US2013137602

Extracted document text. Layout, formulas and symbols may not survive extraction; consult the preserved PDF for the original presentation.

Text extraction covers 27 pages, including 27 transcribed by optical character recognition.

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US 20130137602A1

as) United States
cz) Patent Application Publication (10) Pub. No.: US 2013/0137602 Al
Gang et al. (43) Pub. Date: May 30, 2013
(54) ARBITRARY ASSEMBLY OF NANO-OBJECTS (52) US.CL
INTO DESIGNED 1D AND 2D ARRAYS CPC ... see C12Q 1/6837 (2013.01)
USPC soos 506/16; 506/30
(75) Inventors: Oleg Gang, Setauket, NY (US); Daniel
van der Lelie, Chapel Hill, NC (US)
(87) ABSTRACT
(73) Assignee: Brookhaven Science Associates, LLC,
Upton, NY (US) The present invention is directed to nanoscale fabrication of
nano-materials with application in electronics, energy con-
(21) Appl. No.: 13/583,517 version, bio-sensing and others. Specifically, the invention is
directed to arbitrary, that is periodic and non-periodic, assem-
(22) PCT Filed: Mar. 7, 2011 bly of nano-objects on I D and 2D arrays. The present inven-
tion utilizes self-organization properties of nanoscale bio-
(86) PCT No.: PCT/US11/27393 encoded building blocks, programmability of biomolecular
§ 371 (c)(1), interactions, and simple processing techniques for providing
(2),(4) Date: Jan. 7, 2013 arbitrary by-design fabrication capability. Specifically, the
present invention utilizes double stranded DNA attached to a
Related U.S. Application Data surface and intercalating PNA-DNA hybrids attached to
(60) Provisional application No. 61/311,991, filed on Mar. nano-objects to bind the nano-objects to the dsDNA in a site
9.2010 . a . specific manner. The present invention allows for an integra-
, ° tion of a large number of nano-components in unified well-
Publication Classification defined systems. Accordingly, the present invention is appli-
cable for fabrication of I D and 2D structures of various
(51) Int. Cl. by-design placements of nano-objects of multiple types,
C12Q 1/68 (2006.01) including metal, semiconducting and organic nano-objects.

4A. Fabrication of DNA array using multiple anchoring points

Addition of specific
intercalators with
recognizable attachment
sites, following by nan
objects binding and D:

alignment

N
SA. Assembly of nano-objects array on

DNA/intercalator “lithographic” array

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US 2013/0137602 Al

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Patent Application Publication May 30,2013 Sheet 9 of 15 US 2013/0137602 Al

1A. Deposition and attachment of double
stranded (ds) “lithographic” DNA

termination

Lithographic DNA contains specifically encoded regions for binding of
recognizable intercalators that also able to attach encoded nano-objects

FIGURE 9

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Patent Application Publication May 30,2013 Sheet 100f15 US 2013/0137602 Al

2A. Binding of intercalators to ds-DNA with free attachment sites:
$s PNA-DNA chimeras invasion of ds-DNA at specific locations

Specific I
region of ds-DNA

*%  { ss-PNA binds to a specific dsDNA region
ds-DN: ss-PNA paat s8-DNA is a specific attachment site for
Helix { Example of intercalator {complementary encoded nano-object
: (PNA-DNA chimera ) 3
Intercalated part with a specific binding

to encoded DNA region.

FIGURE 10

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Patent Application Publication May 30,2013 Sheet11o0f15 US 2013/0137602 Al

3A. Nano-objects bind to specific DNA locations via recognition of free
attachment sites of bound intercalators

Mixture of encoded nano-objects

Nano-objects are
dispersed from a solution

Nano-objects are attached at specific locations via
recognition of specific sites of free ends of intercalators.
DNA is aligned to remove bends.

FIGURE 11

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Patent Application Publication May 30,2013 Sheet 120f15 US 2013/0137602 Al

4A. Fabrication of DNA array using multiple anchoring points

Addition of specific
intercalators with
recognizable attachment
sites, following by nano-

any, ‘ 5A. Assembly of nane-objects array on
‘ DNA/intercalator “lithographic” array

FIGURE 12

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May 30, 2013 Sheet 13 of 15

Patent Application Publication

1B. Deposition fixation of initial “column” DNA (Y-DNA) with

encoded regions for intercalator placements

cit

FIGURE 13

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May 30, 2013 Sheet 14 of 15

Patent Application Publication

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Attachment of mult

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FIGURE 14

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Patent Application Publication May 30,2013 Sheet 15o0f15 US 2013/0137602 Al

3B. Fabrication of nano-objects array using 2D arbitrary
matrix of encoded object-recognizable sites

Addition of specific
intercalators with
recognizable attachment Y
sites, following by nano-
objects binding and DNA Yw
alignment yy

Vy

FIGURE 15

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ARBITRARY ASSEMBLY OF NANO-OBJECTS
INTO DESIGNED 1D AND 2D ARRAYS

[0001] This application is an International PCT applica-
tion, which claims the benefit of U.S. Provisional Application
No. 61/311,991, filed on Mar. 9, 2010 which is hereby incor-
porated by reference in its entirety.

[0002] The present invention was made with government
support under Contract No. DE-AC02-98CH 10886 awarded
by the U.S. Department of Energy. The United States govern-
ment has certain rights in the invention.

BACKGROUND

[0003] I. Field of the Invention

[0004] The invention is directed to nanoscale fabrication
which can be used for the fabrication of broad classes of
nano-materials with application in electronics, energy con-
version, bio-sensing and others. Specifically, the invention is
directed to arbitrary assembly of nano-objects on arrays.
[0005] II. Background of the Related Art

[0006] The nanoscience revolution has led to the rapid
development of a diversity of remarkable nanoscale objects
including metallic and semiconductor nanoparticles, carbon
based nanomaterials and supramolecular organic complexes.
In order to construct complex functional systems from these
nanoscale objects, new methods of material assembly are
required. While conventional lithographic methods have been
proven to provide robust and versatile fabrication approaches,
their limited resolution, increasing cost of fabrication of small
features on large areas, serial nature of fabrication process,
and limited ability to integrate newly developed synthetic
nanoscale functional blocks call for new methods in material
and device fabrication. Conventional self-assembly is prom-
ising for the creation of large scale structures since it relies on
the intrinsic ability of the system’s components to self-orga-
nize in particular structures based on their mutual interactions
and entropic effects. Conventional self-assembly can be
assisted with external fields, stimuli, patterns, and the like.
[0007] Although the conventional approach offers an ease
of fabrication, it often cannot compete with lithographic
methods for a number of reasons. First, there is rarely a
rational design of final structures because of the complex
relationship between component interactions of a system and
the final structure. Second, self-assembly is mostly limited to
assembly of similar components or only few types of different
components, which is a serious drawback for fabrication of
complex structures. Third, and more importantly, structures
fabricated via self-assembly methods are generally periodic,
and therefore cannot compete with flexible and non-periodic
designs offered by lithographic methods.

[0008] In the last decade, a number of diverse biomimetic
approaches have been explored for nanomaterials fabrication.
The central and most promising approaches for nanotechnol-
ogy have been based on (i) specificity of programmable inter-
actions of nanoscale objects due to biomolecular recognition,
(ii) assembly of structures that can direct self-assembly pro-
cesses; and (iii) bio-mineralization or metallization pro-
cesses. A variety of different biological systems have been
suggested for the realization of biomimetic nanoassembly
including viruses, DNAs, peptides and proteins. The validity
of these approaches has recently been demonstrated for the
assembly of semiconductor and metallic nanowires based on
hybridization of DNA oligomers, assembly of the DNA func-
tionalized particles, synthesis of DNA-based ‘nanocrystal

May 30, 2013

molecules’, formation of hierarchical self-assemblies from
lipid-actin complexes, and assembly of 3D DNA guided
superlattices of nanoparticles.

[0009] Among the various biomolecular materials, DNA
has attracted much attention due to its unique recognition
capabilities, mechanical and physicochemical stability, and
synthetic accessibility of practically any desired nucleotide
sequences. The development of structural nucleic acid nano-
technology has been facilitated by the advancement of
nucleic acid synthesis technology. For example, technology
has progressed such that DNA of any desired sequence can be
synthesized up to about 200 bases in a single strand. These
synthetic strands of DNA can self-assemble into complex,
branched structures and mechanical assemblies. The features
of these assemblies can be approximately two nanometers in
size, which is equivalent to the width of a DNA double helix
(ALDAYE, F. A.; SLEIMAN, H. F. Journal of the American
Chemical Society 129(14): 4130-4131 (2007); KUMARA,
M. T.; NYKYPANCHUK, D.; SHERMAN, W. B. Nano Let-
ters 8(7): 1971-1977 (2008); SHIH, W. M.: QUISPE, J. D.;
JOYCE, G. F. Nature 427(6975):618-621 (2004); ZHANG,
X. P.; YAN, H.; SHEN, Z. Y.; SEEMAN, N.C. Journal of the
American Chemical Society 124(44):12940-12941 (2002)).
Accordingly, DNA nanotechnology is one of the premier
techniques for forming structures in the nanometer size range
because of the wide variety of possible structures that can
form through assemblies driven by Watson-Crick base pair-
ing.

[0010] Recently several groups have reported assembly of
nano-objects into arrays using DNA scaffolds (LE, J. D., etal,
“DNA-Templated Self-Assembly of Metallic Nanocompo-
nent Arrays on a Surface”, Nano Letters, 4(12), 2343-2347,
(2004); DENG, Z. X., et al, “DNA-Encoded Self-Assembly
of Gold Nanoparticles into One-Dimensional Arrays”,
Angew. Chem. Int. Ed., 44, 3582-3585, (2005); ZHANG, J.P.,
et al, “Transparent, Conductive, and Flexible Carbon Nano-
tube Films and Their Application in Organic Light-Emitting
Diodes”, Nano Letters, 6(2):248-251, (2006)). Various types
of patterns were are capable of being formed by designing
branched DNA structures. These DNA patterns have the abil-
ity to incorporate DNA binding sites for potential attachment
of DNA coated nano-objects via hybridization (MIRKIN, C.
A, etal, “A DNA-Based Method For Rationally Assembling
Nanoparticles Into Macroscopic Materials”, Nature,
382(6592):607-609, (1996); ALIVISATOS, A. P., et al,
“Organization Of Nanocrystal Molecules’ using DNA”
Nature, 382:609-611, (1996), MAYE, M. M., et al, “DNA-
Regulated Micro- and Nanoparticle Assembly”, Small 3,
1678-1682, (2007)). However, only periodic placement of
nano-objects was possible using this approach as demon-
strated by the regular periodic patterns that were observed.
Additional limitations of this approach include: (i) the com-
plexity of structures and ability to incorporate various types
of elements are restricted because the unit cell of periodic
structures is typically small (e.g., on the order of a few
nanometers to tens of nanometers); (ii) the size of uniform
scaffold area is typically only a few nanometers; (iii) Mag-
nesium ions are required to stabilize DNA scaffolds which
often induce uncontrollable aggregation of DNA coated
nano-objects; (iv) typically mica surfaces are required for
DNA scaffold immobilization that limit a choice of materials
on which structure can be created; and (v) there are techno-
logical limits with the applications and integration with other

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fabrication techniques because the placement or orientation
of scaffold is difficult to control.

[0011] Fabrication of arbitrary shapes has been success-
fully demonstrated by folding genetic single stranded (ss)
DNA into particular predesigned shapes, known as DNA
origami (ROTHEMUND, P. W. K., “Folding DNA to create
nanoscale shapes and pattern”, Nature, 440:297, (2006).
However, using this approach for positioning particles is
somewhat restricted because of factors (ii-v) as discussed
above. Additionally, the size of a DNA origami structure is
restricted to a few hundred nanometers because ss-DNA sig-
nificantly limit design and scalability of the system. Other
recently developed approaches for 3D ordering of nano-ob-
jects using DNA have been limited to periodic structures as
well (NYKYPANCHUK, D., et al, “DNA-guided crystalliza-
tion of colloidal nanoparticles”, Nature, 451(7178):542-552,
(2008)).

[0012] Thus, there is a need for creating an arbitrary assem-
bly of nano-objects on arrays that overcome the limitations
known in the art.

SUMMARY

[0013] The present invention is directed to nanoscale fab-
rication of broad classes of nano-materials with application in
electronics, energy conversion, bio-sensing, and others. Spe-
cifically, the present invention is directed to arbitrary, that is
periodic and non-periodic, assembly of nano-objects on 1D
and 2D arrays. The present invention utilizes self-organiza-
tion properties of nanoscale bio-encoded building blocks,
programmability of biomolecular interactions, and simple
processing techniques for providing arbitrary by-design fab-
rication capability. Moreover, the present invention allows for
an integration of a large number of nano-components and
their types in unified well-defined systems.

[0014] The present invention is applicable for fabrication of
1D and 2D structures of various by-design placements of
nano-objects of multiple types, including metal, semicon-
ducting and organic nano-objects. The present invention pro-
vides nanometer level precision in a registration of nano-
object on a pre-designed site and allow to create structures
with sizes of tens microns or larger.

[0015] Inoneembodiment, the present invention provides a
one dimensional matrix that directs the organization of nano-
objects onto row DNAs. Row DNAsare created by deposition
and attachment of double stranded lithographic DNA onto a
surface through an anchoring point. This allows for a by-
design fabrication of an arbitrary matrix of individually
encoded sites on lithographic ds-DNA. Using specific inter-
calators which bind to pre-determined regions of lithographic
ds-DNA, encoded nano-objects recognize their position with
nm-level accuracy via self-assembly. The present invention
provides versatility of integration of multiple types of objects
over at least tens of microns.

[0016] In another embodiment, the present invention pro-
vides two dimensional matrices that direct organization of
nano-objects onto column and row DNAs. A numberof DNA
anchoring points on column lithographic DNA provide spe-
cific sites for attachment of row DNA. This allows for a
by-design fabrication of arbitrary matrix of individually
encoded sites on lithographic ds-DNA rows. Using specific
intercalators which binds to pre-determined regions of ds-
DNA, encoded nano-objects recognize their position on 2D
matrix with nm-level accuracy via self-assembly. The present

May 30, 2013

invention provides versatility of integration of multiple types
of objects over at least tens of microns.

[0017] The versatility of integration by the present inven-
tion is difficult or nearly impossible to achieve today by any
other methods. The present invention can also be combined
with existing optical lithography methods, which can enable
the fabrication of large scale features tens of microns in size.
The present invention naturally incorporates 1D arbitrary
assembly and ultimately can be extended into 3D.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1A is an illustration of PNA-directed AUNP
assembly according to an embodiment of the present inven-
tion.

[0019] FIG. 1B provides sequences used for PNA-directed
AuNP assembly according to an embodiment of the present
invention.

[0020] FIG. 2A is a TEM micrograph of DNA functional-
ized AuNPs according to an embodiment of the present inven-
tion.

[0021] FIG. 2B provides DLS measurements of AuNPs and
DNA functionalized AuNPs according to an embodiment of
the present invention.

[0022] FIG. 3A is a representative TEM micrograph of an
aggregation of Au nanoparticles based on PNA invasion of
dsDNA according to an embodiment of the present invention.
[0023] FIG. 3B is a statistical analysis of the TEM micro-
graph shown in FIG. 3A.

[0024] FIG. 3C provides DLS measurements of D, (hydro-
dynamic diameter) of single particles (control, red) and
assembled aggregates (black) according to FIG. 3A.

[0025] FIG. 4A is a representative TEM micrograph show-
ing aggregation of Au nanoparticles based on PNA invasion
of dsDNA at 4° C. and the inset TEM micrograph is for the
control sample, in which no PNA-DNA chimera is added
according to an embodiment of the present invention.
[0026] FIG. 4B is a statistical analysis of the TEM micro-
graph shown in FIG. 4A.

[0027] FIG. 4C provides DLS measurements of D, (hydro-
dynamic diameter) of single particles (control, red) and
assembled aggregates (black) according to FIG. 4A.

[0028] FIG. 5A is an illustration showing A-AUNPs mixed
with A' Complementary DNA, A"-PNA-DNA-B', and Cy3-
DNAB (complementary to DNA-B') according to an embodi-
ment of the present invention.

[0029] FIG. 5B is an analysis of the PNA to NP binding
according to FIG. 5A.

[0030] FIG. 6A is a UV-vis melting curve of A"-PNA-
DNA-B' and DNA-B complementary according to an
embodiment of the present invention.

[0031] FIG. 6B is a Dynamic Light Scattering (DLS) melt-
ing curve of DNA A-AuNP, DNA B-AuNP, A'-DNA comple-
mentary, and A"-PNA-DNA-B' according to an embodiment
of the present invention.

[0032] FIG. 7 is an illustration of PNA-directed AuNP
assembly along a dsDNA according to an embodiment of the
present invention.

[0033] FIG. 8A is a representative TEM micrograph of
assembled nanoclusters based on PNA invasion of dsDNA
according to the schematic in FIG. 7 and an embodiment of
the present invention.

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[0034] FIG. 8B illustrates three possible configurations of
assembled trimers along a dsDNA on a flat surface (top:
schematic; bottom: TEM images) based on the TEM micro-
graph depicted in FIG. 8A.

[0035] FIG. 8C provides statistical analysis based on FIG.
8A.

[0036] FIG. 8D provides DLS measurements of D,, of
single particles (control, red) and assembled clusters (black)
based on FIG. 8A.

[0037] FIG. 9 is an illustration showing deposition and
attachment of double stranded lithographic DNA according
to an embodiment of the present invention.

[0038] FIG. 10 is an illustration showing binding of inter-
calators to double stranded DNA with free attachment sites:
ss-PNA-DNA chimeras invasion of ds-DNA at specific loca-
tions according to an embodiment of the present invention.
[0039] FIG. 11 is an illustration showing nano-objects that
bind to specific DNA locations via recognition of free attach-
ment sites of bound intercalators according to anembodiment
of the present invention.

[0040] FIG. 12, top is an illustration showing fabrication of
a DNA array using multiple anchoring points according to an
embodiment of the present invention.

[0041] FIG. 12, bottom, is an illustration showing assembly
of nano-objects array on DNA/intercalator lithographic array
according to an embodiment of the present invention.

[0042] FIG. 13 is an illustration showing deposition fixa-
tion of initial column DNA (Y-DNA) with encoded regions
for intercalators placements according to an embodiment of
the present invention.

[0043] FIG. 14 is an illustration showing fabrication of 2D
arbitrary matrix of encoded sites according to an embodiment
of the present invention.

[0044] FIG. 15 is an illustration showing fabrication of
nano-objects array using 2D arbitrary matrix of encoded
object-recognizable sites according to an embodiment of the
present invention.

DETAILED DESCRIPTION

[0045] _ In the interest of clarity, in describing the invention,
the following terms and acronyms are defined as provided
below.
[0046]
[0047]
[0048]
[0049]
[0050]
[0051]
[0052]
[0053]
[0054]
[0055]

ACRONYMS:
DNA: Deoxyribonucleic acid
RNA: Ribonucleic acid
PNA: Protein Nucleic Acid
NP: Nanoparticle
AuNP: Gold Nanoparticle
1D: One-Dimensional
2D: Two-Dimensional
ds: Double Stranded
ss: Single Stranded

DEFINITIONS

[0056] Intercalator: A molecule having one end that is
capable of binding to a specific site on a DNA, matrix, or
array (intercalator binding site) and second end that serves
as an attachment site for a bio-encoded nano-object (nano-
object binding site).

[0057] Lithographic DNA: Double stranded DNA with
specifically designed regions for intercalator binding (in-
tercalator binding sites).

May 30, 2013

[0058] Arbitrary assembly: Periodic and non-periodic by-
design fabrication of nanoscale bio-encoded building
blocks.

[0059] Periodic: Occurring at regularly spaced intervals.

[0060] Non-periodic: Occurring at non-regularly spaced
intervals.

[0061] Nanoparticle: Any manufactured, naturally, or
chemically produced structure or particle with nanometer-
scale dimensions (i.e., 1 to 100 nm).

[0062] Matrix: A total population of encoded sites for given
polymers or biopolymers with well defined encoded bind-
ing sites (e.g., nucleic acids, peptides, polymer chains with
chemically active groups). The same matrix might be used
for making different arrays depending what sites (on a
matrix) are chosen.

[0063] Array: A structure or architecture of compounds in
the form of an organized matrix that contains a specifically
encoded sites for binding of correspondingly encoded par-
ticles. The array is used to make specific arbitrary assem-
bly.

[0064] Row DNA: DNA that aligns on a surface along an
x-axis.

[0065] Column DNA: DNA that aligns on a surface along a
y-axis.

[0066] The present invention is directed to a method for the
by-design fabrication of arbitrary, non-periodic and periodic,
1D and 2D arrays of nano-objects of multiple types and
compositions. Any arbitrary 1D or 2D structure (array) can be
represented as a matrix with nano-objects positioned in pre-
designed sites, which positions are determined by their hori-
zontal (X) and vertical (Y) coordinates, and each position on
the matrix possesses some chemical, electrical, biological or
other functionality. The present invention can be used to
create any arbitrary 1D or 2D architecture from nano-objects
through the fabrication of a highly specific matrix.

[0067] The arrays and/or matrices of the present invention

can be attached to any surface that can bind arrays and/or

matrices without inhibiting or interfering with the array and/
or matrix structure. For example, the surface can be a solid
surface, a membrane, microscopic beads, a film, or any other
type of surface capable of binding a matrix and/or array. The
surface can be composed of any material, for example, glass,
silicon, silica, mica, metal, plastic, Polyvinylidene Fluoride

(PVDF), nitrocellulose, semiconductor, graphene or combi-

nations thereof. In a preferred embodiment, the surface is a

solid support made of silicon.

[0068] The matrix and/or array of the present invention can

be any chemical or compound that is capable of binding to a

surface and capable of binding to intercalators in a periodic

and non-periodic manner. For example, the matrix and/or
array may be comprised of small molecules or macromol-
ecules used alone or in combination. Examples of macromol-
ecules that can be used include nucleic acids (e.g., DNA,

RNA, and/or combinations thereof), amino acids (e.g., tradi-

tional and modified amino acids, peptides, proteins, amino

acid-nucleic acid hybrids, and/or combinations thereof); car-
bohydrates (e.g., monosaccharides, polysaccharides, oli-

gosaccharides, and/or combinations thereof); or lipids (e.g.,

fatty acids, glycerolipids, glycerophospholipids, sphingolip-

ids, sterol lipids, prenol lipids, saccharolipids, polyketides,
fats, waxes, sterols, fat-soluble vitamins, monoglycerides,
diglycerides, phospholipids, and/or combinations thereof).

[0069] In some embodiments of the present invention, the

matrix and/or array is prepared using nucleic acids. In a

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preferred embodiment, the matrix and/or array is prepared
using DNA. Naturally occurring and/or genetically engi-
neered DNAs of any sequence can be used to encode the
structure of the matrix and/or array. In a more preferred
embodiment, the DNA is lithographic ds-DNA, that is, linear,
ds-DNA designed with pre-determined sequences that pro-
vide specifically encoded locations for attachment of interca-
lators. The specifically designed regions for intercalator bind-
ing can be arbitrary, that is, periodic (separated at regular
intervals) or non-periodic (separated by non-regular inter-
vals).

[0070] The structure of the matrix and/or array is not lim-
ited to any pattern, shape, or size. In some embodiments, the
structure of the matrix and/or array is essentially linear or
one-dimensional. In other embodiments, the structure of the
matrix and/or array is non-linear or two-dimensional. In yet
other embodiments, the structure of the matrix and/or array is
three-dimensional.

[0071] Ina preferred embodiment, when the matrix and/or
array is 2D, the matrix and/or array is prepared by assembling
lithographic DNA into an XxY array containing one or more
than one row and one or more than one column, as illustrated
further below. Alternatively, lithographic DNA can be
arrange in non-linear or non-rectangular patterns, for
example, in circular-like, sinusoid-like, etc.

[0072] The matrix and/or array can be attached to the sur-
face, as described above, by a number of different specific or
non-specific methods. For example, the matrix and/or array
can be attached to the surface by covalent bonds, non-cova-
lent bonds, electrostatic interactions, protein-protein interac-
tion, DNA-DNA interaction, protein-nucleic acid interaction,
protein substrate interaction, and the like. In a preferred
embodiment, the DNA is bound through an anchoring point
via DNA-hybridization or biotin-streptavidin interaction.
[0073] _Intercalators are molecules or compounds that have
at least two ends. One end of the intercalator is capable of
recognizing and binding to specific locations on a matrix
and/or array. A second end of the intercalator is capable of
attaching to a bio-encoded nano-object. Intercalators of the
present invention can be any chemical or compound that is
capable of binding to a matrix and/or array on one end and to
a nano-object on another end. For example, the intercalators
may be comprised of small molecules or macromolecules
used alone or in combination. Examples of macromolecules
that can be used include nucleic acids (e.g., DNA, RNA,
and/or combinations thereof); amino acids (e.g., traditional
and modified amino acids, peptides, proteins, amino acid-
nucleic acid hybrids, and/or combinations thereof); carbohy-
drates (e.g., monosaccharides, polysaccharides, oligosaccha-
rides, and/or combinations thereof); or lipids (e.g., fatty acids,
glycerolipids, glycerophospholipids, sphingolipids, sterol
lipids, prenol lipids, saccharolipids, polyketides, fats, waxes,
sterols, fat-soluble vitamins, monoglycerides, diglycerides,
phospholipids, and/or combinations thereof). In a preferred
embodiment, the intercalator is a protein nucleic acid (PNA).
[0074] Nano-objects of the present invention are not lim-
ited to any type, shape, or size. Examples of nano-objects
include small and macromolecules used alone or in combi-
nation. Examples of macromolecules that can be used include
nanoparticles, nucleic acids (e.g., DNA, RNA, and/or com-
binations thereof); amino acids (e.g., traditional and modified
amino acids, peptides, proteins, amino acid-nucleic acid
hybrids, and/or combinations thereof); carbohydrates (e.g.,
monosaccharides, polysaccharides, oligosaccharides, and/or

May 30, 2013

combinations thereof); or lipids (e.g., fatty acids, glycerolip-
ids, glycerophospholipids, sphingolipids, sterol lipids, prenol
lipids, saccharolipids, polyketides, fats, waxes, sterols, fat-
soluble vitamins, monoglycerides, diglycerides, phospholip-
ids, and/or combinations thereof).

[0075] In a preferred embodiment, the nano-object is a
nanoparticle (NP). Examples of nanoparticles include metal-
lic (e.g.. gold, silver, platinum), semiconductive (e.g., CdSe,
CdTe, CdSeZnS), or magnetic (e.g., Fe,O,, FePt) nanopar-
ticles. Additionally, NPs can be of any shape, such as spheri-
cal, rod-shaped, icosahedral, planar, tubular, etc. As used
herein, unless otherwise noted, “particle” should be con-
strued to include micro-objects (including microspheres,
microrods, etc.) and nano-objects (fullerenes, quantum dots,
nanorods, nanotubes, etc.). In one embodiment the nanopar-
ticle is metallic. In a specific embodiment, the nanoparticle is
a gold nanoparticle (AuNP).

EXAMPLES,

[0076] The following examples and references to the fig-
ures should not be considered limiting in any way. General
materials and techniques are described; however, it should be
understood that variants of the disclosed materials,
sequences, and/or methods have been considered by the
inventors and are deemed as part of the invention.

Materials

[0077] DNA oligonucleotides were purchased from Inte-
grated DNA Technologies, Inc. (www.idtdna.com) as lyo-
philized powders. Unmodified and thiolated oligonucleotides
were purified by gel filtration chromatography. Sequences for
the DNA strands, which are also identified in FIG. 1 were:

A:
(SEQ ID No: 1)

5'-ATT GIT ATT AGC TCC ACG CCT TCT ACA TCT GAC GT-

T15-SH-3"'

Al:

(SEQ ID NO; 2)
5'-TGT AGA AGG CGT GGA GCT AAT AAC AAT-3'

(SEQ ID No: 3)
5'-HS-T1S-TTC AGA AGA GAT GTG-3'
200-bp ssDNA A:

(SEQ ID NO: 4)
5'-TCC GCA AGC TGG CCC TCA CTT CAA CGC ATT
ATT GIT AAT CTT CCA ATG GGC CAC CTA CCG TAG ACA
cGG ACT cT¢ TAC GCG TTA Tec cTc AGC
ATA TTA TTG TTA CTG CGG GAC ATA CGA TAG AGC TIT
GCT AAA ATA AGT CCC TGC CTT TCC ACC AAT AGA
AAT TAT TGT TAC GTA GCC AAT CGA CGT ATT TGG TAC
GT-3'
200-bp ssDNA A’

(SEQ ID NO: 5)

5'-ACG TAC CAA ATA CGT CGA TTG GCT ACG

TAA CAA TAA TTT CTA TTG GIG GAA AGG CAG GGA CTT

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US 2013/0137602 Al

-continued
ATT TTA GCA AAG CTC TAT CGT ATG TC¢ cec

AGT_AAC AAT AAT ATG CTG AGG CAT AAC GCG TAG AGA
GTC CGT GTC TAC GGT AGG TGG CCCATT GGA AGA

TIA ACA ATA ATG CGT TGA AGT GAG GGC CAGCTT GCG GA-
3"

[0078] Three identical anchoring positions are underlined,
which can be “invaded” by the PNA part of the PNA-DNA
chimera

[0079] PNA-DNA chimeras were synthesized and pur-
chased from Bio-Synthesis Inc. as lyophilized powders. The
chimeras can be further purified by HPLC techniques known
in the art. Sequences for the chimeras were:

(15 bp-DNA Chimera): A"-PNA-DNA-B'

(SEQ ID NOs: 6 & 7)
5'-TAA TAA CAA T-linker-T15-CAC ATC TCT TCT GAA-3'

(10-bp DNA Chimera): A"-PNA-DNA-B)':
(SEQ ID NO: 6 & 8)
5'-TAA TAA CAA-Linker-CAC ATC TCT T

[0080] The PNA is underlined and is written from N-C and
the DNA is written from 5'-3'. The linker is: cysteine-SMCC-
C6 amino.

Au Nanoparticle Synthesis

[0081] 10-nm Au nanoparticles were synthesized through a
classic citrate reduction method with slight modifications.
Briefly, 1 mM HAuCL, aqueous solution was first heated to
boil for 20-30 minutes. Subsequently, 10 mL of trisodium
citrate solution with a concentration of 38 nM was added to
the above solution. The reaction was allowed to continue until
the initial color changed to red, and quenched by deionized
water. After the Au nanoparticle solution cooled to room
temperature, it was stored in a glass bottle at ambient condi-
tion for further functionalized with DNA. The particle size
was examined by DLS and TEM and the concentration was
determined through UV-vis absorption at 4=519 nm with an

extinction coefficient of 1.0x10* L-mole™* cm’.

Functionalization of Au Nanoparticles

[0082] The thiol functionality of the DNA was deprotected
by the addition of 0.1 M dithiothretol (DTT) for at least 2 hrs
on ice prior to DNA loading (typically, 10-11 OD of concen-
trated DNA; 200 jl of DTT). The deprotected DNA solutions
were purified using desalting NAP-5 columns (Sephadex
G-25, Amersham Biosciences). Au nanoparticles were func-
tionalized with deprotected thiol-oligonucleotides following
methods for high DNA coverage reported by Mirkin and
co-workers (Mirkin, C. A., etal., Nature, 1996. 382(6592): p.
607-609; which is incorporated herein by reference). In a
typical experiment with 10 nm gold nanoparticles, an aliquot
(1-50 ul) ofa purified DNA 50-300 uM solution was added to
a 1 mL aliquot of gold particles (10-30 nM). The ssDNA and
particle solutions were incubated at room temperature in a
non buffered solution for at least 3 hr before adding phosphate
buffer to bring its concentration to 10 mM (pH=7.4). The
solution was left to anneal at 25° C. for 4 hr before the
addition of NaCl (0.025M). The salt concentration was then
increased gradually from 0.025 to 0.3 M NaCl over 24 hr, and

May 30, 2013

left to anneal for an additional 24 hr at 0.3M. The excess DNA
next was removed from the solutions by centrifugation for 30
minutes at 4,500 g.

Characterization of Aggregates and Trimers.

[0083] Dynamic Light Scattering (DLS):

[0084] DLS measurements were performed on a Malvern
Zetasizer ZS instrument. The instrument was equipped with 1
633 nm laser source and a backscattering detector at 173°.
[0085] Transmission Electron Microscopy (TEM):

[0086] TEM micrographs of DNA-functionalized Au NPs
and assembled aggregates and nanoclusters were collected
using a JEOL 1300 transmission electron microscope oper-
ated at 120 kV. Samples were prepared by placing a droplet of
the aqueous solution onto a 400-mesh carbon-coated copper
grid, followed by drying at room temperature for overnight
before imaging.

Example 1

PNA-Directed Assembly of Aggregates

[0087] A specific PNA-DNA chimera was used to direct the
formation of macroscopic aggregates of DNA functionalized
AuNPs, as depicted in FIG. 1A. Gold nanoparticles, 10 nm in
diameter, were functionalized with two types of non-comple-
mentary single-stranded (ss) DNA, A and B (A-AuNPs and
B-AuNP5), respectively (described above and shown in FIG.
1B). The molar concentration of AuNP probes were measured
by UV-vis spectroscopy (molar extinction coefficient 1.0x10*
M™ cm" at 524 nm). An equimolar concentration of DNA-
functionalized AuNPs (A-DNA AuNPs and B-DNA AuNPs)
were mixed with a 10-fold excess of A'-DNA, and A"-PNA-
DNA-B'. The solution was heated to 65° C. for 10 minutes,
and slowly cooled to room temperature in 0.1 M PBS (0.1M
sodium chloride, 10 mM sodium phosphate buffer, pH 7.0).
The aggregates were characterized without further purifica-
tion.

[0088] In this process, a tertiary complex is formed
between the A-DNA sequences on the A-AuNPs, comple-
mentary A'-DNA, and the A"-PNA-DNA-B' chimera. The
oligonucleotides on A-AuNPs partially hybridized to A'
through a 27-base-pair (bp) A-A' DNA-DNA sequence rec-
ognition. The higher affinity of PNA to SSDNA due to the
lack of charge of the PNA backbone, allow the A"-PNA
sequence of the A"-PNA-DNA-B' chimera to “invade” and
form a 10-bp duplex at the end of the A-DNA sequence
immobilized on the AuNP. Meanwhile, the B' strands in the
A"-PNA-DNA-B' chimera hybridize to B-AuNPs through a
15-bp B-B' DNA-DNA sequence recognition. In sucha fash-
ion, nanoparticle aggregates are formed between non-
complementary A-AuNPs and B-AuNPs through PNA-di-
rected assembly.

Aggregation of AuNPs Based on PNA Invasion of dsDNA.
[0089] PNA-directed aggregation of non-complementary
AuNPs was monitored using transmission electron micros-
copy (TEM) and dynamic light scattering (DLS) without any
further purification.

[0090] As an initial control, AUNPs and DNA functional-
ized AuNPs were evaluated in the absence ofa linker. FIG. 2A.
shows a representative TEM micrograph of DNA functional-
ized AuNPs. The TEM shows single nanoparticles that do not
assemble to form clusters. DLS profiles characterizing the
volume-averaged hydrodynamic diameter (Dh) population of

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the DNA functionalized AuNPs and control sample (non-
functionalized AuNPs) are shown in FIG. 2B. The DNA
functionalized AuNPs exhibit a single population at Dh=25
nm, which is shifted in comparison to the non-functionalized
AuNPs.

[0091] FIG. 3A shows a representative TEM micrograph,
illustrating the formation of nanoparticle clusters by an
embodiment of the present invention. Specifically, a linker
was added to AuNPs functionalized with DNA according to
the method described above, and in FIG. 1 (Similar to FIG.
2A, a control was also conducted without using the PNA-
DNA chimera linker (FIG. 3A inset) which again showed no
connection between nanoparticles). Statistical analysis based
on the TEM observations in FIG. 3A revealed that ~77% of
nanoparticles were assembled into larger aggregates (n—1035
particles) (FIG. 3B). DLS profiles characterizing the volume-
averaged hydrodynamic diameter (Dh) population of the
assembled aggregates and control sample are shown in FIG.
3C. The control sample exhibits a single population at Dh=25
nm, similar to the DNA-functionalized AuNPs (FIG. 2B).
However, the sample using the PNA “invasion” approach
shows an additional population at Dh=100-2000 nm, suggest-
ing the existence of the larger-scale aggregates. Moreover, a
statistical analysis based on the DLS profile reveals a yield of
82% of assembled aggregates, agreeing well with the TEM
analysis.

[0092] A similar experiment was also conducted using a
PNA-DNA chimera with 10-bp PNA and 10-bp DNA (A"-
PNA-DNA-B,') at 4° C. to compare with the experiments
performed at room temperature (the PNA-DNA chimera used
in the room temperature experiments has 10-bp PNA and
15-bp DNA). Nanoparticles also assembled into large aggre-
gates in this scenario (FIG. 4).

Quantitation of Hybridized PNA-DNA Chimeras

[0093] The extent of PNA-DNA chimera binding to
A-AuNPs was determined according to the method outlined
in (FIG. 5). Similar to the PNA-directed aggregation experi-
ments, A-AuNPs were mixed with a 10-fold excess of
complementary A'-DNA, A"-PNA-DNA-B' chimera, and
Cy3-DNA-B was used to replace B-AuNPs. The solution was
heated to 65° C. for 10 minutes and slowly cooled to room
temperature in 0.1M PBS. Unhybridized Cy3-DNA-B was
removed by centrifugation, and amount of hybridized DNA
was determined by fluorescence spectroscopy using a Varian
Fluorimeter. The change of fluorescence of the supernatant
shows that approximately 2-3 PNA-DNA chimeras bind per
nanoparticle (FIG. 5). The relative low efficiency of PNA
“invasion” indicates a low accessibility to the A-AUNP sur-
face, and supports the formation of the smaller macroscopic
aggregates observed in TEM images and DLS data

Melting Profiles

[0094] Duplex DNA structures formed between target
DNA and DNA on nanoparticles typically exhibit sharp melt-
ing profiles and increased melting temperatures in aggregate
assemblies compared to single complementary DNA strands.
The UV melting curve of a 1 uM solution of 15-bp DNA
duplex formed between A"-PNA-DNA-B' and B-DNA shows
a broad melting curve and the duplex melts with a Tm=48° C.
(FIG. 6A). Temperature dependent dynamic light scattering
was used to determine the melting transition of the PNA-
directed aggregate assemblies (FIG. 6B). Here, the concen-

May 30, 2013

tration of DNA is 200-fold less than in UV-vis melting experi-
ments. The melting transition observed is sharper than that
observed for the duplex in the absence of nanoparticles, how-
ever, the melting temperature of the nanoparticle-linked
15-bp DNA duplex (Tm=45° C.), is in agreement with that
obtained for the DNA duplex alone. The similar melting
temperatures indicate the temperature-dependent change in
size observed is due to thermal dissociation of the PNA-
directed aggregates.

[0095] This example demonstrates a new strategy to
assemble DNA-functionalized nanoparticles by the concept
of PNA “invasion” of dsDNA by specifically polymerizing
dsDNA-modified AuNPs into aggregates.

Example 2

PNA-Directed AuNP Assembly on dsDNA (PNA
“Invasion” for Trimer Formation)

[0096] The PNA “invasion” strategy was also used to
assemble AuNPs into well-defined nanoclusters along a
dsDNA template which is shown in (FIG. 7). A 200-bp ds-
DNA was designed that contains three identical 10-bp frag-
ments which are complementary to the 10-bp PNA part (A")
of the PNA-DNA chimera. 10-nm AuNPs were functional-
ized with assDNA B (B-AuNPs) that is complementary to the
15-bp DNA part (B’) of the PNA-DNA chimera. A mixture
was prepared containing 200-bp ssDNA A, 200-bp ssDNAA’,
A"-PNA-DNA-B', and B-AuNPs ina molar ratio of 1:1:3:3 in
0.1 M PBS. The mixture was then heated to 65° C. for 10
minutes, and cooled to room temperature for overnight. In
this process, the PNA-DNA chimera “invaded” the 200-bp
dsDNA duplex at the designed locations to create three
anchors, and then the DNA-functionalized AuNPs can recog-
nize these anchors on the dsDNA duplex through DNA-DNA.
base-pairing hybridization. In this manner, nanoparticle tri-
mers assembled along the dsDNA duplex.

[0097] The assembled nanoparticle trimers were character-
ized by TEM and DLS. The TEM image in FIG. 8A reveals a
mixture of single particles, dimers, trimers, and larger clus-
ters from the sample. The circles around the clusters indicate
assembled trimers. FIG. 8B illustrates three possible configu-
rations of assembled trimers along a dsDNA on a flat surface
(top: schematic; bottom: TEM images). A statistical analysis
based on the TEM observation (FIG. 8C) suggests that the
sample contained 40% of single nanoparticles, 20% of
dimers, 22% of trimers and 18% of larger clusters (4-10
particles). DLS profiles of the assembled and control (without
adding PNA-DNA chimera) solutions in FIG. 8D also dem-
onstrate the formation of nanoparticle clusters by the PNA
“invasion”. A statistical analysis on the DLS result suggests a
yield of 58% nanoparticle clusters, which was consistent with
the TEM analysis (60% in total for dimers, trimers, and larger
clusters).

[0098] The impurities that result from the design can be
attributed to several factors. The formation of larger clusters
may be due to the fact that after linked to the PNA-DNA
anchor on one dsDNA duplex, the surface of the DNA-func-
tionalized AuNPs has not been passivated so that they can also
hybridize with other dsDNA duplexes “invaded” by the PNA-
DNA chimera. Therefore, larger nanoparticle clusters are
formed using the present invention. The presence of single
nanoparticles and dimers, could be due to the “invasion”
efficiency of the PNA-DNA chimera into the 200-bp dsDNA
duplex which were demonstrated in Example 1.

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US 2013/0137602 Al

[0099] This example demonstrates a new strategy to
assemble DNA-functionalized nanoparticles by the concept
of PNA “invasion” of dsDNA by specifically organizing
ssDNA-functionalized AuNPs along dsDNA duplex.

Example 3

Formation of an Individual Row of a 1D Array

[0100] An individual row of a matrix can be fabricated in
the manner shown in FIGS. 9-11 using a lithographic DNA.
The steps described can be performed in any order. In a
preferred embodiment, the steps are performed as set forth
below.

[0101] First, ds-DNA is deposited on a surface containing
an anchoring point (FIG. 9). The anchoring point can include
any fabricated nano-structure, nanoparticle, or surface fea-
ture capable of binding to a DNA end. The DNA/anchoring
point binding can be non-specific (thiol, silaine, etc.) or spe-
cific (e.g., DNA-hybridization, biotin-streptavidin, etc).
Lithographic DNA should be designed or chosen from natural
or genetic material so that the DNA sequences are known.
Specific pre-determined DNA regions, called intercalator
binding sites, are located along the lithographic ds-DNA for
nano-object attachment (FIG. 9, bottom panel, locations
identified as X,, X;, etc.). The specific uniqueness of each
intercalator binding site is determined by DNA base pairs
(bp) sequence and nucleotide length. All specific intercalator
binding sites can be pre-determined and encoded via by
sequences. The length of the intercalator binding sites can be
any length that allows efficient intercalator binding. In some
embodiments the intercalator binding site comprises 12-15
bp, which provides a robust encoding and sufficient thermal
stability for intercalator/nano-object attachments.

[0102] In the next step (FIG. 10), intercalators with a spe-
cific recognition to the intercalator binding sites on the matrix
are added. The intercalators are capable of recognizing and
specifically binding to the intercalator binding sites. The
intercalators bind to the matrix at one end, and the unbound
end is free to serve as an encoded recognizable attachment
site for complementary encoded nano-object.

[0103] In some embodiments, the intercalator is a protein,
DNA, or RNA. In preferred embodiments, the intercalator is
a single stranded peptide nucleic acid (PNA) chain or a PNA-
DNA chimera. A ss-PNA molecule can specifically interact
with ss-DNA using Watson-Crick base pairing. The absence
of charge on ss-PNA peptide backbone results in a stronger
interaction between complementary sequences of ss-DNA
and ss-PNA compared to ss-DNA/ss-DNA case. Single
stranded PNA has the ability to interact with ds-DNA, which
results ina local de-hybridization and PNA intercalation. This
phenomenon is known as PNA invasion (LOHSE, J., et al,
“Double duplex invasion by peptide nucleic acid: A general
principle for sequence-specific targeting of double-stranded
DNA”, PNAS, 96(21):11804-11808, (1999)). When a PNA-
DNA chimera is added to a lithographic ds-DNA, the PNA
end will bind to the intercalation binding site and the ss-DNA
end will serve as a recognition site for nano-object containing
a complementary functionalized strand.

[0104] In the next step (FIG. 11), various types of nano-
objects that encode nucleic acid strands complementary to the
nano-object binding site on the intercalator are added. The
nano-objects then recognize and specifically interact with the
nano-object binding site on the intercalator, which results ina
self-assembly of a 1D structure according to the instruction

May 30, 2013

provided by a lithographic DNA and intercalators. Alterna-
tively, intercalators can be directly embedded with or bound
to nano-objects. A design or choice of specific sites on DNA
allows for arbitrary placement of various types of nano-ob-
jects on a DNA row through the intercalators.

[0105] The accuracy of nano-object positioning can be
determined by a base-pair formation and by nucleic acid
chain flexibility at the attachment site. Base-pairs have a
fraction of nanometer of co-localization precision, while
chain flexibility can be minimized to several bases. Together
this will provide 1-2 nm precision of positioning with mini-
mum distances between sites on an order of 2-5 nm. The
minimum distance between sites is determined by the length
of PNA-DNA invasion region. The use of other, stronger
binding intercalators may allow reducing minimum site-site
separation to 1-2 nm. The use of designed and genetic DNA
allows for a precise positioning of nano-objects at least on the
scale of tens of microns, which allow for assembly of thou-
sands of objects in one row. The distance between nano-
objects can be between about 100 nm to 1 mm, preferably
between about 1 to about 100 microns, and more preferably
between about 3 to about 20 microns.

[0106] The DNA can be aligned in order to minimize its
large scale bends. The DNA straightening step can be per-
formed at any stage. In a preferred embodiment, the DNA is
straightened after all nano-objects are assembled on litho-
graphic ds-DNA.

[0107] The DNA can be straightened using a fluid flow, an
electric field, or by optical tweezers (ALLEMAND, J. F., et
al, “Stretching DNA and RNA to probe their interactions with
proteins”, Current Opinion in Structural Biology, 13:266,
(2003); which is incorporated herein by reference). In a pre-
ferred embodiment, the straightening is performed using the
fluid flow method.

Example 4

Formation of Multiple Individual Rows of a 2D
Array

[0108] Multiple rows of a 2D array can be fabricated in the
manner shown in FIG, 12 using a lithographic DNA. The
steps described can be performed in any order. In a preferred
embodiment, the steps are performed as set forth below.

[0109] Using multiple anchoring points and following a
similar approach as described above, 2D arrays can be also
fabricated (FIG. 12, top). Regular, periodic and non-periodic
1D patterns can be fabricated using anchoring points aligned
in one line with designed separation (Dy) or shifted relative
each other (Dx) (FIG. 12, top). The placement of anchoring
points using traditional lithographic methods can be per-
formed with tens of nm precision routinely. This is typically
done using e-beam writer that burn (“write”) a defined area in
a resist polymer layer in the pre-determined positions. In the
next step material (typically metal, like gold) is deposited on
asurface, and then a polymer layer is removed. This leaves the
metal (gold) deposited spot, which is used as anchoring point.
[0110] This technique allows for programmable fabrication
of large DNA encoded arrays containing a large number (up
to about a million or more) of various nano-objects using only
very simple fabrication of a relatively small number (about
one hundred to about one thousand) of identical anchoring
sites. These arrays can be on the scale of tens of microns in
size. The size of arrays is determined by the choice of DNA.
Tt can range from tens on nanometers to hundred of microns,

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US 2013/0137602 Al

or may be even larger. The preferable scale from character-
ization perspective and integration with other technologies is
few microns and more. The upper limit is probably deter-
mined only by computation power required to choose suitable
attachment sites and by easy available DNA. This approach is
highly suitable for deposition of similar lithographic DNA if
the same binding motif is used for attachment to anchoring
points. Additionally, the specificity of interactions between
the anchoring point and a DNA end can be designed thereby
allowing multiple types of lithographic DNA to be used. For
example, this can be accomplished by using DNA and pro-
teins which allows for fabrication of significantly more com-
plex structures due to incorporation at various DNA “rows”.
[0111] Inasubsequent step (FIG. 12, bottom), intercalators
can be added which recognize intercalator binding sites on
the row DNA. This can then be followed by nano-objects
binding and DNA alignment as discussed previously.

[0112] An advantage of the method (in particular FIG. 12,
top and bottom) is the absence of any kind of conventional
nano-fabrication except for fabrication of a first anchoring
point. This method permits a fabrication of arbitrary place-
ment of a large number of multiple types of nano-objects on
at least tens of micron array with nm level-precision.

[0113] The DNA can be aligned in order to minimize its
large scale bends. The DNA straightening step can be per-
formed at any stage. In a preferred embodiment, the DNA is
straightened after all nano-objects are assembled on litho-
graphic ds-DNA.

[0114] The DNA can be straightened using a fluid flow, an
electric field, or by optical tweezers (ALLEMAND, J. F., et
al, “Stretching DNA and RNA to probe their interactions with
proteins”, Current Opinion in Structural Biology, 13:266,
(2003); which is incorporated herein by reference). In a pre-
ferred embodiment, the straightening is performed using the
fluid flow method.

Example 5

Formation of a 2D Matrix Comprising a Column and
Rows

[0115] A 2D matrix comprising a column and rows can be
fabricated in the manner shown in FIGS. 13-15 using a litho-
graphic DNA. The steps described can be performed in any
order. In a preferred embodiment, the steps are performed as
set forth below.

[0116] Specifically, in an embodiment of the present inven-
tion, the need for fabrication multiple anchoring points (i.e.,
multiple individual rows of DNA attached by anchoring
points) is eliminated. This design allows for a full scale 2D
matrix formed by self-assembly. In this embodiment, the
positioning of the individual DNA rows can be encoded by an
appropriate choice of a column DNA (FIG. 13).

[0117] In the first step (FIG. 13), an initial lithographic
ssDNA having a pre-designed sequence is vertically aligned
with one end attached to the surface through an anchoring
point and the other end attached through a fixation point. The
fixation points can be chemically different from the anchor-
ing points. Additionally, the termination sites on DNA that
responsible for attachment to the points can also be chemi-
cally different. In some embodiments, it is not necessary to fix
the second end since after straightening and drying the DNA
is immobilized on the surface. In a specific embodiment, the
full 2-side fixation is utilized when other in-liquid manipula-
tions will be performed, for example, adding some perpen-

May 30, 2013

dicular DNA lines. The initial lithographic ssDNA forms the
first column of the matrix. Different regions of the column
DNA encode positions (i.e., pre-designed sequences) where
row DNA will later be attached using intercalators (e.g.,
PNA).

[0118] In some embodiments, the ends of row DNA are
complementary (sticky) to the free tails intercalators that are
attached to the column DNA. The row DNA will entropically
recognize the correct positions on the column by binding to
specific intercalators through Watson-Crick interactions due
to the presence of the ssDNA end at the termination, which
can be achieved either via intercalators with free ss DNA ends,
or by biochemical cleavage DNA end. The column DNA can
also contain intercalators with free ssDNA ends which are
complimentary to those on row DNAs. Finally, intercalators
and encoded nano-objects (e.g., nanoparticles) are introduced
and find their programmed placed on row DNAs, whereby
arbitrary matrix of nanoparticles is formed.

[0119] In a preferred embodiment, an initial lithographic
DNA is attached to anchoring point with one end and a
fixation point at the other end to form the column DNA (FIG.
13). The DNA can be either single stranded or double
stranded. In a preferred embodiment the DNA is double
stranded. In the next step (FIG. 14, top panel), an assembly of
intercalators bind to the intercalator binding sites on the litho-
graphic DNA at the pre-designed locations. Then, a set of
various ds-DNAs, which contain either the same or different
1D positional encoding sites, is added (FIG. 14, bottom
panel). One end of each of these row DNAs indirectly
attaches to the column DNA by directly binding to the nano-
object binding site on the intercalator, for example via PNA
invasion. This results in assembly of 2D arbitrary matrix of
DNA encoded binding sites. The position and binding speci-
ficity of the row DNA are also determined by design, as
described previously. The accuracy of placement of row
DNAs is similar to or less than the accuracy of nano-objects,
as described previously.

[0120] Ina subsequent step (FIG. 15), additional intercala-
tors can be added to the 2D array which recognize intercalator
binding sites on the row DNA. The addition of specific inter-
calators, as discussed before in FIGS. 10 and 12, will allow
for precise placement of multiple types of nano-objects on the
row DNA. A simultaneous alignment of all row DNA with
attached nano-objects can be performed at the final stage
using known methods, as discussed above (e.g., FIG. 11).
This will result in the formation of arbitrary arrays with
various nano-objects on a fully designed architecture.
[0121] The DNA can be aligned in order to minimize its
large scale bends. The DNA straightening step can be per-
formed at any stage. In a preferred embodiment, the DNA is
straightened after all nano-objects are assembled on litho-
graphic ds-DNA.

[0122] The DNA can be straightened using a fluid flow, an
electric field, or by optical tweezers (ALLEMAND, J. F., et
al, “Stretching DNA and RNA to probe their interactions with
proteins”, Current Opinion in Structural Biology, 13:266,
(2003); which is incorporated herein by reference). In a pre-
ferred embodiment, the straightening is performed using the
fluid flow method.

[0123] It will be appreciated by persons skilled in the art
that the present description is not limited to what has been
particularly shown and described in this specification. Rather,
the scope is defined by the claims which follow. It should
further be understood that the above description is only rep-

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US 2013/0137602 Al

resentative of illustrative examples of embodiments. For the
reader’s convenience, the above description has focused on a
representative sample of possible embodiments, a sample that
teaches the principles of the present invention. Other embodi-
ments may result from a different combination of portions of
different embodiments. The description has not attempted to
exhaustively enumerate all possible variations. That alternate
embodiments may not have been presented for a specific
portion of the invention, and may result from a different

May 30, 2013

combination of described portions, or that other undescribed
alternate embodiments may be available for a portion, is not
to be considered a disclaimer of those alternate embodiments.
It will be appreciated that many of those undescribed embodi-
ments are within the literal scope of the following claims, and
others are equivalent. Furthermore, all references, publica-
tions, U.S. patents, and U.S. patent Publications cited
throughout this specification are incorporated by reference in
their entireties as if fully set forth in this specification.

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 8

<210> SEQ ID NO 1

<211> LENGTH: 50

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence
<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide
<220> FEATURE:

<221> NAME/KEY: misc feature

<222> LOCATION: (50}).. (50)

<223> OTHER INFORMATION: Thiolated

<400> SEQUENCE: 1

attgttatta getccacgee ttctacatct gacgtttttt tttttttttt 50

<210> SEQ ID NO 2

<211> LENGTH: 27

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence
<220> PEATURE:

<223> OTHER INFORMATION: Oligonucleotide

<400> SEQUENCE: 2

tgtagaagge gtggagctaa taacaat 27
<210> SEQ ID NO 3

<211> LENGTH: 30

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)..(1)

<223> OTHER INFORMATION: Thiolated

<400> SEQUENCE: 3

tttttttttt tttttttcag aagagatgtg 30
<210> SEQ ID NO 4

<211> LENGTH: 200

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide

<400> SEQUENCE: 4

teogeaaget ggceetcact teaacgeatt attgttaate ttccaatggg ecacetaceg 60

tagacacgga ctctctacge gttatgectc agcatattat tgttactgcg ggacatacga 120

tagagetttg ctaaaataag tecctgectt tecaccaata gaaattattg ttacgtagee «180

aategacgta tttggtacgt

200

Page 26

US 2013/0137602 Al

May 30, 2013

-continued

<210> SEQ ID NO 5
<211> LENGTH: 200

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence
<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide

<400> SEQUENCE: 5

acgtaccaaa tacgtegatt ggctacgtaa caataatttc tattggtgga aaggcaggga 60

cttattttag caaagetcta tegtatgtcc cgcagtaaca ataatatget gaggcataac = _-:120

gegtagagag tecgtgtcta eggtaggtgg cccattggaa gattaacaat aatgcgttga 180

agtgagggee agettgegga

<210> SEQ ID NO 6
<211> LENGTH: 10

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence
<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide

<400> SEQUENCE: 6

taataacaat

<210> SEQ ID NO 7
<211l> LENGTH: 30

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence
<220> FEATURE:

<223> OTHER INFORMATION: Oligonucleotide

<400> SEQUENCE: 7

trtttttttt tttttcacat ctcttctgaa

<210> SEQ ID NO 8
<211> LENGTH: 10
<212> TYPE: DNA
<213> ORGANISM: Artificial Sequence
<220> FEATURE
<223> OTHER INFORMATION: Oligonucleotide

<400> SEQUENCE: 8

cacatetett

200

10

30

10

1, An array comprising:

a surface having an anchoring point;

a strand of nucleic acids attached to the surface at the
anchoring point;

an intercalator; and

a nano-object,

wherein one end of the intercalator binds to a specific
sequence on the strand of nucleic acids anda second end
of the intercalator binds to the nano-object.
2. The array according to claim 1, wherein the surface is a
solid support made of silicon.
3. The array according to claim 1, wherein the anchoring,
point is a nucleic acid sequence, biotin, or streptavidin.
4, The array according to claim 1, wherein the strand of
nucleic acids is DNA.

5. The array according to claim 1, wherein the strand of
nucleic acids is a lithographic DNA.

6. The array according to claim 1, wherein the intercalator
is astrand ofnucleic acids, a protein, an organic compound, or
a combination thereof.

7. The array according to claim 1, wherein the intercalator
is a PNA-DNA chimera.

8. The array according to claim 1, wherein the nano-object
is a nanoparticle, nanohorn, nanotube, or nanosphere.

9. The array according to claim 1, wherein the nano-object
is a DNA-functionalized gold nanoparticle.

10. The array according to claim 1, wherein the surface is
made of silicon, the strand of nucleic acids is a lithographic
DNA, the intercalator is a PNA-DNA chimera, and the nano-
object is a DNA-functionalized nanoparticle.

11. A method for assembling nano-objects on the array
comprising:

Page 27

US 2013/0137602 Al

preparing an array that comprises a surface having an

anchoring point;

binding a strand of nucleic acids to the anchoring point on

the surface; and

attaching a nano-object to a specific sequence on the strand

of nucleic acids through an intercalator;

wherein one end of the intercalator binds to a specific

sequence on the strand of nucleic acids anda second end
of the intercalator binds to the nano-object.

12. The method according to claim 11, wherein the surface
is made of silicon, the strand of nucleic acids is a lithographic
DNA, the intercalator is a PNA-DNA chimera, and the nano-
object is a DNA-functionalized nanoparticle.

13. The method according to claim 11, wherein the anchor-
ing point is DNA and the strand of nucleic acids is bound to
the anchoring point through DNA-DNA hybridization.

14. The method according to claim 11, wherein the strand
of nucleic acids is bound to the surface by biotin-streptavidin
interaction, thiointeration, or nucleic acid hybridization.

11

May 30, 2013

15. The method according to claim 11, wherein more than
one nano-object is bound to the strand of nucleic acids at
periodic or non-periodic intervals.

16. The method according to claim 11, wherein the array is
1D or 2D.

17. The method according to claim 11, wherein the surface
is a solid support made of silicon.

18. The method according to claim 11, wherein the anchor-
ing point is a nucleic acid sequence, biotin, or streptavidin.

19. The method according to claim 11, wherein the inter-
calator is a strand of nucleic acids, a protein, an organic
compound, or a combination thereof.

20. The method according to claim 11, wherein the nano-
object is a nanoparticle, nanohorn, nanotube, or nanosphere.

21. The method according to claim 11, wherein the nano-
object is a DNA-functionalized gold nanoparticle.

ek Rk