> <212> «213> <220> <223> SEQ ID NO 223 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-50 <400> SEQUENCE: 223 aagggaaaat tgtgeggaga tttgtatcag caccaatgaa «210> SEQ ID NO 224 <211> LENGTH: 42 «212> TYPE: DNA <213> ORGANISM: Artificial sequence DNA staple sequence,, name 42 DNA staple sequence,, name: 42 DNA staple sequence,, name: 42 DNA staple sequence,, name 42 DNA staple sequence,, name 42 DNA staple sequence,, name: 42
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US 12,548,243 B2 159 160 -cont inued <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic Cub-staple-51 <400> SEQUENCE: 224 tgggattttt ttcacgttga aaatgtttcc gaattttotg «210> «21Ll> <212> «213> <220> <223> SEQ ID NO 225 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-52 <400> SEQUENCE: 225 gagtagtatt atactttege aaatggtcte aattctacta «210> <21l> <212> <213> <220> <223> SEQ ID NO 226 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-53 <400> SEQUENCE: 226 teaaagegeg gattcctgac tattatagtt catcaacatt <210> <21l> <212> <213> <220> <223> SEQ ID NO 227 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-54 <400> SEQUENCE: 227 gagctaaage tcataacgtt aatattttaa aacagaggeg <210> <211> <212> <213> <220> <223> SEQ ID NO 228 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-55 <400> SEQUENCE: 228 ttegegteca ttogecaget ttceggeaac gacgagtgta «210> «<21l> <212> <213> «220> <223> SEQ ID NO 229 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-56 <400> SEQUENCE: 229 aacagtaaag agaacagtac cttttacaaa tcgegcagag <210> SEQ ID NO 230 <211> LENGTH: 42 <212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE: DNA staple sequence,, name 42 DNA staple sequence,, name 42 DNA staple sequence,, name: 42 DNA staple sequence,, name gt 42 DNA staple sequence,, name ga 42 DNA staple sequence,, name 42
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US 12,548,243 B2 161 162 -cont inued <223> OTHER INFORMATION: Description: Cubic Cub-staple-57 <400> SEQUENCE: 230 coggaacggt catagtageg cgttttcacg gotgtetttc «210> «21Ll> «212> «213> <220> <223> SEQ ID NO 231 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-58 <400> SEQUENCE: 231 getattactt ttttcattta acaatttcca gctggegaaa «210> «21Ll> <212> <213> <220> <223> SEQ ID NO 232 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-59 <400> SEQUENCE: 232 taaccctgta aaatctccaa aaaaaaggat ttcttaaaca <210> <21l> <212> <213> <220> <223> SEQ ID NO 233 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-60 <400> SEQUENCE: 233 ctggctcaaa ttgggacgag aaacaccaca atagtagtag <210> <211> <212> <213> <220> <223> SEQ ID NO 234 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-61 <400> SEQUENCE: 234 toggttggaa ccctcggaat acccaaaaaa tacatacata <210> «21Ll> <212> <213> <220> «223> SEQ ID NO 235 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-62 <400> SEQUENCE: 235 cttttgatce teatgecttg atattcactt gaggcaaaag «210> <21l> «212> <213> «220> <223> SEQ ID NO 236 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic DNA staple sequence,, name 42 DNA staple sequence,, name: 42 DNA staple sequence,, name: 42 DNA staple sequence,, name 42 DNA staple sequence,, name 42 DNA staple sequence,, name 42 DNA staple sequence,, name
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US 12,548,243 B2 163 164 -cont inued Cub-staple-63 <400> SEQUENCE: 236 tagctatata ataacatata ttttaaatag acagtcaaat «210> «21Ll> «212> «213> <220> <223> SEQ ID NO 237 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-64 <400> SEQUENCE: 237 taaatgegaa ccgecacect cagagecaac tgagtttegt «210> «21Ll> «212> <213> <220> <223> SEQ ID NO 238 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-65 <400> SEQUENCE: 238 accaacgcag atgaagaaac caccagaatt taaaataacg «210> <211> <212> <213> <220> <223> SEQ ID NO 239 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-66 <400> SEQUENCE: 239 ccaaagatca ccgtgaccaa ctttgaaaca agagtaatct <210> <211> <212> <213> <220> <223> SEQ ID NO 240 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-67 <400> SEQUENCE: 240 tacgagetge tattectece gacttgegtt atccggaate <210> <21l> «212> <213> <220> <223> SEQ ID NO 241 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-68 <400> SEQUENCE: 241 cattttcege aaatcagata tagaaggegg aggttttgaa <210> «211> <212> «213> <220> «223> SEQ ID NO 242 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-69 aa 42 DNA staple sequence,, name: 42 DNA staple sequence,, name: 42 DNA staple sequence,, name 42 DNA staple sequence,, name 42 DNA staple sequence,, name gg 42 DNA staple sequence,, name
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US 12,548,243 B2 165 166 -cont inued <400> SEQUENCE: 242 ateggtgegg aagetgtgtg aaattgttcc gagctccaag <210> «21Ll> «212> «213> «220> <223> SEQ ID NO 243 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-70 <400> SEQUENCE: 243 ggtaatacgt ttacgtaaga gcaacactac gttagtaaat «210> «21Ll> «212> «213> <220> <223> SEQ ID NO 244 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-71 <400> SEQUENCE: 244 atttgecaaa atagacegte tatcaaatgg agcggaatta «210> «211> <212> <213> <220> <223> SEQ ID NO 245 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-72 <400> SEQUENCE: 245 tttagegeca ccagacecte agagcegega gecgegecac <210> <21l> <212> <213> <220> <223> SEQ ID NO 246 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-73 <400> SEQUENCE: 246 tatttttttg cccccacege ctggeectct tttcacatat <210> <21l> <212> «213> <220> <223> SEQ ID NO 247 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-74 <400> SEQUENCE: 247 atttttatac caaatcagag cataaagege aaggtggcaa <210> «21l> «212> <213> «220> <223> SEQ ID NO 248 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-75 42 DNA staple sequence,, name: 42 DNA staple sequence,, name: ga 42 DNA staple sequence,, name: ca 42 DNA staple sequence,, name gt 42 DNA staple sequence,, name 42 DNA staple sequence,, name
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US 12,548,243 B2 167 168 -cont inued <400> SEQUENCE: 248 acgatctgee gacatgettt cgaggtgact ccaaattgeg <210> <21l> «212> «213> «220> «223> SEQ ID NO 249 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-76 <400> SEQUENCE: 249 aatcaatatt accctggetg accttcatga ggacattaaa <210> «21Ll> «212> «213> «220> <223> SEQ ID NO 250 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-77 <400> SEQUENCE: 250 aataataaac egttgtgaga aaggeeggge aatgcacega «210> «211> «212> <213> <220> <223> SEQ ID NO 251 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-78 <400> SEQUENCE: 251 tgggegeece gtegtectgt agecagette ccggaaacca <210> «211l> <212> <213> <220> <223> SEQ ID NO 252 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-79 <400> SEQUENCE: 252 aacaaaattt atcagacgct gagaagagct tagaaaateg <210> <21l> <212> <213> «220> <223> SEQ ID NO 253 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-80 <400> SEQUENCE: 253 toaaagggag atagecctta taaatcaacc cagagggtaa <210> <21l> <212> «213> <220> «223> SEQ ID NO 254 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-81 <400> SEQUENCE: 254 42 DNA staple sequence,, name gg 42 DNA staple sequence,, name: ag 42 DNA staple sequence,, name: gg 42 DNA staple sequence,, name: 42 DNA staple sequence,, name 42 DNA staple sequence,, name
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US 12,548,243 B2 169 -cont inued 170 tacaaattac ctgaatacca agttacaatc gggagttcga <210> <21l> <212> «213> «220> «223> <400> SEQ ID NO 255 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-82 SEQUENCE: 255 aggecgegga ctaaggagtg tactggtaaa tgcccectge <210> <21l> «212> «213> «220> «223> <400> SEQ ID NO 256 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-83 SEQUENCE: 256 aattacgttt aaactattca ttgaatecag actggcagag «210> «211> «212> «213> <220> <223> <400> SEQ ID NO 257 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-84 SEQUENCE: 257 geaaggeetg caggtegact aattttecte gggggatgtg <210> «211l> <212> <213> <220> <223> <400> SEQ ID NO 258 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-85 SEQUENCE: 258 getttgattt tgcgaggett gcagggagaa ctatttegga <210> <21l> <212> <213> <220> «223> <400> SEQ ID NO 259 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-86 SEQUENCE: 259 gtegagagee acectcagac ctaaatttca cggataagtg <210> <21l> <212> «213> «220> <223> <400> SEQ ID NO 260 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-87 SEQUENCE: 260 DNA staple sequence,, name DNA staple sequence,, name gg 42 DNA staple sequence,, name: DNA staple sequence,, name: DNA staple sequence,, name DNA staple sequence,, name
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US 12,548,243 B2 171 -cont inued 172 ttgegtattt ccagtaattg cgttgegcag attaaatttt <210> <21l> <212> <213> «220> «223> <400> SEQ ID NO 261 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-88 SEQUENCE: 261 gtaacaaate gtaacegtga ccagacegga aaatgtgage <210> <21l> <212> «213> «220> «223> <400> SEQ ID NO 262 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-89 SEQUENCE: 262 aatcaggatt tttgtaattg ctecttttga agcaategag <210> «211> «212> «213> «220> <223> <400> SEQ ID NO 263 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-90 SEQUENCE: 263 tgaattacaa aaggtcatat ggtttaccat tgacaagaac <210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 264 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-91 SEQUENCE: 264 cteeggetaa ttactaaata agaataaaaa tggtttaatt <210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 265 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-92 SEQUENCE: 265 gaccattegg tgtcatgttt taaatatyga atcagttgag <210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 266 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-93 SEQUENCE: 266 aaataaatga tacaagactt tttcatgace taaaacgaaa tg 42 DNA staple sequence,, name ga 42 DNA staple sequence,, name DNA staple sequence,, name: DNA staple sequence,, name: DNA staple sequence,, name: DNA staple sequence,, name
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US 12,548,243 B2 173 -cont inued 174 <210> SEQ ID NO 267 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic Cub-staple-94 <400> SEQUENCE: 267 atagccegeg aaaacagect ttacagagec tgaacaaagt <210> SEQ ID NO 268 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic Cub-staple-95 <400> SEQUENCE: 268 tgccegettg ggcgtcagaa aagececagt taaaattoge <210> SEQ ID NO 269 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic Cub-staple-96 <400> SEQUENCE: 269 tgeatgegat taagettege tattacgeat ttccacacaa <210> SEQ ID NO 270 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic Cub-staple-97 <400> SEQUENCE: 270 acaatagett ctgaattatt tgeacgtagg tttaaaaagt <210> SEQ ID NO 271 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic Cub-staple-98 <400> SEQUENCE: 271 tagtgaatta attaaatgga aacagtactt ctgtatectt <210> SEQ ID NO 272 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic Cub-staple-99 <400> SEQUENCE: 272 agaaccatca gactgecece ttattagcaa ccagacacce DNA staple DNA staple gt DNA staple DNA staple aattetgtce DNA staple gaaaacatag DNA staple teagaacege sequence, , sequence, , sequence, , sequence, , agac sequence, , egat sequence, , cace name 42 name 42 name 42 name 54 name: 54 name: 54
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US 12,548,243 B2 175 176 -cont inued <210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 273 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-100 SEQUENCE: 273 agaggtctct ttacgcatca aaaagatttt ttaatactcc <210> <21l> <212> <213> <220> «223> <400> SEQ ID NO 274 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-101 SEQUENCE: 274 tagttgeaaa gtttacaact ttcaacagaa aggaaaggag <210> <211l> <212> «213> «220> «223> <400> SEQ ID NO 275 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-102 SEQUENCE: 275 gatattcaga gcaaaagece tttttaagga aggaactgag <210> <211l> <212> <213> <220> <223> <400> SEQ ID NO 276 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-103 SEQUENCE: 276 tgattateta acgaaaataa acagecattt ttgttgtttg <210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 277 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-104 SEQUENCE: 277 caaatgeagg catacagacg acgataaagt tttgeatage <210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 278 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-105 SEQUENCE: 278 ttaaatecte acatteggga aacctgtecg gggaggaaga DNA staple aacaggtcag DNA staple cotttaattg DNA staple taatgtgtag DNA staple agtaacatta DNA staple gtecaatact DNA staple ttgtataage sequence, , name: gatt 54 sequence, , name tate 54 sequence, , name gtaa 54 sequence, , name teat 54 sequence, , name: gegg 54 sequence, , name aaat 54
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US 12,548,243 B2 177 -cont inued 178 <210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 279 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-106 SEQUENCE: 279 ttaggaagct caactggaag tttcattcag tagataaate <210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 280 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-107 SEQUENCE: 280 ctattatgte getgggatcg tcaccctcca acggettaac <210> <211l> <212> <213> «220> <223> <400> SEQ ID NO 281 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-108 SEQUENCE: 281 catcgatagt acaatcgaaa tecgegacag acggtaatta <210> <211l> <212> <213> <220> <223> <400> SEQ ID NO 282 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-109 SEQUENCE: 282 gatattcaga aaatgegaca ttcaacegtt attcagatga <210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 283 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-110 SEQUENCE: 283 ttaacatttg cectgettga gatggttttg cgatttgttt «210> <211l> <212> <213> <220> <223> <400> SEQ ID NO 284 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-111 SEQUENCE: 284 tttcaattct taccattgag aatcgecaag gcattaaaca <210> SEQ ID NO 285 DNA staple tacgttaata DNA staple ggggtcagtg DNA staple gagecagcaa DNA staple acggtgtaca DNA staple agetatattt DNA staple ataacggatt sequence, , aaac sequence, , cett sequence, , aate sequence, , gace sequence, , teat sequence, , egee name: 54 name: 54 name 54 name 54 name: 54 name: 54
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US 12,548,243 B2 179 180 -cont inued <21l> <212> <213> <220> <223> <400> LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-112 SEQUENCE: 285 caaagegetg gectgattct cogtgggaca cgttgeagta <210> «21l> <212> <213> <220> <223> <400> SEQ ID NO 286 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-113 SEQUENCE: 286 gagegetgea aaatggttga gtgttgtteg tggaccataa <210> <211l> <212> <213> <220> <223> «<400> SEQ ID NO 287 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-114 SEQUENCE: 287 ggaatcatta ggttaateca atcgcaagtt ttagttgaaa <210> <211l> <212> <213> <220> <223> <400> SEQ ID NO 288 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-115 SEQUENCE: 288 tataaaataa agccaacatt atgacectac gcaageatga <210> <211l> <212> <213> <220> <223> <400> SEQ ID NO 289 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-116 SEQUENCE: 289 ctacaacatt aggatataag tatageccag tacegeattt <210> «211> <212> <213> <220> <223> <400> SEQ ID NO 290 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Cubic Cub-staple-117 SEQUENCE: 290 gattagtatg tagaaccaag taccgcacat cgtaggtatt «210> <211l> SEQ ID NO 291 LENGTH: 54 DNA staple teggectcag DNA staple aaacagggaa DNA staple tacegacegt DNA staple ttaagactcc DNA staple teagggatag DNA staple ctaagaacge sequence, , name: gaag BA sequence, , name: gege BA sequence, , name gtga 54 sequence,, name ttat 54 sequence,, name caag 54 sequence,, name: gagg 54
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US 12,548,243 B2 181 182 -cont inued <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic DNA staple sequence,, name: Cub-staple-118 <400> SEQUENCE: 291 tacactaacg attgtaaage cagaatggag cgtcatccat taaacgggta aaat BA <210> SEQ ID No 292 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic DNA staple sequence,, name: Cub-staple-119 <400> SEQUENCE: 292 tacgageegg gectttgggt aacgecagge cagtgegaat tegtaatcat ggte BA <210> SEQ ID NO 293 <211> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Cubic DNA staple sequence,, name: Cub-staple-120 <400> SEQUENCE: 293 egetggtgag agatctggag caaacaagtg tcaatccagt gagacgggca acag 54 <210> SEQ ID NO 294 <211l> LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct element: gold nanoparticle attachment sequence <400> SEQUENCE: 294 teactteata ctctacgttg ttgttgttgt tgttgttgtt gttgtt 46 <210> SEQ ID NO 295 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 295 accagtagca cccegtaate agtagcatta tacatgttac tttttttttt ttttatcegt 60 ta 62 <210> SEQ ID NO 296 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 296 attagacggg aggagagata acccacttga tggggaacaa gatttttttt ttttatecgt 60 ta 62
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US 12,548,243 B2 183 184 -cont inued <210> SEQ ID NO 297 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 297 aggegcatag gcaaatcaac gtaacagttt attgagggaa ggtttttttt ttttatecgt 60 ta 62 <210> SEQ ID NO 298 <211> LENGTH: 62 <212> TYPE: DNA «213> ORGANISM: Artificial sequence «220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 298 cccaatagga acgcattcca cagacactya gacgtgtate actttttttt ttttatcegt 60 ta 62 «210> SEQ ID NO 299 <211> LENGTH: 62 «212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 299 agettagatt aaaaatcata ggtctgacaa acaaatatat gttttttttt ttttateegt 60 ta 62 <210> SEQ ID NO 300 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 300 acgtaatgec accatctttg accccccagg aggagtctct gatttttttt ttttatecgt 60 ta 62 <210> SEQ ID NO 301 <21l> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 301 cteagageca ccagecgecg ccagcagaat caaatctttt catttttttt ttttatecgt 60 ta 62 <210> SEQ ID NO 302 <21l> LENGTH: 62 <212> TYPE: DNA
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US 12,548,243 B2 185 186 -cont inued <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 302 atagetgttt ccataaagtg taaagetgtt gggecagtca cgtttttttt ttttatecgt 60 ta 62 <210> SEQ ID NO 303 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 303 gagtaacagt gecatgaaag tattaacacg cataaagaca getttttttt ttttatecgt 60 ta 62 <210> SEQ ID NO 304 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 304 agattcaaaa ggctagctga taaattattg agtagcagat agtttttttt ttttatccgt 60 ta 62 <210> SEQ ID NO 305 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 305 gaactaacgg aattcaacta atgeagatty ctgcagttga tttttttttt ttttatecgt 60 ta 62 <210> SEQ ID NO 306 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence «220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 306 tttgeggaac aatggcaatt catcaattat cctatcccaa tetttttttt ttttatcegt 60 ta 62 <210> SEQ ID NO 307 <211> LENGTH: 62 <212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1
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US 12,548,243 B2 -cont inued <400> SEQUENCE: 307 tacgeagtat gtaaagacac cacggaagaa ttattttgeg ggtttttttt ttttatecgt 60 ta 62 «210> SEQ ID NO 308 <211> LENGTH: 62 «212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 308 tgattgettt gagcaaaaga agatgatatc atacaacgce aatttttttt ttttatcogt 60 ta 62 <210> SEQ ID NO 309 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 309 ttggggegeg agatcataca ggcaagtget catactttaa tetttttttt ttttatccgt 60 ta 62 <210> SEQ ID NO 310 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 310 ctgattgece ttageaggeg aaaatccegg agaatgaacg gttttttttt ttttatcegt 60 ta 62 <210> SEQ ID NO 311 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 311 ategeactce agecatteag getgeggeca teageggatt gatttttttt ttttatecgt 60 ta 62 <210> SEQ ID NO 312 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 312 aategtcata aaagttcaga aaacgaataa cgcatagega gatttttttt ttttateegt 60
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US 12,548,243 B2 189 -cont inued 190 <210> SEQ ID NO 313 <211> LENGTH: 62 <212> TYPE: DNA «213> ORGANISM: Artificial sequence «220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 313 egttttageg aattgcacce agctacatcc catgaacaag catttttttt ttttatecgt 60 ta 62 «210> SEQ ID NO 314 <211> LENGTH: 62 «212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 314 gacgacaata aactgaacaa gaaaaaatcc tgaaataaag aatttttttt ttttateegt 60 ta 62 <210> SEQ ID NO 315 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 315 taaataagge gttagaaaaa goctgtacta cctacgegag aatttttttt ttttatecgt 60 ta 62 <210> SEQ ID NO 316 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 316 ggtttatcag ctatgacaac aaccattcat agtggagtga gatttttttt ttttatccgt 60 ta 62 <210> SEQ ID NO 317 <21l> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 317 atttaaattg tatttttaac caatagggtg cctctgcatt aatttttttt ttttatecgt 60 ta 62
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US 12,548,243 B2 191 192 -cont inued <210> SEQ ID NO 318 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-1 <400> SEQUENCE: 318 agagagtace tteggatgge ttagaggace ataagecega aatttttttt ttttatccgt 60 ta 62 <210> SEQ ID NO 319 <211l> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence «220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 319 accagtagca cccegtaate agtagcatta tacatgttac tttttttttt tttttaacgg 60 at 62 «210> SEQ ID NO 320 <211> LENGTH: 62 «212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 320 attagacggg aggagagata acccacttga tggggaacaa gatttttttt tttttaacgg 60 at 62 «210> SEQ ID NO 321 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 321 aggegcatag gcaaatcaac gtaacagttt attgagggaa ggtttttttt tttttaacgg 60 at 62 <210> SEQ ID NO 322 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 322 cecaatagga acgcattcca cagacactga gacgtgtate actttttttt tttttaacgg 60 at 62 <210> SEQ ID NO 323 <211> LENGTH: 62 «212> TYPE: DNA <213> ORGANISM: Artificial sequence
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US 12,548,243 B2 193 194 -cont inued <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 323 agcttagatt aaaaatcata ggtctgacaa acaaatatat gttttttttt tttttaacgg 60 at 62 <210> SEQ ID NO 324 <211> LENGTH: 62 «212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 324 acgtaatgce accatctttg accccecagg aggagtetct gatttttttt trtttaacgg 60 at 62 <210> SEQ ID NO 325 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 325 ctcagageca ccagecgeeg ccagcagaat caaatetttt catttttttt tttttaacgg 60 at 62 <210> SEQ ID NO 326 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 326 atagetgttt ccataaagtg taaagetgtt gggecagtca cgtttttttt tttttaacgg 60 at 62 <210> SEQ ID NO 327 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 327 gagtaacagt gecatgaaag tattaacacg cataaagaca getttttttt tttttaacgg 60 at 62 <210> SEQ ID NO 328 <211l> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence «220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2
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US 12,548,243 B2 195 196 -cont inued <400> SEQUENCE: 328 agattcaaaa ggctagctga taaattattg agtagcagat agtttttttt tttttaacgg 60 at 62 «210> SEQ ID NO 329 <211> LENGTH: 62 «212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 329 gaactaacgg aattcaacta atgcagattg ctgcagttga tttttttttt tttttaacgg 60 at 62 «210> SEQ ID NO 330 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 330 tttgeggaac aatggeaatt catcaattat cctatcccaa tetttttttt tttttaacgg 60 at 62 <210> SEQ ID NO 331 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 331 tacgcagtat gtaaagacac cacggaagaa ttattttgeg ggtttttttt tttttaacgg 60 at 62 <210> SEQ ID NO 332 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 332 tgattgettt gagcaaaaga agatgatate atacaacgee aatttttttt tttttaacgg 60 at 62 <210> SEQ ID NO 333 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 333 ttggggegeg agatcataca ggcaagtget catactttaa tetttttttt trtttaacgg 60
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US 12,548,243 B2 197 -cont inued 198 at 62 <210> SEQ ID NO 334 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence «220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 334 ctgattgece ttagcaggeg aaaatccegg agaatgaacg gttttttttt tttttaacgg 60 at 62 <210> SEQ ID NO 335 <211> LENGTH: 62 «212> TYPE: DNA «213> ORGANISM: Artificial sequence «220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 335 ategeactee agecattcag getgeggcca tcageggatt gatttttttt tttttaacgg 60 at 62 «210> SEQ ID NO 336 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 336 aatcgtcata aaagttcaga aaacgaataa cgcatagega gatttttttt tttttaacgg 60 at 62 <210> SEQ ID NO 337 <211l> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 337 egttttageg aattgcacce agetacatcc catgaacaag catttttttt tttttaacgg 60 at 62 <210> SEQ ID NO 338 <21l> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 338 gacgacaata aactgaacaa gaaaaaatcc tgaaataaag aatttttttt tttttaacgg 60 at 62 <210> SEQ ID NO 339
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US 12,548,243 B2 199 200 -cont inued <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 339 taaataagge gttagaaaaa gectgtacta cctacgegag aatttttttt trtttaacgg 60 at 62 <210> SEQ ID NO 340 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 340 ggtttatcag ctatgacaac aaccatteat agtggagtga gatttttttt tttttaacgg 60 at 62 <210> SEQ ID NO 341 <211> LENGTH: 62 <212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 341 atttaaattg tatttttaac caatagggtg cctctgeatt aatttttttt tttttaacgg 60 at 62 «210> SEQ ID NO 342 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Construct component: DNA sticky end for cube-2 <400> SEQUENCE: 342 agagagtace ttcggatgge ttagaggacc ataageccga aatttttttt tttttaacgg 60 at 62 <210> SEQ ID NO 343 <211l> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-1 <400> SEQUENCE: 343 attgtgtage cgtcccegaa cataccgaac gaacccage 39 <210> SEQ ID NO 344 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-2
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US 12,548,243 B2 201 202 -cont inued <400> SEQUENCE: 344 atttgtattg aggacaactc ggaagata 28 <210> SEQ ID NO 345 <21l> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-3 <400> SEQUENCE: 345 aggacagate ttgagaataa cataaaaaaa cacccgaat 39 <210> SEQ ID NO 346 <21l> LENGTH: 38 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-4 <400> SEQUENCE: 346 acgtageegg aacgaggece acaatgaaca atttaatg 38 <210> SEQ ID NO 347 <21l> LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-5 <400> SEQUENCE: 347 caggegeata ccgegacctg ctccaccaat aagggagaac ctaaaa 46 <210> SEQ ID NO 348 <211l> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-6 <400> SEQUENCE: 348 ctcatctaaa atacaaagaa accaccagaa ggageggaat t 41 <210> SEQ ID NO 349 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-7 <400> SEQUENCE: 349 getgacgege attagaaaag tttgagtcaa cctaaaagge tg 42 <210> SEQ ID NO 350 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-8
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US 12,548,243 B2 -cont inued <400> SEQUENCE: 350 aggaagttte cattaaacgg gtttgaccca acggag 36 <210> SEQ ID NO 351 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, <400> name: tet-staple-9 SEQUENCE: 351 aaaatgaaaa tagcagectt tgggtaattg age <210> «21Ll> «212> «213> «220> <223> <400> SEQ ID NO 352 LENGTH: 22 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: name: tet-staple-10 SEQUENCE: 352 ggaacttcat caagagtaat ga «210> «211> «212> <213> <220> <223> <400> SEQ ID NO 353 LENGTH: 48 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: name: tet-staple-11 SEQUENCE: 353 33 Tetrahedral DNA staple strand, 22 Tetrahedral DNA staple strand, catttttect ttgaatagat taaaatatct ttagtgaace ttgaaaaa 48 <210> «211l> <212> <213> <220> <223> <400> SEQ ID NO 354 LENGTH: 31 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: name: tet-staple-12 SEQUENCE: 354 aacgtaatge cactacgaat acacctgata a <210> <21l> <212> <213> «220> <223> «<400> SEQ ID NO 355 LENGTH: 32 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description name: tet-staple-13 SEQUENCE: 355 tttacaaaca attcgattta gaagtattag ac <210> <21l> <212> «213> <220> «223> <400> SEQ ID NO 356 LENGTH: 31 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description name: tet-staple-14 SEQUENCE: 356 Tetrahedral DNA staple strand, 31 Tetrahedral DNA staple strand, 32 Tetrahedral DNA staple strand,
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US 12,548,243 B2 205 -cont inued 206 getaatatca gacgaagece tttttaagaa a 31 <210> SEQ ID NO 357 <21l> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-15 <400> SEQUENCE: 357 tgaaaatage aatagctcca gaagaagact 31 <210> SEQ ID NO 358 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-16 <400> SEQUENCE: 358 gattagegaa atcgaaagag gcaaaagaag gcacaacatt at 42 <210> SEQ ID NO 359 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-17 <400> SEQUENCE: 359 atggttaage tgttactgtg tacagac 27 <210> SEQ ID NO 360 <211l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-18 <400> SEQUENCE: 360 ataacgtcac cttgegagca ctaacaaaga gcaagagcaa ta 42 <210> SEQ ID NO 361 <21l> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-19 <400> SEQUENCE: 361 teaacgetga gagccagace ageatattaa agegg 35 <210> SEQ ID NO 362 <211l> LENGTH: 53 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-20 <400> SEQUENCE: 362
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207 US 12,548,243 B2 -cont inued 208 cttattaatt agtcagtcag aacagagaca agaaccggat attcattacc caa <210> <21l> <212> <213> «220> «223> <400> SEQ ID NO 363 LENGTH: 36 TYPE: DNA ORGANISM: Artificial sequence FEATURE: tet-staple-21 SEQUENCE: 363 OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tctaaagatt aaaagttatt aattttacta ctaata <210> <21l> <212> «213> «220> «223> <400> SEQ ID NO 364 LENGTH: 45 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description name: tet-staple-22 SEQUENCE: 364 Tetrahedral DNA staple strand, aaacaaatat caaacecggt tatcaataca ttcategeta aaaca <210> «211> «212> «213> «220> <223> <400> SEQ ID NO 365 LENGTH: 39 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: name: tet-staple-23 SEQUENCE: 365 Tetrahedral DNA staple strand, gacaatattt ttgaatgget cgcagtatgt tagcaaacg <210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 366 LENGTH: 32 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: name: tet-staple-24 SEQUENCE: 366 egegaaceat gattgaaace gaggaaacaa ca <210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 367 LENGTH: 33 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: name: tet-staple-25 SEQUENCE: 367 aaacagacag tgecatatct ggtcagttgg caa <210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 368 LENGTH: 39 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description name: tet-staple-26 SEQUENCE: 368 Tetrahedral DNA staple strand, Tetrahedral DNA staple strand, Tetrahedral DNA staple strand, taatttgatt tacgacteat cegcegecag cattggaaa 53 36 45 39 32 33 39
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US 12,548,243 B2 209 -cont inued 210 <210> SEQ ID NO 369 <21l> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-27 <400> SEQUENCE: 369 aacgagcaat caatgggtat tgttgagg <210> SEQ ID NO 370 <21l> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-28 <400> SEQUENCE: 370 teaacgtaac aaagetgcac gattttttgt ttaacgt <210> SEQ ID NO 371 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-29 <400> SEQUENCE: 371 gtgaataggt ttaattatac cagtcaggga actaaccac <210> SEQ ID NO 372 <211l> LENGTH: 38 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-30 <400> SEQUENCE: 372 ttgttatece aatccaaagg aatacggaac aagecace <210> SEQ ID NO 373 <211l> LENGTH: 40 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-31 <400> SEQUENCE: 373 ttagttgtct aagagegece aatageaage aaatcagata <210> SEQ ID NO 374 <211> LENGTH: 82 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-32 <400> SEQUENCE: 374 geacceaget acaatttttt tegetgagge ttgcagggag teategecca cgcataaceg 28 37 39 38 40 60
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US 12,548,243 B2 211 -cont inued 212 atttttttat cctgaatctt ac 82 <210> SEQ ID NO 375 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-33 <400> SEQUENCE: 375 tagtaagaag aaaaagecgt ttttattctc ccgactaacg ag 42 <210> SEQ ID NO 376 <21l> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-34 <400> SEQUENCE: 376 ttggattggg cttgagatag ge 22 <210> SEQ ID NO 377 <211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-35 <400> SEQUENCE: 377 aggaatgtac cgcageatgt agataagtce tgaaaagegt ccagtete 48 <210> SEQ ID NO 378 <211l> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-36 <400> SEQUENCE: 378 accacgegag gegttttgee ttaacagage © a1 <210> SEQ ID NO 379 <21l> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-37 <400> SEQUENCE: 379 ttatcattce aagaacaate ggetgtcttt cc 32 <210> SEQ ID NO 380 <21l> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-38 <400> SEQUENCE: 380 gattcateag ttacactate ataacecteg tt 32
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US 12,548,243 B2 213 -cont inued 214 <210> SEQ ID NO 381 <21l> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-39 <400> SEQUENCE: 381 attttacgag gcatagtata geccaccace g <210> SEQ ID NO 382 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-40 <400> SEQUENCE: 382 atectaccag ttacgggagg ttttgaaage gaacttcate gt <210> SEQ ID NO 383 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-41 <400> SEQUENCE: 383 tgttcageta atgcagaacg cgectgttta tatga <210> SEQ ID NO 384 <21l> LENGTH: 52 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-42 <400> SEQUENCE: 384 gegtttteat eggeatttte ggtcaagage agagatttat aagaatcatt ca <210> SEQ ID NO 385 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-43 <400> SEQUENCE: 385 ttttcacgtt ccagtcaaga aaaataaata acgecageca te <210> SEQ ID NO 386 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-44 <400> SEQUENCE: 386 tacttaaage cagaatgaca ggagaaacca acatt 31 42 35 52 42 35
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US 12,548,243 B2 215 216 -cont inued <210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 387 LENGTH: 54 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-45 SEQUENCE: 387 gaacegeece tcagacatta ttggctcatt teaactttaa tcattgtgaa ttac 54 <210> <21l> <212> <213> <220> «223> <400> SEQ ID NO 388 LENGTH: 36 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-46 SEQUENCE: 388 tgaatttcca gagegagaac aagcaatcac tatcce 36 <210> <211l> <212> «213> «220> «223> <400> SEQ ID NO 389 LENGTH: 45 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-47 SEQUENCE: 389 gegatacatg gettttgeaa caatagaaac cgtctttcat caaga 45 <210> <211l> <212> <213> <220> <223> <400> SEQ ID NO 390 LENGTH: 32 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-48 SEQUENCE: 390 acecteacag agececttat tagegtttaa ag 32 <210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 391 LENGTH: 45 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-49 SEQUENCE: 391 accaccaact aatcaaaate accggaacga gccgeataaa acacg 45 <210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 392 LENGTH: 37 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-50 SEQUENCE: 392 caggteaate ctcaaggagt gtactggtaa taagttt 37
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US 12,548,243 B2 217 218 -cont inued <210> SEQ ID NO 393 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-51 <400> SEQUENCE: 393 gaatgacate accgaacgte atctgygccaa cagagaaac 39 <210> SEQ ID NO 394 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-52 <400> SEQUENCE: 394 tttaaaccat ttggattace accttctg 28 <210> SEQ ID NO 395 <211l> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-53 <400> SEQUENCE: 395 acttcaaaac aggtggatgg cttagagctg ttttattcc 39 <210> SEQ ID NO 396 <211> LENGTH: 38 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-54 <400> SEQUENCE: 396 egegactatt atagtcacag ttgaaatatg ctggcaga 38 <210> SEQ ID NO 397 <21l> LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-55 <400> SEQUENCE: 397 gagettcaag gcaaaaatca ggtctaacga gtyctgtagg accagt 46 <210> SEQ ID NO 398 <211l> LENGTH: 41 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-56 <400> SEQUENCE: 398 gegtccaata gegatcaagt ttgectttag egtcagactg t al <210> SEQ ID NO 399
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US 12,548,243 B2 219 220 -cont inued <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-57 <400> SEQUENCE: 399 agagtacctt taattgettt tgaategeca tatttaacaa cggecaa aT <210> SEQ ID NO 400 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-58 <400> SEQUENCE: 400 ccagacctga atatatcgat agcageattg ccagagageg aa 42 <210> SEQ ID NO 401 <211l> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-59 <400> SEQUENCE: 401 cagacgacga taaaaaccaa aatactgoca aatge 35 <210> SEQ ID NO 402 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-60 <400> SEQUENCE: 402 attgeggaag caaactecat at 22 <210> SEQ ID NO 403 <211l> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-61 <400> SEQUENCE: 403 agtageggce ggatcacega ggtaaatatt gacgtgtcac aaagacac 48 <210> SEQ ID NO 404 <211l> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-62 <400> SEQUENCE: 404 gaagaggett ttgcaaagtt tagaaaaacg a 31 «210> SEQ ID NO 405 <211> LENGTH: 32
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221 US 12,548,243 B2 -cont inued 222 <212> <213> <220> <223> <400> TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: name: tet-staple-63 SEQUENCE: 405 aatcaccagt agcaccgaat tagagecage aa <210> «21l> <212> <213> <220> <223> <400> SEQ ID NO 406 LENGTH: 31 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: name: tet-staple-64 SEQUENCE: 406 gaagtttcat teaataacet gtttagetat a <210> <21l> <212> <213> <220> <223> «<400> SEQ ID NO 407 LENGTH: 31 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: name: tet-staple-65 SEQUENCE: 407 caaagataca tttegcaggt ggcagcaaag a <210> <211l> <212> <213> <220> <223> <400> SEQ ID NO 408 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description name: tet-staple-66 SEQUENCE: 408 Tetrahedral DNA staple strand, Tetrahedral DNA staple strand, Tetrahedral DNA staple strand, Tetrahedral DNA staple strand, tgaattcata aatggtaata gtaaaataga agttcegtaa to <210> <211l> <212> <213> <220> <223> <400> SEQ ID NO 409 LENGTH: 27 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description name: tet-staple-67 SEQUENCE: 409 attttctgeg ttaccctgtt ttaatte <210> «<21l> «212> <213> <220> <223> <400> SEQ ID NO 410 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description name: tet-staple-68 SEQUENCE: 410 Tetrahedral DNA staple strand, Tetrahedral DNA staple strand, agcattagtt tatttgaaat tattcatatt tagtttagta gt <210> «21Ll> <212> SEQ ID NO 411 LENGTH: 35 TYPE: DNA 32 31 31 42 27 42
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US 12,548,243 B2 223 224 -cont inued <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-69 <400> SEQUENCE: 411 tgggcaacat ataaaagata gaacttagca agaca 35 <210> SEQ ID NO 412 <211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-70 <400> SEQUENCE: 412 attageattt acataactaa atttttgeca ggattag 37 <210> SEQ ID NO 413 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-71 <400> SEQUENCE: 413 cacggaaggg acatccaatg aaaccaatag taaagg 36 <210> SEQ ID NO 414 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-72 <400> SEQUENCE: 414 gcaatcaata gaaaattgag ggaacttgag cagttcagct ggata 45 <210> SEQ ID NO 415 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-72 <400> SEQUENCE: 415 agaaaataca tacatagegt aagaatacgt ggca 34 <210> SEQ ID NO 416 <21l> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-74 <400> SEQUENCE: 416 caatcgtctg aaatggatta aaattaagca ataaagect 39 <210> SEQ ID NO 417 <211> LENGTH: 32 «212> TYPE: DNA <213> ORGANISM: Artificial sequence
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US 12,548,243 B2 225 -cont inued 226 <220> <223> <400> FEATURE: OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-75 SEQUENCE: 417 ttcaccaagg caagtcaatt ctactaatga cc «210> «21Ll> <212> «213> <220> <223> <400> SEQ ID NO 418 LENGTH: 33 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-76 SEQUENCE: 418 acctgaaaaa ggtgtttace agegecaaag aca «210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 419 LENGTH: 39 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-77 SEQUENCE: 419 cacgacgtta catctttcaa teggaacgag ggtagcaat <210> <21l> <212> <213> <220> <223> <400> SEQ ID NO 420 LENGTH: 28 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-78 SEQUENCE: 420 taacgecegg attcegegca gtacagag <210> <211> <212> <213> <220> <223> <400> SEQ ID NO 421 LENGTH: 39 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-79 SEQUENCE: 421 gteatageeg gaaggecctc atagttagac gttagggag «210> «<21l> <212> <213> «220> <223> <400> SEQ ID NO 422 LENGTH: 38 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-80 SEQUENCE: 422 ttegeatgee tgcaggtttt cagctaaatg acgetttt <210> «21Ll> <212> «213> <220> SEQ ID NO 423 LENGTH: 46 TYPE: DNA ORGANISM: Artificial sequence FEATURE: 32 33 39 28 39 38
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US 12,548,243 B2 227 228 -cont inued <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-81 <400> SEQUENCE: 423 ttgttatect tacgacggce agtgcaagga actgtegtce tcagea 46 <210> SEQ ID NO 424 <21l> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-82 <400> SEQUENCE: 424 getattaagg caaaattaat tacatttaac aatttcattt gaatt 45 <210> SEQ ID NO 425 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-83 <400> SEQUENCE: 425 aattccatct aaagtagaag atgatgageg caactygctc ac 42 <210> SEQ ID NO 426 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-84 <400> SEQUENCE: 426 acegcttetg gtgcceggaaa cccgecagca gttggg 36 <210> SEQ ID NO 427 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-85 <400> SEQUENCE: 427 actacaacge ctgtagcatt cogtatggga tttt 34 <210> SEQ ID NO 428 <21l> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-86 <400> SEQUENCE: 428 acgacacaac atacgagctg t 21 «210> SEQ ID NO 429 <211> LENGTH: 48 «212> TYPE: DNA <213> ORGANISM: Artificial sequence «220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand,
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229 US 12,548,243 B2 230 -cont inued <400> name: tet-staple-87 SEQUENCE: 429 toaagattat toagggagaa aggtttaacg tcagtgaata aatcctga 48 «210> «21Ll> «212> «213> <220> <223> <400> SEQ ID NO 430 LENGTH: 31 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: name: tet-staple-8¢ SEQUENCE: 430 aaagegecat tegecatgtg cgggtcecag t «210> «21Ll> «212> <213> <220> <223> <400> SEQ ID NO 431 LENGTH: 32 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: name: tet-staple-89 SEQUENCE: 431 taccaagtta caaaatgect gattgctttg aa «210> <211> <212> <213> <220> <223> <400> SEQ ID NO 432 LENGTH: 31 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description name: tet-staple-90 SEQUENCE: 432 gctaaacaac ttaatcteca aaaaaaagge t <210> <211> <212> <213> <220> <223> <400> SEQ ID NO 433 LENGTH: 31 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description name: tet-staple-91 SEQUENCE: 433 tgataataat tttttcattt atcaacaatg a <210> <21l> «212> <213> <220> <223> <400> SEQ ID NO 434 LENGTH: 42 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description name: tet-staple-92 SEQUENCE: 434 Tetrahedral DNA staple strand, 31 Tetrahedral DNA staple strand, 32 Tetrahedral DNA staple strand, 31 Tetrahedral DNA staple strand, 31 Tetrahedral DNA staple strand, taccttttgt aaagggaagg gogatcgtca ggctaacaaa ca 42 <210> «211> <212> «213> <220> «223> SEQ ID NO 435 LENGTH: 27 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description name: tet-staple-93 Tetrahedral DNA staple strand,
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US 12,548,243 B2 231 232 -cont inued <400> SEQUENCE: 435 gaagaataga caagettetg tgtgaaa 27 <210> SEQ ID NO 436 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-94 <400> SEQUENCE: 436 tettaattat acttcatgaa tatacagcta aaggaatgaa tt 42 <210> SEQ ID NO 437 <21l> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-95 <400> SEQUENCE: 437 ccatgatgge aattcatcaa cggcaggega aaaac 35 <210> SEQ ID NO 438 <21l> LENGTH: 53 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-96 <400> SEQUENCE: 438 caacaactaa aggcatttte tacagacaca taaagtgtaa agectggggt geo 53 <210> SEQ ID NO 439 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-97 <400> SEQUENCE: 439 ttgtttgaca gcattacctg agcaaattaa taacag 36 <210> SEQ ID NO 440 <21l> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-98 <400> SEQUENCE: 440 atatggaagg gttagaagat tttcacaata aagggtttce tette 45 <210> SEQ ID NO 441 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-99
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US 12,548,243 B2 -cont inued <400> SEQUENCE: 441 atcatattce tgatggacta aagacttttt c 31 <210> SEQ ID NO 442 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, <400> name: tet-staple-100 SEQUENCE: 442 gegggattge goeggettge tttegaggtt ge 32 <210> «21Ll> «212> «213> «220> <223> <400> SEQ ID NO 443 LENGTH: 33 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-101 SEQUENCE: 443 getttgatat cagatatcaa aattatttge acg 33 «210> «211> «212> <213> <220> <223> <400> SEQ ID NO 444 LENGTH: 39 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-102 SEQUENCE: 444 acagetgata geccctcaga gataaagtac cgacaaaca 39 <210> «211l> <212> <213> <220> <223> <400> SEQ ID NO 445 LENGTH: 28 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-103 SEQUENCE: 445 ttttcacace gtacgecace caagtaat 28 <210> <21l> <212> <213> «220> <223> «<400> SEQ ID NO 446 LENGTH: 39 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-104 SEQUENCE: 446 gttccgagtt gttctcaaaa tcataggtgt tgggtegag 39 <210> <21l> <212> «213> <220> «223> <400> SEQ ID NO 447 LENGTH: 38 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-105 SEQUENCE: 447
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US 12,548,243 B2 -cont inued ggcagttgca gcaagegaag aacgtatata agtaattt 38 <210> SEQ ID NO 448 <211> LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-106 <400> SEQUENCE: 448 ataaatcaaa attcaccgee tggcccaaat ataacctect cgagec 46 <210> SEQ ID NO 449 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-107 <400> SEQUENCE: 449 ggccaacaat tgcggtaceg taacactgag tttcgtcace agtac 45 <210> SEQ ID NO 450 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-108 <400> SEQUENCE: 450 agecegacta cettttttte agggatacag tegggaaaga at 42 <210> SEQ ID NO 451 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-109 <400> SEQUENCE: 451 aatgagtgag ctaactcaca ttgcgegggg tttttc 36 <210> SEQ ID NO 452 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-110 <400> SEQUENCE: 452 gacgetgaga agagtcaata gttaaatget gatg 34 <210> SEQ ID NO 453 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-111 <400> SEQUENCE: 453
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US 12,548,243 B2 237 -cont inued 238 gagagatagg gttgagtaat c <210> SEQ ID NO 454 <21l> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-112 <400> SEQUENCE: 454 aataggegee accggaatag ccaggeggat aagttattat taatgece <210> SEQ ID NO 455 <21l> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-113 <400> SEQUENCE: 455 catttgeget cactgectge attaacggge a <210> SEQ ID NO 456 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-114 <400> SEQUENCE: 456 egecacecte agaaccteag gaggtttagt ac <210> SEQ ID NO 457 <211l> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-115 <400> SEQUENCE: 457 caaatecaat egttgaaata cegacegtgt g <210> SEQ ID No 458 <21l> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-116 <400> SEQUENCE: 458 aaattctgac ctaaattaca ceggettace a <210> SEQ ID NO 459 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-117 <400> SEQUENCE: 459 ataagtattg ceccetgteg tgecageege tttegeaage ce 21 4g 31 32 a1 31 42
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US 12,548,243 B2 239 -cont inued 240 <210> SEQ ID No 460 <21l> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-118 <400> SEQUENCE: 460 atcacttttt ctgagagaaa atccctt <210> SEQ ID NO 461 <21l> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-119 <400> SEQUENCE: 461 geetgttteg gaacegeegt cgagaggttt agttaagaaa aa <210> SEQ ID NO 462 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-120 <400> SEQUENCE: 462 aagacagtge cegtataaaa ggtatcagaa caace <210> SEQ ID NO 463 <21l> LENGTH: 51 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-121 <400> SEQUENCE: 463 egetcaacag tagggeaatt tecttttgat aagaggtcag tacggtgtct g <210> SEQ ID NO 464 <211> LENGTH: 53 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-122 <400> SEQUENCE: 464 gtataaacca acatetatat ggaatttaca gtttggaaca agagtccact att <210> SEQ ID NO 465 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-123 <400> SEQUENCE: 465 ectgectaga gaatccacca cecteattat gttgat 27 42 35 51 53 36
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US 12,548,243 B2 -cont inued <210> SEQ ID No 466 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-124 <400> SEQUENCE: 466 gttctgaaac atgaaagetc agtagtgtat ccagtgagat gaatc 45 <210> SEQ ID NO 467 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-125 <400> SEQUENCE: 467 aacggggtca gtgcecagacg acgacaataa acaa 34 <210> SEQ ID NO 468 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-126 <400> SEQUENCE: 468 aggcagaaca aattaatcat aattactatt tc 32 <210> SEQ ID NO 469 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-127 <400> SEQUENCE: 469 totgteettg agtaaggetg agactectca aga 33 <210> SEQ ID NO 470 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-128 <400> SEQUENCE: 470 accttgegca ttaagecage taatcaccat caatacagy 39 <210> SEQ ID NO 471 <211> LENGTH: 28 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-129 <400> SEQUENCE: 471 atcaataage teatttegeg teaacegt 28
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US 12,548,243 B2 243 244 -cont inued <210> SEQ ID NO 472 <21l> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-130 <400> SEQUENCE: 472 ettttttaat ggaaacagea gecagettte cog 33 <210> SEQ ID NO 473 <211l> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-131 <400> SEQUENCE: 473 aagaacgtgg actccaacca tagegatage ttagatt 37 <210> SEQ ID NO 474 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-132 <400> SEQUENCE: 474 ggegaaagat tttacacega gtaaaagaaa tacttcctt 39 <210> SEQ ID NO 475 <211> LENGTH: 38 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-133 <400> SEQUENCE: 475 gtettccctt agaatcecta teggetttga ttaggtaa 38 <210> SEQ ID NO 476 <21l> LENGTH: 40 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-134 <400> SEQUENCE: 476 ggaagatggt gtagegtggy aacaaacyge ggattgaceg 40 <210> SEQ ID NO 477 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-135 <400> SEQUENCE: 477 aacaggeate acgcaaaatg tgagegaate tgccaggage ta 42 <210> SEQ ID NO 478
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US 12,548,243 B2 245 246 -cont inued <211l> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-136 <400> SEQUENCE: 478 tgtcaggecg attaaaggaa co 22 <210> SEQ ID No 479 <211> LENGTH: 48 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-137 <400> SEQUENCE: 479 cegtegtect gtaatttttg atatttaaat tgtagagaat cttgectg 48 <210> SEQ ID NO 480 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-138 <400> SEQUENCE: 480 cteatgggeg categtacag tategtgaat a 31 <210> SEQ ID NO 481 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-139 <400> SEQUENCE: 481 acgecatcaa aaataatttt taaccaatag ga 32 <210> SEQ ID NO 482 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-140 <400> SEQUENCE: 482 ctgagtagaa gatggaaata cctacatttt gacg 34 <210> SEQ ID NO 483 <211l> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-141 <400> SEQUENCE: 483 getttgcaac aggaaaacca aaaaataaaa a 31 «210> SEQ ID NO 484 <211> LENGTH: 42
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US 12,548,243 B2 247 -cont inued 248 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-142 <400> SEQUENCE: 484 aaattcttct gtatgagggg acgacgaace gtgegtaaca ac <210> SEQ ID NO 485 <211> LENGTH: 53 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-143 <400> SEQUENCE: 485 agagcataaa gctaaatcgg ttgtaacgct caactcaaat tgaaaagtca aag <210> SEQ ID NO 486 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-144 <400> SEQUENCE: 486 tttgegagca aacaaaacgt taatattata ttaccgaata ct <210> SEQ ID NO 487 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-145 <400> SEQUENCE: 487 aactctacaa aggctattga tattetggec tgatt <210> SEQ ID NO 488 <211> LENGTH: 84 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-146 <400> SEQUENCE: 488 teatatgtac cccggggtta acaccgectg caaggtgagg cggtcagtgg cagaaaagec ccaaaaacag gaagattgta gtaa <210> SEQ ID NO 489 <211l> LENGTH: 54 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-147 <400> SEQUENCE: 489 tttttagtaa tgtgtagtaa tgtgaggega caggaacygt acgecagaat cctg <210> SEQ ID NO 490 42 53 42 35 60 84 54
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US 12,548,243 B2 249 250 -cont inued <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-148 <400> SEQUENCE: 490 agagtctaca gtcattcate aacattaaca ttgtta 36 <210> SEQ ID NO 491 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-149 <400> SEQUENCE: 491 teagatgaac ggtaatctaa gcaattaaat ctatgtgagg cctca 45 <210> SEQ ID NO 492 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-150 <400> SEQUENCE: 492 agattcacge aaggeattat gacectgtcc ag 32 <210> SEQ ID NO 493 <211l> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-151 <400> SEQUENCE: 493 geeggaggag gagaagectt tatttcaaaa agggtttgta gegte 45 <210> SEQ ID NO 494 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-152 <400> SEQUENCE: 494 tetagettga gagatageat gtcaa 25 <210> SEQ ID NO 495 <211> LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-153 <400> SEQUENCE: 495 aaacaccagt gcaaaataaa cagectgcag attacgttac accaga 46 «210> SEQ ID NO 496 <211> LENGTH: 27
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US 12,548,243 B2 251 -cont inued 252 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-154 <400> SEQUENCE: 496 gaacaactaa atattatece tgacgag <210> SEQ ID No 497 <211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-155 <400> SEQUENCE: 497 agageggtta atcgtegeta ttatccagaa ttaaccggag aaag <210> SEQ ID NO 498 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-156 <400> SEQUENCE: 498 ccaggtaata attaatttat catagaatc <210> SEQ ID NO 499 <211> LENGTH: 65 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-157 <400> SEQUENCE: 499 egtaatgaac teaatatttt categeccag aatacccaaa agaactggtg atagettaac tgcag <210> SEQ ID NO 500 <211l> LENGTH: 65 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-158 <400> SEQUENCE: 500 egtaatgaac teaatatttt aataaaataa acatccaata aatcatacgt cacacctcaa catta <210> SEQ ID NO 501 <211> LENGTH: 66 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-159 <400> SEQUENCE: 501 egtaatgaac teaatatttt gegaaaggaa cagcttgata cegatagtcg teacctttce 27 44 29 60 65 60 65 60
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US 12,548,243 B2 253 -cont inued 254 agegta 66 <210> SEQ ID NO 502 <21l> LENGTH: 66 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Tetrahedral DNA staple strand, name: tet-staple-160 <400> SEQUENCE: 502 cgtaatgaac tcaatatttt agtaataatt tagtatcata tgegttatgg catttggett 60 agetga 66 <210> SEQ ID NO 503 <211> LENGTH: 62 «212> TYPE: DNA «213> ORGANISM: Artificial sequence «220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 503 aggaagagtt tttttttttt tttttttttt gegegaaaca aagtacccag egattatace 60 aa 62 «210> SEQ ID NO 504 <211> LENGTH: 65 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 504 aggaagagtt tttttttttt tttttttttt tttacegaac tgaccaactg gtcaatcata 60 aggga 65 <210> SEQ ID NO 505 <211> LENGTH: 63 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 505 aggaagagtt tttttttttt tttttttttt tttatcaaca gttgaaagga attgaggaat 60 caa 63 <210> SEQ ID NO 506 <211> LENGTH: 84 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: «223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 506 aggaagagtt tttttttttt tttttttttt agtaagcaga tagccgaaca aagttaatct 60 tacgagataa cgcagacttg aaag 84 <210> SEQ ID No 507 <21l> LENGTH: 66 <212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE:
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US 12,548,243 B2 255 -cont inued 256 <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 507 aggaagagtt tttttttttt tttttttttt ttttagaagg cttatccggt atctattttc aacget «210> SEQ ID NO 508 «211> LENGTH: 61 «212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 508 aggaagagtt tttttttttt tttttttttt cttatgegat tttaagaact acaggtagaa a <210> SEQ ID NO 509 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 509 aggaagagtt tttttttttt tttttttttt attcacaaac aaataagacg attggecttg at <210> SEQ ID NO 510 <211> LENGTH: 65 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 510 aggaagagtt tttttttttt trtttttttt tttaccctca gaaccgecac tecctcagag cogee <210> SEQ ID NO 511 <211l> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 511 aggaagagtt tttttttttt tttttttttt ttcattgaat cccectggaa togtcataaa ta <210> SEQ ID NO 512 <211> LENGTH: 65 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 512 aggaagagtt tttttttttt tttttttttt tttaagatta agaggaagca geggattgca teaaa 60 66 60 61 60 62 60 65 60 62 60 65
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US 12,548,243 B2 257 258 -cont inued <210> SEQ ID NO 513 <211> LENGTH: 63 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 513 aggaagagtt trtttttttt tttttttttt tttaaaggge gacattcaac cgattgagge 60 ata 63 <210> SEQ ID NO 514 <211> LENGTH: 84 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: «223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 514 aggaagagtt tttttttttt tttttttttt ttttcatttg gggcgegage tgaaaaaatg 60 gtecatataa gaagcaaceg aaag 84 <210> SEQ ID NO 515 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: «223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 515 aggaagagtt tttttttttt tttttttttt tgctgcaagg cgattatgge gaaaggggga 60 tg 62 <210> SEQ ID NO 516 <211> LENGTH: 65 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 516 aggaagagtt tttttttttt tttttttttt ttteegaget cgaattegta gaggatecce 60 gggta 65 <210> SEQ ID NO 517 <211> LENGTH: 63 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: «223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 517 aggaagagtt tttttttttt tttttttttt ttttaaaaca gaaataaaga aattgegtac 60 cta 63 <210> SEQ ID NO 518 <211> LENGTH: 84 «212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: «223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 518
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US 12,548,243 B2 -cont inued aggaagagtt tttttttttt tttttttttt ccaaaaggag cctttaattg tateggegtt 60 gaatcaacag cgactctaat catg 84 <210> SEQ ID NO 519 <211> LENGTH: 62 «212> TYPE: DNA «213> ORGANISM: Artificial sequence «220> FEATURE: «223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 519 aggaagagtt tttttttttt tttttttttt attgggegce agggtggaga ggcggtttge 60 gt 62 «210> SEQ ID NO 520 «211> LENGTH: 65 «212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 520 aggaagagtt tttttttttt tetttttttt tttggegaaa atcctgttte tggtttgece 60 cagea 65 «210> SEQ ID NO 521 <211> LENGTH: 63 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 521 aggaagagtt tttttttttt tttttttttt tttgaaggat taggattage ggggttttgt 60 att 63 <210> SEQ ID NO 522 <211> LENGTH: 84 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 522 aggaagagtt tttttttttt tttttttttt ataaataagg cgttaaataa gaataataat 60 ggtcaagaca gtccacggat ggtg 84 <210> SEQ ID NO 523 <211l> LENGTH: 66 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 523 aggaagagtt tttttttttt tttttttttt ttttaatggg ataggtcacg ttcgeactct 60 acataa 66 <210> SEQ ID NO 524 <211> LENGTH: 61 «212> TYPE: DNA <213> ORGANISM: Artificial sequence
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US 12,548,243 B2 261 262 -cont inued <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 524 aggaagagtt tttttttttt tttttttttt agaagtgttt ttataatcaa acatcacttg 60 «210> SEQ ID NO 525 <211l> LENGTH: 62 «212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 525 aggaagagtt tttttttttt tttttttttt agagggtage tattttgata aattaatgee 60 gg 62 <210> SEQ ID NO 526 <211> LENGTH: 65 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 526 aggaagagtt tttttttttt tttttttttt tttaaatgea atgectgaga accctcatat 60 atttt 65 <210> SEQ ID NO 527 <211l> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 527 ctettcettt tttttttttt tttttttttt gegegaaaca aagtacccag cgattatace 60 aa 62 <210> SEQ ID NO 528 <211l> LENGTH: 65 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 528 ctettecttt trtttttttt trttttttrt tttaccgaac tgaccaactg gtcaatcata 60 aggga 65 <210> SEQ ID NO 529 <21l> LENGTH: 63 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 529 ctettecttt trtttttttt tttttttttt trtatcaaca gttgaaagga attgaggaat 60 caa 63
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US 12,548,243 B2 263 -cont inued 264 <210> SEQ ID NO 530 <211> LENGTH: 84 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 530 etettecttt trtttttttt tttttttttt agtaagcaga tagecgaaca aagttaatct 60 tacgagataa cgcagacttg aaag 84 <210> SEQ ID NO 531 <211l> LENGTH: 66 <212> TYPE: DNA <213> ORGANISM: Artificial sequence «220> FEATURE: «223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 531 ctettecttt tttttttttt tttttttttt ttttagaagg cttatccggt atctattttc 60 aacget 66 <210> SEQ ID NO 532 <211> LENGTH: 61 <212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 532 ctettecttt tttttttttt tttttttttt cttatgegat tttaagaact acaggtagaa 60 a 61 <210> SEQ ID NO 533 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 533 ctettecttt tttttttttt tttttttttt attcacaaac aaataagacg attggecttg 60 at 62 <210> SEQ ID NO 534 <211> LENGTH: 65 <212> TYPE: DNA <213> ORGANISM: Artificial sequence «220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 534 ctettecttt tttttttttt tttttttttt tttaccetca gaacegecac tecetcagag 60 cegee 65 <210> SEQ ID NO 535 <211l> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence «220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 535
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US 12,548,243 B2 265 -cont inued 266 ctettecttt tttttttttt tttttttttt ttcattgaat ccccctggaa tegtcataaa 60 ta 62 <210> SEQ ID NO 536 «<211> LENGTH: 65 «212> TYPE: DNA «213> ORGANISM: Artificial sequence «220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 536 ctetteettt tttttttttt tttttttttt tttaagatta agaggaagea geggattgea 60 toaaa 65 «210> SEQ ID NO 537 «211l> LENGTH: 63 «212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 537 ctettecttt trtttttttt tttttttttt tttaaaggge gacattcaac egattgagge 60 ata 63 <210> SEQ ID NO 538 <211> LENGTH: 84 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 538 ctettecttt tttttttttt tttttttttt ttttcatttg gggegegage tgaaaaaatg 60 gtccatataa gaagcaaceg aaag 84 <210> SEQ ID NO 539 <211l> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 539 ctettcettt tttttttttt tttttttttt tgctgeaagg cgattatgge gaaaggggga 60 tg 62 <210> SEQ ID NO 540 <211> LENGTH: 65 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 540 ctettcettt tttttttttt tttttttttt trtcegaget cgaattcgta gaggatccce 60 gggta 65 <210> SEQ ID NO 541 <211l> LENGTH: 63 <212> TYPE: DNA
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US 12,548,243 B2 267 -cont inued 268 <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 541 ctettecttt tttttttttt tttttttttt ttttaaaaca gaaataaaga aattgegtac 60 eta 63 <210> SEQ ID NO 542 <211> LENGTH: 84 «212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 542 ctettecttt tttttttttt tttttttttt ccaaaaggag cctttaatty tateggegtt 60 gaatcaacag cgactctaat catg 84 <210> SEQ ID NO 543 <211> LENGTH: 62 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 543 ctettecttt tttttttttt tttttttttt attgggegce agggtggaga ggcggtttge 60 gt 62 <210> SEQ ID NO 544 <211> LENGTH: 65 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 544 ctettcettt tttttttttt tttttttttt trtggegaaa atectgttte tggtttgece 60 cagea 65 <210> SEQ ID NO 545 <211> LENGTH: 63 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 545 etettecttt trtttttttt ttrttttttt trtgaaggat taggattage ggggttttgt 60 att 63 <210> SEQ ID NO 546 <211> LENGTH: 94 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 546 ctettecttt tttttttttt tttttttttt ataaataagg cgttaaataa gaataataat 60 ggtcaagaca gtccacggat ggtg 84
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US 12,548,243 B2 269 270 -cont inued <210> SEQ ID NO 547 <211> LENGTH: 66 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: «223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 547 ctettccttt tttttttttt tttttttttt ttttaatggg ataggtcacg ttcgeactct 60 acataa 66 <210> SEQ ID NO 548 <211> LENGTH: 61 <212> TYPE: DNA «213> ORGANISM: Artificial sequence «220> FEATURE: «223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 548 ctettecttt tttttttttt tttttttttt agaagtgttt ttataatcaa acatcacttg 60 c 61 <210> SEQ ID NO 549 <211> LENGTH: 62 «212> TYPE: DNA «213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 549 ctettecttt tttttttttt tttttttttt agagggtage tattttgata aattaatgee 60 gg 62 «210> SEQ ID NO 550 <211> LENGTH: 65 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: DNA sticky end for tetrahedron <400> SEQUENCE: 550 ctettecttt tttttttttt tttttttttt tttaaatgca atgectgaga accctcatat 60 atttt 65 <210> SEQ ID NO 551 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Modified DNA sequence attached on QDs (525, 605) and streptavidin for octahedral system (HPLC purified) <220> FEATURE: <221> NAME/KEY: modified base <222> LOCATION: 18 <223> OTHER INFORMATION: biotinylated <400> SEQUENCE: 551 tatgaagtga tggatgat 18 <210> SEQ ID NO 552 «<211> LENGTH: 18 <212> TYPE: DNA
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US 12,548,243 B2 271 272 -cont inued <213> <220> <223> <220> <221> <222> «223> <400> ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Modified DNA sequence attached on QDs (705) for octahedral system (HPLC purified) FEATURE: NAME/KEY: modified base LOCATION: 18 OTHER INFORMATION: biotinylated SEQUENCE: 552 taggtatgat ggtagtag 18 <210> <21l> <212> <213> <220> «223> «220> «221> <222> <223> <400> SEQ ID NO 553 LENGTH: 18 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Modified DNA sequence attached on gold nanoparticle for octahedral system (HPLC purified) FEATURE: NAME/KEY: modified_base LOCATION: 33 OTHER INFORMATION: thiolated SEQUENCE: 553 tatgaagtga tggatgat 18 «210> «211> «212> <213> <220> <223> <220> <221> <222> <223> <400> SEQ ID NO 554 LENGTH: 33 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Modified DNA sequence attached on gold nanoparticle for cubic system (HPLC purified) FEATURE: NAME/KEY: modified base LOCATION: 33 OTHER INFORMATION: thiolated SEQUENCE: 554 gtagagtatg aagtgatgga tgatgatgat gat 33 <210> <211> <212> <213> <220> <223> <220> <221> <222> <223> <400> SEQ ID NO 555 LENGTH: 36 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Modified DNA sequence attached on gold nanoparticle for tetrahedral system (HPLC purified) FEATURE: NAME/KEY; modified base LOCATION: 36 OTHER INFORMATION: thiolated SEQUENCE: 555 tattgagtte attaegtttt tttttttttt ttteee 36 «210> <211l> <212> <213> <220> <223> <220> «221> <222> «223> <400> SEQ ID NO 556 LENGTH: 23 TYPE: DNA ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Description: Modified DNA sequence attached on glucose oxidase for enzyme lattice (standard desalting) FEATURE: NAME/KEY: modified_base LOCATION: 2 OTHER INFORMATION: thiolated SEQUENCE: 556
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US 12,548,243 B2 273 274 -cont inued tttttgtaga tggtagtatg gat 23 <210> SEQ ID NO 557 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Description: Modified DNA sequence attached on horseradish peroxidase for enzyme lattice (standard desalting) <220> FEATURE: <221> NAME/KEY: modified base <222> LOCATION: 1 <223> OTHER INFORMATION: thiolated <400> SEQUENCE: 557 tttttgatta ggtagtgaag tat 23 What is claimed is: 1. A voxel, comprising a plurality of frames including at least one single stranded (ss) DNA motif with at least one free base, wherein the at least one ssDNA motif hybridizes with a comple- mentary strand fragment of other frames through ver- tex-to-vertex hybridization; and at least one object, wherein the at least one object is located within the voxel. 2. The voxel of claim 1, wherein the plurality of frames comprises stand-alone DNA frames, polyhedral frames, or a combination thereof. 3. The voxel of claim 1, wherein the plurality of frames forms a tetrahedra framework, an octahedra framework, a cubic framework, or a combination thereof. 4. The voxel of claim 1, wherein the at least one object comprises a gold nano particle, a streptavidin, a protein, a quantum dot (QD), an enzyme, or a combination thereof. 20 w 30 5. The voxel of claim 4, wherein the object is a function- alized object. 6. The voxel of claim 1, wherein the voxel forms a three-dimensional (3D) origami lattice. 7. The voxel of claim 6, wherein the 3D origami lattice is a body-centered-cubic (BCC) lattice, a simple cubic (SC) lattice, or a cubic diamond lattice. 8. The voxel of claim 1, wherein the voxel is a DNA- prescribed voxel. 9. The voxel of claim 1, wherein the voxel is a valence- controlled voxel. 10. The voxel of claim 1, wherein the at least one object comprises a metallic particle, a semiconductor particle, a protein superlattice, or combinations thereof. ee OR Rk
Source notes & attribution
- https://rexresearch.com/GangDNASilica/US12548243B2.pdf