US8729012

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.

Page 1

a2) United States Patent
Lee et al.

US008729012B2

US 8,729,012 B2
May 20, 2014

(10) Patent No.:
(45) Date of Patent:

(54) CONTROLLABLE ASSEMBLY AND
DISASSEMBLY OF NANOPARTICLE
SYSTEMS VIA PROTEIN AND DNA AGENTS

(75) Inventors: Soo-Kwan Lee, Seoul (KR); Oleg
Gang, Setauket, NY (US); Daniel van
der Lelie, Shoreham, NY (US)

(73) Assignee: Brookhaven Science Associates, LLC,
Upton, NY (US)

(*) Notice: — Subject to any disclaimer, the term of this

patent is extended or adjusted under 35

U.S.C. 154(b) by 542 days.

(21) Appl.No.: — 12/995,919

(22) PCT Filed: Jun. 2, 2009

(86) PCT No.: PCT/US2009/045983
§371 (0),
(2), (4) Date: Apr. 25, 2011

(87) PCT Pub. No.: WO2009/149091
PCT Pub. Date: Dee. 10, 2009

(65) Prior Publication Data
US 2011/0196130Al — Aug. 11, 2011
Related U.S. Application Data

(60) Provisional application No. 61/058,037, filed on Jun.
2, 2008.

(51) Int. Cl.
CO7K 14/00
(52) U.S. Cl.
USPC + 514/2; 424/491; 977/728
(58) Field of Classification Search
None
See application file for complete search history.

(2006.01)

(56) References Cited
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7,045,285 BL 5/2006 Kayyem et al.

2002/0172953 AL
2003/0198956 Al
2003/0215903 AL
2005/0130174 AL
2007/0054337 Al
2007/0134420 Al

11/2002 Mirkin et al.

10/2003 Makowski et al.

11/2003 Hyman et al.
6/2005 Bao etal.
3/2007 Ferning et al.
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(Continued)

Primary Examiner — Anand Desai
(74) Attorney, Agent, or Firm — Dorene M. Price; Lars O.
Husebo

67) ABSTRACT

The invention relates to the use of peptides, proteins, and
other oligomers to provide a means by which normally
quenched nanoparticle fluorescence may be recovered upon
detection of a target molecule. Further, the inventive technol-
ogy provides a structure and method to carry out detection of
target molecules without the need to label the target mol-
ecules before detection. In another aspect, a method for form-
ing arbitrarily shaped two- and three-dimensional protein-
mediated nanoparticle structures and the resulting structures
are described. Proteins mediating structure formation may
themselves be functionalized with a variety of useful moi-
eties, including catalytic functional groups.

13 Claims, 16 Drawing Sheets

Page 2

US 8,729,012 B2
Page 2

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* cited by examiner

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1
CONTROLLABLE ASSEMBLY AND
DISASSEMBLY OF NANOPARTICLE
SYSTEMS VIA PROTEIN AND DNA AGENTS

This application is a national phase entry under 35 U.S.C.
371 of International Application Number PCT/US2009/
045983, filed on Jun. 2, 2009, entitled “CONTROLLABLE
ASSEMBLY AND DISASSEMBLY OF NANOPARTICLE
SYSTEMS VIA PROTEIN AND DNA AGENTS”, which
claims priority to U.S. Provisional Patent Application Num-
ber 61/058,037, filed on Jun. 2, 2008, all of which are hereby
incorporated by reference.

This invention was made with Government support under
contract number DE-AC02-98CH10886, awarded by the
US. Department of Energy. The Government has certain
rights in the invention.

BACKGROUND OF THE INVENTION

The invention relates to the use of peptides, proteins, and
other oligomers to provide a means by which normally
quenched nanoparticle fluorescence may be recovered upon.
detection of a target molecule. Further, the invention provides
a structure and method to carry out detection of target mol-
ecules without the need to label the target molecules before
detection.

Nanotechnology research and its findings have been stud-
ied in many different research areas and are being applied for
the development of scientific and industrial technologies,
such as nano electronics, sensors, and catalysts. Frontier
energy, medical, and security-related nanotechnology will
depend on the integration and optimization of nanoparticle-
based technologies and biological sciences to design hybrid
materials for increasingly cleaner and more efficient energy
conversion and storage, as well as biological sensors having
increased sensitivity. Such integration requires not only the
precise control of nanoparticle size, shape, composition, and
surface properties, but also the ability to self-assemble and
dissociate nanoparticles with controlled kinetics and final
assembly morphology under conditions tolerated by biologi-
cal systems.

Currently, the self-assembly of nanoparticles with metallic
(Au, Ag, Pt), semiconductive (CdSe, CdS, ZnS, GaAs), and
magnetic (Fe,O5) properties using biological building blocks
is achieved by two main approaches: (i) DNA-based systems
and (ii) protein- (peptide-) based systems. The remarkable
specificity and programmable interactions of DNA allows
self-assembly of DNA-conjugated nanoparticles and con-
struction of complex architectures. Their complexity and

functionality may be extended via the incorporation of pro- 5

teins that function as biological sensors or as organizers of
complex scaffolds. The DNA-based self-assembly systems
have shown great potential as biological sensors. Specific
binding of peptides to inorganic surfaces has been demon-
strated, and these peptides can be selected by using phage
display system. Also, the peptides have been successfully
used for the construction of nanostructures and self-assembly
of inorganic nanoparticles. Due to the functional variety of
proteins, the combination of DNA- and protein-based sys-
tems is receiving considerable attention to design functional
hybrid nanomaterials.

The self-assembly of nanoparticles and programmed com-
plex architectures using DNA have been demonstrated.
DNA-induced self-assembly of nanoparticles was first intro-
duced in 1996 by pioneering papers of Mirkin and co-work-
ers, “A DNA-based method for rationally assembling nano-
particles into macroscopic materials,” Nature, 382(6592): p.

S

a

w
8

w

8

40

2

607-609, 1996, and Alivisatos and co-workers, “Organization
of ‘nanocrystal molecules’ using DNA,” Nature, 382(6592):
p. 609-611, 1996, both of which articles are hereby incorpo-
rated by reference in their entirety. The ability of specific
hybridization of DNA was utilized for self-assembly of nano-
particles on which single stranded DNA (ssDNA) is chemi-
cally immobilized. Aggregation was obtained by adding a
single stranded linker DNA whose ends were complementary
to the ssDNAs conjugated to the particles. The aggregation of
ssDNA conjugated nanoparticles is accompanied by changes
of physical and optical properties, and was applied to detect
DNA. Addition of a DNA fragment complementary to the
linker results in dissociation of the aggregates. Alternatively,
the system can be designed such that the DNA fragments to be
detected function as the linker. The melting properties of
DNA, which depend on sequence and length, make it possible
to reverse the self-assembly of the aggregates by increasing
temperatures (FIG. 1). The sequence-dependent melting
properties allow detection of single point mutation using
ssDNA-conjugated nanoparticles.

Gold-nanoparticle quenched fluorescent oligonucleotides,
that are designed for complementary binding at their 3' and 5'
ends to form a hairpin structure, have been used as molecular
beacons for detecting target DNA that hybridizes to the hair-
pin structure, resulting in emission of the quenched fluores-
cence,

Microarray technology is used on a routine basis for high
through-put quantification of large numbers of different DNA
or RNA fragments. The array-based systems require labeling
of target DNA, or RNA, via synthesis of C-DNA. ssDNA-
conjugated nanoparticles have been used as candidates to
probe their target molecules.

In addition, the specific interaction of DNA has been used
to create DNA building blocks, and assembly of the building
blocks to construct sophisticated geometries and morpholo-
gies.

SUMMARY

While the above mentioned systems require hybridization
of complementary DNA strands as a prerequisite for correct
assembly, and disassembly is obtained by melting of the DNA
at elevated temperatures, the processes will often result in the
irreversible inactivation of functional moieties, such as pro-
teins. The inventive method allows for controllable assembly
of non-complementary single stranded DNA-(ssDNA-) con-
jugated nanoparticles, using the gene 5 protein (g5p) as a
molecular “glue” binding to two anti-parallel ssDNA strands.
Control of assembly kinetics of particle aggregates can be
obtained via sequence-specific hybridization with comple-
mentary ssDNA (C-ssDNA), while the size of aggregates is
controlled by adjustment of the g5p concentration. The con-
trollable disassembly of the g5p-ssDNA complex may be
triggered by hybridization with C-ssDNA, which allows
regulation of assembly kinetics and effective decom-position
of particle aggregates at room or physiological temperatures.
In addition, the g5p protein allows for the construction of
functional, recombinant derivatives that contain poly-histi-
dine or other affinity tags that allow these proteins to specifi-
cally bind to surfaces of nanoparticles. This allows control of
bothassembly and disassembly of nanoparticles without ther-
mal treatment, and easy incorporation of proteins into DNA-
based nanostructures, conferring the potential to design com-
plex nanomaterials.

The invention relates to the use of peptides, proteins, and
other oligomers to provide a means by which normally
quenched nanoparticle fluorescence may be recovered upon

Page 21

US 8,729,012 B2

3

detection of a target molecule. Further, the inventive technol-
ogy provides a structure and method to carry out detection of
target molecules without the need to label the target mol-
ecules before detection. In another aspect, a method for form-
ing arbitrarily shaped two- and three-dimensional protein-
mediated nanoparticle structures and the resulting structures
are described. Proteins mediating structure formation may
themselves be functionalized with a variety of useful moi-
eties.

In some embodiments of the invention, a controllable and
reversible assembly of nanoparticles using the gSp protein or
similar DNA binding proteins is provided. In some variants,
the nanoparticles are encapsulated in DNA and/or RNA and.
the nanoparticles are assembled with a nucleotide binding
protein. In other variants, the nanoparticles are encapsulated
with a nucleotide binding protein, and the nanoparticles are
assembled with DNA and/or RNA. Some embodiments pro-
vide a multi-dimensional structure comprising branches of
single stranded DNA and/or RNA, nucleotide binding pro-
tein, and nanoparticles. Some embodiments of the invention
also provide a method or process for preparing a controllable
and reversible assembly of nanoparticles using a nucleotide
binding protein.

Other embodiments of the invention provide a nucleic acid
binding protein-mediated DNA assemblage that comprises
non-complementary DNA or RNA strands bound to a nucle-
otide binding protein.

Still other embodiments of the invention provide a molecu-
lar switch which comprises a fluorescent quenched “off”
position anda fluorescent emission “on” position, comprising
ssDNA bound to fluorescein and a second ssDNA bound to
DABCYL. The switch can be turned “on” by hybridizing a
complementary ssDNA or ssRNA to either the fluorescein-
ssDNA strand or the ssDNA-DABCYL strand.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a prior art method for self-assembly and
disassembly of ssDNA-conjugated nanoparticles.

FIG. 2 depicts a controllable g5p-mediated assembly and
disassembly technique.

FIG. 3 shows a sketch of ssDNA-mediated assembly and
disassembly of g5p-conjugated nanoparticles.

FIG. 4 depicts the functioning ofa g5p-mediated molecular
switch.

FIG. 5 shows self-assembly of nanoparticles at ordered
peptide-conjugated g5p and DNA.

FIGS. 6A and 6B show the results of electrophoretic
mobility shift assays.

FIGS. 7A, 7B, and 7C are data from g5p-mediated assem- 5

bly of ssDNA-conjugated gold particles.

FIGS. 8A and 8B show results of dynamic light scattering,
and ultraviolet-visible analyses of ssDNA-conjugated gold
particles.

FIGS. 9A and 9B show a schematic of a g5p protein-
mediated assembly of ss DNA-conjugated gold particles, and
changes of the hydrodynamic diameter value of such assem-
blies, respectively.

FIGS. 10A, 10B, and 10C depict the decomposition of
g5p-mediated ssDNA-conjugated gold particle aggregates.

FIG. 11 shows the effect of the addition of C-ssDNA on
gold particle aggregates.

DETAILED DESCRIPTION

Incontrast to DNA, proteins have not only specific binding
properties, but also abundant catalytic functional properties.

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The interactions of protein and peptide have been utilized for
self-assembly and ordering of nanoparticles. Peptides that
have specificity for inorganic compounds (metallic, semicon-
ductive, and magnetic compounds) have been selected using
peptide libraries, and have been used successfully for self-
assembly of inorganic nanoparticles and biomineralization.
Noting the specific interactions between peptides and inor-
ganic surfaces, the inventors suggest that the inorganic nano-
particles may be linked with proteins by genetic engineering
of proteins to display peptides.

The combination of the DNA- and protein-based systems is
attractive for extension of their applicability as biosensors or
organizers of complex scaffolds. Biotin-conjugated DNA has
been utilized as a building block for assembly by binding to
streptavidin that can cross-link the biotin-conjugated DNA.
Antibodies have been used for protein-assisted self-assembly
of DNA-conjugated nanoparticles that contain the antibod-
ies’ target molecules.

Despite these advances in using both DNA and proteins for
the construction of nano-scale materials, there are still many
limitations and technical hurdles. DNA requires the proper
conditions for the desired hybridization. There are inherent
limitations of DNA hybridization such as G-C content, salt
concentration, and temperature. The distribution of proteins
in the composed structure allows for identical functionality
throughout the entire structure with control of kinetics and
size of assemblies only when homogenous. During self-as-
sembly of ssDNA-conjugated nanoparticles induced by
DNA-hybridization, it is difficult to introduce proteins with-
out chemical modification of the oligonucleotides, e.g. bioti-
nylation, to allow for binding of the protein. The increased
temperature can induce irreversible inactivation of biological
molecules, especially proteins. Disassembly of the aggre-
gates formed by DNA hybridization requires increases of
temperature above the melting temperature, often around 55°
C. or above, which can cause limitations of the system to be
used in biological environments. To detect target molecules
(DNA, RNA) using array-based systems, labeling is a critical,
costly, and time-consuming step. Labeling of target mol-
ecules requires additional time for sample preparation, limit-
ing quick detection or diagnosis of biological molecules.

Using the gene 5 protein (g5p) as a molecular “glue” bind-
ing two anti-parallel non-complementary singled stranded
DNA (ssDNA) overcomes many of the limitations for fabri-
cating nanoscale materials by combination of DNA and pro-
teins mentioned above. Additional hybridization with
complementary ssDNA (C-ssDNA), which triggers the g5p-
ssDNA complex to dissociate, may also be used.

An aspect of the invention provides for controllable and
reversible assembly of nanoparticles and methods for prepar-
ing the same.

In some embodiments of the invention, the nanoparticle is
metallic. In preferred embodiments, the metal is gold, silver,
or platinum. In more preferred embodiments, the nanopar-
ticle is gold. In other embodiments, the nanoparticle is a
semiconductor. In some variants, the semiconductor is cad-
mium selenide, cadmium sulfide, zine sulfide, or gallium
arsenide. In other embodiments, the nanoparticle is magnetic.
In some variants, the magnetic nanoparticle comprises iron
oxide.

In some embodiments of the nanoparticle assembly, the
DNA and/or RNA are single stranded or double stranded. In
preferred embodiments, the DNA is single stranded.

In some embodiments the nucleotide binding protein is any
protein that can bind to DNA or RNA. In preferred embodi-
ments, the nucleotide binding protein is gene 5 protein (g5p).

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In some embodiments, a controllable and reversible assem-
bly of nanoparticles using the g5p protein or similar DNA
binding proteins is provided.

In some embodiments, the controllable and reversible
nanoparticle assembly comprises nanoparticles encapsulated
with non-complementary DNA and/or RNA. In preferred
embodiments, nanoparticles encapsulated with non-comple-
mentary DNA and/or RNA are bound together by a nucleotide
binding protein to form a nanoparticle assembly. In this man-
ner, a controllable and reversible nanoparticle assembly is
formed.

In further embodiments of the controllable and reversible
nanoparticle assembly, the nanoparticle assembly is disas-
sembled by further combining the nanoparticle assembly
with DNA and/or RNA complementary to the non-comple-
mentary DNA and/or RNA. In this manner, a controllable and
reversible nanoparticle disassembly is achieved.

The inventive method provides for controllable assembly
of non-complementary ssDNA-conjugated nanoparticles
using the gSp protein or similar DNA binding proteins.
Hybridization with low concentrations of C-ssDNA during
the assembly phase may be used to regulate assembly kinet-
ics. In addition, C-ssDNA has been used to achieve effective
decomposition of particle aggregates at room temperature
(FIG. 2) or physiological temperatures. This approach makes
it possible to control both assembly and disassembly of nano-
particles without thermal treatment, and to easily incorporate
g5p-based hybrid proteins, e.g., containing affinity tags or
additional catalytic domains, into DNA-based nanostruc-
tures, conferring the potential to design complex nanomate-
rials.

In some embodiments of the method, a controllable nano-
particle assembly and disassembly process is prepared by
encapsulating nanoparticles with DNA and/or RNA; combin-
ing the encapsulated nanoparticles with a nucleotide binding
protein; and binding the DNA and/or RNA to the nucleotide
binding protein. In this manner, a controllable nanoparticle
assembly process is prepared.

In some variants of the method, the assembly of nanopar-
ticles is disassembled by further combining the nanoparticle
assembly with nucleotides complementary to the non-
complementary DNA and/or RNA. In this manner, a control-
lable nanoparticle disassembly process is achieved.

In some embodiments of the method, a nanoparticle assem-
bly is prepared by functionalizing a plurality of nanoparticles
with non-complementary ssDNA; exposing the functional-
ized nanoparticles to gene 5 protein; and linking at least two
strands of the ssDNA with the g5p.

To achieve further sensitive assembly and disassembly of

nanoparticles in response to target DNAs, the invention con- 5

templates the use of g5p-conjugated nanoparticles that can be
bridged by non-complementary ssDNA and dissociated by
sensing its complementary ssDNA (FIG. 3). Poly-histidine
tagged g5p will be immobilized on Ni-NTA-conjugated gold
nanoparticles to achieve oriented g5p in which the DNA
binding sites are active by facing the outer direction of the
nanoparticles. Length and shape of DNA may affect the size
or morphology of the aggregates. This DNA-mediated con-
trol of g5p-conjugated nanoparticles may lead to the design of
a smart materials sensor for DNA at biological conditions.

In some embodiments, an assembly of nanoparticles is
prepared by functionalizing a plurality of nanoparticles with
a nucleotide binding protein; incubating the functionalized
nanoparticles with non-complementary DNA and/or RNA;
and linking at least two strands of the DNA and/or RNA with
the nucleotide binding protein. In this manner, an assembly of
nanoparticles is prepared.

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To achieve precise self-assembly of nanoparticles in mul-
tiple dimensions, the complex of structured DNA and pep-
tide-tagged g5p is proposed, in which the peptide can specifi-
cally bind to nanoparticles. The organization of g5p by
binding to a ssDNA region may result in the formation of a
DNA scaffold from simple building blocks, while the affinity
tag can be used for further ordering of nanoparticles on the
DNA scaffold (FIG. 5). This method may make possible the
design of complex heterogeneous nano-structures.

In some variants of the multi-dimensional structure, the
multi-dimensional structure of DNA and nanoparticle-fune-
tionalized gSp comprises a DNA structure having multiple
branches of ssDNA, a peptide conjugated to g5p, and nano-
particles bound specifically to the peptide. In preferred
embodiments the g5p is bound to the ssDNA. In this manner,
a complex structure of DNA and nanoparticle-functionalized
g5p is formed.

In some variants of the multi-dimensional scaffold, the
multi-dimensional DNA scaffold comprises two strands of
ssDNA (strands 1 and 2) mutually bound by sequences of
C-ssDNA at a first locus; two other strands of ssDNA (strands
3 and 4) mutually bound by sequences of C-ssDNA at a
second locus; strands 2 and 3 are mutually bound by
sequences of C-ssDNA at the second locus; and g5p bound to
at least two of the strands of ssDNA at one or more points
between the bound loci.

In preferred embodiments of the multi-dimensional scaf-
fold, the multi-dimensional scaffold further contains at least
one nanoparticle bound to at least one of the g5p. In more
preferred embodiments of the multi-dimensional scaffold, at
least one nanoparticle is bound to at least one region of DNA.

In some embodiments a goal is to improve DNA- and.
protein-induced nano-scale materials and sensors. In some
embodiments this may be achieved by: association of two
anti-parallel ssDNA by g5p; and dissociation of g5p from
ssDNA by hybridization of complementary DNA or nucleic
acids.

In some embodiments, a g5p-mediated DNA assemblage
comprises non-complementary DNA or RNA strands bound
to anucleotide binding protein. In this manner, an assemblage
is formed.

g5p is encoded by filamentous bacteriophages where it
cooperatively binds to ssDNA to form precursors for the
assembly of phage particles. In vitro, g5p forms a homodimer
which will non-specifically bind two anti-parallel ssDNAs,
inducing helical rod-like structures with 8~9 nm outer diam-
eter. The number of nucleotides bound per g5p monomer is
2~4 and depends on binding conditions, including the protein
to nucleotide ratio. The binding affinity of g5p to ssDNA,
about 10° to about 10° M~', depends on the sequence of
ssDNA and the salt concentration, with preferential binding
to structured DNA such as hairpins and G-quadruplexes. The
g95p protein’s DNA binding properties for assembly and dis-
assembly of ssDNA-conjugated nanoparticles are thus
exploited.

To study the distinct binding properties of g5p and its
preferential DNA topology, an exemplary DNA structure
with three distinct regions has been designed: a 19 base paired
double-stranded DNA (dsDNA) region, followed by a 32
nucleotide anti-parallel poly-T ssDNA region, and a 3'
extended ssDNA tail of 15 nucleotides. The DNA structure (1
lum) was titrated with g5p (in 10 mM Tris-HCl, pH=7.4, 200
mM NaCl), and g5p binding was studied using the electro-
phoretic mobility shift assay. Three distinct stages of g5p
binding were observed, suggesting its sequential binding
(FIG. 6A). Since g5p has a much lower affinity to dsDNA than
to ssDNA at high salt concentration, the following order of

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occupancy is hypothesized: binding to the anti-parallel
ssDNA region with a large shift of the DNA band at 10 uM.
g5p, followed by binding to the ssDNA tail, and finally to
dsDNA at g5p concentrations over 35 1M. Thus, g5p binds to
ssDNA regions before it binds to the dsDNA region, and its
binding affinity for the anti-parallel region is slightly higher
than for the ssDNA tail. To study the effect of g5p association
on forming dsDNA heteroduplexes, and confirm the pro-
posed binding order to the ssDNA regions, an equal amount
(1 uM) of C-ssDNA that can hybridize to the ssDNA tail was
added. After C-ssDNA addition, the two different stages of
band shift observed with 20 1M and 30 1M g5p changed to an
identical position with higher mobility (FIG. 6B), indicating
that the first stage of the band shift results from binding of g5p
to the anti-parallel ssDNA region. Smearing of the band after
C-ssDNA addition at 45 uM g5p supports g5p binding to
dsDNA at this concentration. By comparing the intensity of
the band in the presence of 30 1M g5p to the band under
control conditions without g5p, the hybridization efficiency
for C-ssDNA to the g5p-complexed ssDNA tail was deter-
mined to be approximately 82%, indicating effective DNA
hybridization without significant inhibition by g5p.

To exploit g5p's ability to assemble ssDNA-conjugated
nanoparticles, gold nanoparticles (Au) encapsulated with
approximately 50 copies of ssDNA (5'-HS-C3H6-(T),5-
TAACCTAACCTTCAT-3') (SEQ ID NO. 1) were synthe-
sized, and g5p-mediated assembly of the ss DNA-conjugated
gold nanoparticles (ssDNA-Au) was tested. Dynamic light
scattering (DLS) was used to measure changes of the hydro-
dynamic diameter value (D,), which is related to aggregate
size, interparticle interactions, and geometry. FIG. 7A shows
that the initial assembly rate of D,, during incubation of g5p
and the ssDNA-Au for 20 minutes was highly sensitive to the
g5p concentration, indicating g5p-dependent assembly of
ssDNA-Au. No assembly was observed in the absence of g5p.
To investigate assembly of ssDNA-Au (20 nM) during pro-
longed incubation (~24 hours) with a series of g5p concen-
trations, the ssDNA-Au surface plasmon (SP) resonance band.
was studied using ultraviolet-visible spectrophotometry (UV-
vis). The SP band is associated with isolated Au and
assembled nanostructures. After adding increased g5p con-
centrations, the SP band at 525 nm of isolated ssDNA-Au was
red-shifted with band broadening, indicative of either a
decrease in interparticle distances or an increase of aggregate
size (FIG. 7B). The higher extinction intensity observed over
10 uM g5p is due to a decrease of the solution’s turbidity
resulting from the formation of larger aggregates. Both DLS
and UV-vis results confirm the assembly of ssDNA-Au by
g5p. The sizes and morphologies of the gSp-mediated

ssDNA-Au assemblies were studied using transmission elec- 5

tron microscopy (TEM) (FIG. 7C). The aggregate sizes were
found to increase with increased g5p concentrations, as sug-
gested by DLS and UV-vis, whereas the numbers of non-
assembled particles decreased, suggesting that the size of
aggregates can be simply controlled by the g5p concentration.
Polyhistidine-tagged YieF protein (MW=20 KDa) was used
as a negative control, for which no aggregation of particles
was observed during DLS and UV-vis studies (FIGS. 8A and
8B). These results clearly illustrate that 25p can be used for
the controlled assembly of ssDNA-Au by changing the pro-
tein-nanoparticle ratios.

To investigate inhibitory effects of dsDNA on aggregate
formation, the ssDNA-capping of the particles was first par-
tially hybridized with C-ssDNA (5'-ATGAAGGTTAG-
GTTA-3') (SEQ ID NO. 2) before g5p-mediated assembly
was initiated (FIG. 9A). Changes in assembly rate in response
to C-ssDNA concentrations were studied using DLS (FIG.

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9B). The assembly rate dramatically dropped when the frac-
tion of C-ssDNA to ssDNA on Au (f,) was larger than about
0.05. This large inhibitory effect caused by a low density of
dsDNA suggests large steric hindrance for g5p-mediated
assembly of ssDNA-Au. A non-complementary ssDNA (NC-
ssDNA; 5'-AATATTGATAAGGATAGC-3') (SEQ ID NO. 3)
was used as a control to eliminate any inhibitory titration
effects caused by binding of g5p to ssDNA in solution. The
effect of the NC-ssDNA on the assembly rate was found to be
statistically insignificant (FIG. 9B).

g5p bound to ssDNA can be replaced by C-ssDNA hybrid-
ization (FIG. 6B). This should result in dissociation of the
aggregates, which can be used to detect the presence of C-ss-
DNA. To prove this, aggregates were prepared by incubation
of ssDNA-Au (10 nM) and g5p (5 |M) for about 24 hours,
after which the aggregates were incubated with C-ssDNA for
about 12 hours at room temperature. Colorimetric changes
indicative of aggregate size changes were monitored using
UV-vis. The peak position blue-shifted over approximately
200 nM of C-ssDNA (f¢=~0.4) (FIG. 10A), indicating disso-
ciation of small clusters and release of individual particles. In
the NC-ssDNA control the peak intensity decreased slightly
but no shift in peak position was observed (FIG. 10B), NC-
ssDNA caused the aggregates to divide into smaller clusters,
but not to the level of individual particles. A TEM image of the
sample with 200 nM C-ssDNA clearly shows the dissociation
with dispersed particles (FIG. 10C), whereas this dissociation
was not observed for the sample with NC-ssDNA.

DNA-controlled dissociation of aggregates is a useful
property of the g5p-ssDNA system. To demonstrate this, gold
nanoparticles were assembled into aggregates by a conven-
tional hybridization method incubating ssDNA-Au particles
and their target particles (C-ssDNA-Au) that were encapsu-
lated by C-ssDNA (5'-HS-C3H,-(T),s-ATGAAGGTTAG-
GTTA-3') (SEQ ID NO. 4). Once formed, dissociation of the
aggregates was studied as a function of the addition of
increased C-ssDNA concentrations. However, no aggregate
dissociation was observed till {,, approached about 16 (FIG.
11).

For the correct assembly of ssDNA-Au and C-ssDNA-Au
particles, properties inherent to the DNA sequences, such as
G-C content, often control the assembly process. The inven-
tive method for controllable assembly of non-complementary
ssDNA-conjugated nanoparticles, using gSp as the driving
force, is novel. Control of assembly kinetics and dissociation
of particle aggregates can be obtained via sequence-specific
hybridization with C-ssDNA, while the size of aggregates is
controlled by adjustment of the g5p concentration.

Another aspect of the invention includes a molecular
switch. In a variant of the molecular switch comprises a
fluorescent quenched “off” position and a fluorescent emis-
sion “on” position. In preferred embodiments of the fluores-
cent quenched “off” position, a first ssDNA is bound to fluo-
rescein at the 5' end forming a fluorescein-ssDNA strand, a
second ssDNA bound to DABCYL at the 3' end to form a
ssDNA-DABCYL strand, the fluorescein-ssDNA strand and
the ssDNA-DABCYL strand bound together with g5p. The
switch can be turned “on” by hybridizing a complementary
ssDNA or ssRNA to either the fluorescein-ssDNA strand or
the ssDNA-DABCYL strand. In this manner, the fluorescein
quencher is removed and fluorescence detection is enabled,
turning the switch from an “off” position to an “on” position.

In another variant of the molecular switch, an activated,
“on”, switch comprises a segment of dsDNA: a first strand of
ssDNA bound to one of the two strands of the dsDNA and to
DABCYL at respective ends. The switch also includes a
second strand of ssDNA bound to the other of the two strands

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of the dsDNA and to fluorescein at respective ends, the two
strands of ssDNA non-complementary each to the other. The
switch is turned “off” by attaching g5p to at least one pair of
DABCYL-tipped and fluorescein-tipped ssDNA strands,
such that fluorescence from the fluorescein is quenched dur-
ing attachment via g5p.

Peptides have been successfully used for the construction
of nanostructures and self-assembly of inorganic nanopar-
ticles. Genetic engineering of the g5p protein to display pep-
tides provides further opportunities to design sophisticated
nanomaterials. The sensitive disassembly of the aggregates in
the presence of C-ssDNA without the need for thermal treat-
ment, as would be the case for nanoparticle aggregates based.
on complementary DNAs, demonstrates the great potential of
this approach for the design of biologically functional, hybrid
materials and DNA-based biosensors; while thermal treat-
ment of these hybrid materials will often result in the irre-
versible inactivation of functional moieties, such as proteins,
the addition of a C-ssDNA should not affect their activities.

We also note that this new assembly approach, based on gyp 2

and ssDNA, can be extended to other kinds of nanomaterials,
including carbon nanotubes, semi-conductors, and magnetic
nanoparticles.

FIG. 1 shows a prior art method for self-assembly and
disassembly of ssDNA-conjugated nanoparticles. This tradi-
tional method for self-assembly of ss DNA-conjugated nano-
particles by hybridization with complementary DNA, and
disassembly by increase of the temperature above the melting
temperature (T,,,) of the hybridized DNA. Nanoparticles 110
are functionalized with ssDNA sequences 112, at least some
of which are complementary to each other. The complemen-
tary DNA sequences 114 of the nanoparticles are allowed to
hybridize 116, resulting in self-assembly of the nanoparticles.
The assembled nanoparticles 118 then form an aggregate 120,
which may be disassembled 122 by increasing the tempera-
ture above the melting temperature 124 of the dsDNA strands
126 resulting in disaggregation and disassembly 128 of the
functionalized nanoparticles.

FIG. 2 depicts a controllable gyp-mediated assembly and
disassembly of non-complementary ssDNA-conjugated
nanoparticles Inhibition of assembly and induction of disas-
sembly by hybridization with complementary DNA may be
observed. Nanoparticles 210 are functionalized with non-
complementary DNA 252. g5p 254 is added and the nanopar-
ticles self-assemble into an assemblage 256 and, if desired,
into an aggregate 220. The addition of DNA complementary
to the sequences functionalizing the nanoparticles 258 results
in disaggregation and disassembly 260 of the nanoparticles.

These assembly/disassembly processes may also work in

reverse. FIG. 3 shows a sketch of ss DNA-mediated assembly 5

and disassembly of g5p-conjugated nanoparticles. Here
nanoparticles 310 are functionalized with g5p 354. Single-
stranded DNA 312 is added to the solution and the function-
alized nanoparticles self-assemble. When C-ssDNA 314 is
added, the nanoparticles disassemble.

FIG. 4 depicts the functioning of a g5p-mediated molecular
switch. Binding of g5p 454 to an anti-parallel ssDNA region
462 causes DABCYL (Q) 464 to move close to fluorescein
466, resulting in quenching of fluorescence. This may be seen
as the fabrication of a molecular switch, in a normally “off”
position, or detector. The fluorescent emission can be recov-
ered by hybridization of complementary RNA (C-RNA) or
C-DNA 468 with either of the anti-parallel strands. This
process corresponds to detection of the target or turning “on”
the molecular switch.

FIG. 5 shows self-assembly of nanoparticles at ordered
peptide-conjugated g5p and DNA. A DNA structure 570,

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shown here with four branches of ssDNA, is assembled by
peptide-conjugated g5p 554, and nanoparticles 510 may be
ordered by binding specifically to the peptides. This may
result in arbitrarily structured nanoparticle architectures.

FIGS. 6A and 6B show the results of electrophoretic
mobility shift assays. Titration of a DNA fragment with three
regions of distinct topology is shown in FIG. 6A. The regions
are 19 base pairs of dsDNA, anti-parallel poly-T ssDNA (32
bases), and a ssDNA tail of 15 bases. A model of the binding
is depicted in the right panel. The bottom of the illustration
shows the structured DNA with the three regions. FIG. 6B
depicts the mobility shift of the g5p complexes upon binding
of C-ssDNA to the ssDNA tail, with a model of the binding in
the right panel.

FIGS. 7A, 7B, and 7C are data from g5p-mediated assem-
bly of ssDNA-conjugated gold particles. The chart in FIG. 7A
shows the change in the hydrodynamic diameter value (D,,)
during incubation of ssDNA-Au (5 nm) and g5p, as a function
of the concentration of g5p (in uM) using a DLS analysis
technique. D,, is seen to increase monotonically with the
concentration of g5p. FIG. 7B shows changes in the UV-vis
spectra as the concentration of g5p changes from zero to 15
uM ina solution of 10 mM Tris-HCl, pH 7.44, 200 mM NaCl.
Not only does the intensity change, but the peak also shifis to
longer wavelengths (experiences a red-shift) as the concen-
tration of gp increases.

FIG. 7C is a series of TEM images of the morphology of
nanoparticle assemblages as the concentration of g5p is
increased from zero to 10 uM in a solution of 10 mM Tris-
HCI, pH 7.44, 200 mM NaCl. With no g5p the nanoparticles
are sparsely and relatively uniformly spaced over the sub-
strate. Increasing the concentration to 2.5 jtM results in larger,
more densely spaced nanoparticle assemblies. Further
increasing the concentration of g5p to 5 4M induces the
beginning of agglomeration of the particles. Virtually com-
plete agglomeration of nanoparticles is shown for a concen-
tration of 10 uM.

FIGS. 8A and 8B show results of DLS and UV-vis analyses
of ssDNA-conjugated gold particles. In FIG. 8A DLS shows
D,, for 5 nm ssDNA-Au incubated with 2.5 uM g5p and 2.5
uMVieF. D,, forssDNA-Au in the presence of YieF showed no
change, while that incubated with gSp increased to nearly 1.5

5 yum. FIG. 8B shows UV-vis spectra after incubation for

approximately 24 hours of 20 nm ssDNA-Au with 15 uM
YieF, showing a peak position of about 525 nm.

FIGS. 9A and 9B show a schematic of a g5p-mediated
assembly of ssDNA-conjugated gold particles, and changes
of the hydrodynamic diameter value of such assemblies,
respectively. FIG. 9A is a schematic illustration of preferen-
tial binding of g5p 954 to anti-parallel ssDNA 912 over
dsDNA 952 FIG. 9B shows the change in D,, per minute as a
function of f; (the number fraction of C-ssDNA in the solu-
tion, i.e., the number of C-ssDNA divided by the total number
of ssDNA on the gold nanoparticles 910). The figure shows
the changes of D, during (2.5 M) g5p protein-mediated
assembly of ssDNA-conjugated gold particles (ssDNA-Au~5
nM) which were prehybridized with C-ssDNA.

FIGS. 10A, 10B, and 10C depict the decomposition of
g5p-mediated ssDNA-conjugated gold particle aggregates
when C-ssDNA is added. FIGS. 10A and 10B show UV-vis
data from nanoparticle aggregates (10 nM ssDNA-Au and 5
uM g5p) after about 12 hours’ incubation with C-ssDNA
(FIG. 10A) or NC-ssDNA (FIG. 10B). As seen in the figures,
ssDNA-Au complexes incubated with C-ssDNA experience a
shift in the peak absorbance wavelength while those incu-

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a
bated with NC-ssDNA do not. FIG. 10C shows TEM images
of the sample after incubation with 100 nM (f,.=~0.2) and 200
nM C-ssDNA (f--~0.4).

FIG. 11 shows the effect of the addition of C-ssDNA on
gold particle aggregates. Gold nanoparticles were assembled 5
into aggregates by a conventional hybridization method incu-
bating ssDNA-Au and their target particles (C-ssDNA-Au)
that were encapsulated with the complementary ssDNA.
Single-stranded DNA-Au (10 nM) and C-ssDNA-Au (10
nM) were incubated for ~24 hours in 10 mM Tris-HCl, pH
7.4, 200 mM NaCl, causing a peak shift from ~525 nm (con-
trol 1130) to ~548 nm (0 UM 1132). Recovery of the peak shift
was not observed after incubation with C-ssDNA (8 uM
1134).

Example Methods

The following precise descriptions are provided merely as
concrete examples. No endorsement of any product or manu-
facturer is implied by its inclusion herein. Specific trade
names, models, and manufacturers are provided only for
specificity and may be substituted for by equipment of similar
capability and reagents of similar quality. In addition, all
measurements of time, quantity, concentration, etc. are given
to within experimental and human error.

Protein Preparations

The g5p gene of bacteriophage M13 (New England
Biolabs (NEB)) was amplified with polymerase chain reac-
tion (PCR) using the primers 5'-TAATTCCATATGAT-
TAAAGTTGAAATTAAACCA-3' (SEQ ID NO. 5) and
5'-TAGCTTGCTCTTCCGCACTTAGCCG-
GAACGAGGCG-3' (SEQ ID NO. 6). This generally results
ina DNA fragment flanked by Ndel and Sap] restriction sites.
The PCR product was digested with NdeI and SapI (NEB)
and ligated into the pET-30b vector (Novagen). After the
confirmation of the sequence, the recombinant plasmid was
introduced into BL21-DE3 electroporation competent cell,
using the Gene Pulser Xcell System (Bio-Rad). G5p protein
expression was induced by addition of Isopropyl B-D-1-
thiogalactopyranoside (IPTG) (Sigma) in lysogeny broth
(LB) medium with Kanamycin (100 ug/ml) (Sigma). After
the expression period, cells were sonicated, and the His-
tagged g5p protein was purified using a Ni-NTA column
(Qiagen). Further g5p purification was obtained using fast
protein liquid chromatography (FPLC) (AKTA explorer, GE
Healthcare) with a Sephacryl S-200 high resolution sizing
column (Amersham Biosciences). A purity of >95% was
obtained, as was determined by analysis of the protein bands
using the Quantity One software (Bio-Rad), after staining
with Coomassie Blue on a 15% SDS-polyacrylamide gel.
Molar extinction coefficient of 7450 M~! em7! was used to
determine protein concentration. The YieF protein was pre-
pared as described previously by Zhang, Y. B., et al., Func- ~
tionalized carbon nanotubes for detecting viral proteins.

40

&

12

Nano Letters, 7(10): pp. 3086-3091, 2007, which is hereby
incorporated by reference in its entirety.
Electrophoretic Mobility Shift Assay

The DNA for the binding study was obtained by annealing
of two ssDNA with sequences, 5'-GACCACATACCGCAC-
CATC(T);,CTGCTACGAGACTTC-3' (SEQ ID NO. 7) and
5'-(T)3.GATGGTGCGGTATGTGGTC-3' (SEQ ID NO. 8),
respectively. The annealed DNA (1 1M) was incubated with
g5p at 37° C. for 10 minutes. The DNA was visualized by
ethidium bromide staining after agarose gel (2.5%) electro-
phoresis. The C-ssDNA, with sequence 5'-GAA GTC TCG
TAG CAG-3' (SEQ ID NO. 9), was added to the complex of
g5p and the annealed DNA, and was incubated at 37°C. for 15
minutes before the electrophoresis. Relative intensities of the
DNA bands were analyzed after ethidium bromide staining
Characterization

UV-Visible spectra were obtained using a PerkinElmer
Lambda 35 spectrometer. DLS was measured using a Malv-
erm Zetasizer ZS instrument that is equipped with a 633 nm
laser and a backscattering detector at 173°. To visualize
ssDNA-Au, a JEOL 1300 TEM was operated at 120 kV. TEM
samples were prepared by incubation of samples ona carbon-
coated copper grid for 10 minutes and washed with distilled
water two times.

Given the teaching herein, the inventors and other practi-
tioners in the art may expand the approach to more complex
DNA-g5p structures for assembly of nanoparticles. Display
of functional peptide at C-terminal of g5p may allow for
biologically active nanomaterials. In addition, the use of other
classes of nanoparticles (semiconductive, magnetic) is con-
sidered for more potential applications. The work described
may be further exploited for the development of biosensors,
including the development of approaches for microarray
design.

This method makes it possible to control both assembly
and disassembly of DNA-conjugated nanoparticles at bio-
logical conditions without thermal treatments, to easily incor-
porate proteins into DNA based nanostructures implying
great potential to design complex nanomaterials, and to
design approaches for the detection of nucleotide sequences
(DNA, RNA) without the need for their labeling.

While the foregoing description has been made with refer-
ence to individual embodiments of the invention, it should be
understood that those skilled in the art, making use of the
teaching herein, may propose various changes and modifica-
tions without departing from the invention in its broader
aspects. For example gene proteins other than g5p may be
used. In another embodiment, the invention may be used for
detecting chemical species for security applications.

The foregoing description being illustrative, the invention
is limited only by the claims appended hereto.

SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS: 9
<210>
<211>
<212>
<213>
<220>
<223>
<220>
<221>
<222>
<223>

SEQ ID NO 1

LENGTH: 30

TYPE: DNA

ORGANISM: Artificial Sequence

FEATURE:

OTHER INFORMATION: Artificial ss-DNA Sequence
FEATURE:

WAME/KEY: misco_feature

LOCATION: (1)..(1)

OTHER INFORMATION: 5'-HS-C3H6-

Page 26

US 8,729,012
13

-cont inued

14

<400> SEQUENCE: 1

tttttttttt ttttttaace taaccttcat

<210> SEQ ID NO 2

<211> LENGTH: 15

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: complementary single-stranded DNA

<400> SEQUENCE: 2

atgaaggtta ggtta

<210> SEQ ID NO 3

<211> LENGTH: 18

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: non-complementary single-stranded DNA

<400> SEQUENCE: 3

aatattgata aggatage

<210> SEQ ID NO 4
<211> LENGTH: 30

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: complementary single-stranded DNA
<220>
<221> misc feature

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

<223> OTHER INFORMATION: 5! -HS-C3Hé

<400> SEQUENCE: 4

tttttttttt tttttatgaa ggttaggtta

<210> SEQ ID NO 5

<211> LENGTH: 33

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: bacteriophage M13 g5p gene primer

<400> SEQUENCE: 5

taattccata tgattaaagt tgaaattaaa cca

<210> SEQ ID NO 6

<21l> LENGTH: 35

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: bacteriophage M13 g5p gene primer

<400> SEQUENCE: 6
tagettgete ttccgcactt agecggaacy aggceg
<210> SEQ ID NO 7

<211> LENGTH: 66

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: single-stranded DNA

<400> SEQUENCE: 7

gaccacatac egeaccatct tttttttttt tttttttttt tttttttttt tetgetacga

30

15

18

30

33

35

60

Page 27

US 8,729,012 B2

15

16

-cont inued

gactte

<210> SEQ ID NO 8

<211> LENGTH: 51

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: single-stranded DNA

<400> SEQUENCE: 8

tttttttttt tttttttttt tttttttttt ttgatggtge ggtatgtggt

<210> SEQ ID NO 9

<211> LENGTH: 15

<212> TYPE: DNA

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

<223> OTHER INFORMATION: complementary single-stranded

<400> SEQUENCE: 9

gaagtctegt agcag

o

66

51

15

The invention claimed is:

1. A controllable and reversible nanoparticle assembly
comprising, nanoparticles encapsulated with non-comple-
mentary DNA and/or RNA, the noncomplementary DNA
and/or RNA bound by a nucleotide binding protein to form a
nanoparticle assembly, wherein the nucleotide binding pro-
tein is gene-5 protein of bacteriophage M13.

2. The controllable and reversible nanoparticle assembly
according to claim 1, wherein the nanoparticle assembly is
disassembled by further combining the nanoparticle assem-
bly with DNA and/or RNA complementary to the non-
complementary DNA and/or RNA.

3. The controllable and reversible nanoparticle assembly
according to claim 1, wherein the nanoparticle is a metal.

4. The controllable and reversible nanoparticle assembly
according to claim 3, wherein the metal is selected from the
group consisting of gold, silver, and platinum.

5. The controllable and reversible nanoparticle assembly
according to claim 4, wherein the metal is gold.

6. The controllable and reversible nanoparticle assembly
according to claim 1, wherein the nanoparticle is a semicon-
ductor.

w

40

45

7. The controllable and reversible nanoparticle assembly
according to claim 6, wherein the semicondutor is selected
from the group consisting of cadmium selenide, cadmium
sulfide, zinc sulfide, and gallium arsenide.

8. The controllable and reversible nanoparticle assembly
according to claim 1, wherein the nanoparticle is magnetic.

9. The controllable and reversible particle assembly
according to claim 8, wherein the nanoparticle is iron oxide.

10. The controllable and reversible nanoparticle assembly
according to claim 1, wherein the DNA and/or RNA are
single stranded.

11. The controllable and reversible nanoparticle assembly
according to claim 1, wherein the DNA and/or RNA include
a double stranded region.

12. A g5p-mediated DNA assemblage comprising, non-
complementary DNA or RNA strands, the non-complemen-
tary DNA or RNA strands bound and a nucleotide binding
protein, thereby forming an assemblage, wherein the nucle-
otide binding protein is gene-5 protein of bacteriophage M13.

13. The g5p-mediated DNA assemblage according to
claim 12, wherein the DNA or RNA is single stranded.

ce