U.S. patent application number 15/363172 was filed with the patent office on 2017-07-27 for nanoparticles having predetermined shapes.
This patent application is currently assigned to President and Fellows of Harvard College. The applicant listed for this patent is President and Fellows of Harvard College. Invention is credited to Wei Sun, Peng Yin.
Application Number | 20170209926 15/363172 |
Document ID | / |
Family ID | 46551879 |
Filed Date | 2017-07-27 |
United States Patent
Application |
20170209926 |
Kind Code |
A1 |
Sun; Wei ; et al. |
July 27, 2017 |
NANOPARTICLES HAVING PREDETERMINED SHAPES
Abstract
Articles and methods for forming nanostructures having unique
and/or predetermined shapes are provided. The methods and articles
may involve the use of nucleic acid containers as structural molds.
For instance, a pre-designed nucleic acid container including a
cavity may be used to control the shape-specific growth of
nanoparticles. Growth of the nanoparticles within the cavities may
be confined by the specific shape of the nucleic acid container. In
some embodiments, the resulting nucleic acid-nanoparticle
structures can be used to control the orientation and numbers of
surface ligands on the surface of nanoparticles. The addressability
of the surface ligands can be used to form higher ordered
assemblies of the structures.
Inventors: |
Sun; Wei; (Brookline,
MA) ; Yin; Peng; (Brookline, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
President and Fellows of Harvard College |
Cambridge |
MA |
US |
|
|
Assignee: |
President and Fellows of Harvard
College
Cambridge
MA
|
Family ID: |
46551879 |
Appl. No.: |
15/363172 |
Filed: |
November 29, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14128028 |
Mar 31, 2014 |
9598690 |
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PCT/US12/44846 |
Jun 29, 2012 |
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15363172 |
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61504066 |
Jul 1, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B22F 1/0044 20130101;
B22F 1/02 20130101; B82B 3/0033 20130101; C12N 9/98 20130101 |
International
Class: |
B22F 1/02 20060101
B22F001/02; B22F 1/00 20060101 B22F001/00 |
Goverment Interests
GOVERNMENT SPONSORSHIP
[0002] This invention was made with U.S. Government support under
N00014-10-1-0827 awarded by the U.S. Office of Naval Research under
N66001-11-1-4136 awarded by the U.S. Department of Defense/SPAWAR.
The Government has certain rights in the invention.
Claims
1. (canceled)
2. An article comprising: a nanoparticle positioned inside a
nucleic acid container having a predetermined three-dimensional
structure, wherein the nanoparticle comprises at least one surface
portion having a shape that is complementary at the sub-nanometer
level to a shape of an inner surface portion of the nucleic acid
container.
3-5. (canceled)
6. An article comprising: an inorganic nanoparticle comprising an
isolated nucleic acid strand attached to a surface of the inorganic
nanoparticle, wherein the inorganic nanoparticle has a
non-spherical shape.
7. (canceled)
8. An article comprising: an inorganic nanoparticle coated with a
nucleic acid container, wherein the nucleic acid container
comprises pores; and a nucleic acid strand attached to a surface of
the inorganic nanoparticle, and extending from the surface of the
inorganic nanoparticle, through a pore of the nucleic acid
container.
9-22. (canceled)
23. An article as in claim 1, wherein the nanoparticle has a shape
that is complementary to a shape of the inner surfaces of the
nucleic acid container.
24. An article as in claim 1, wherein the nanoparticle is formed
from a template-assisted synthesis inside the nucleic acid
container.
25. An article as in claim 1, wherein the nanoparticle has a
three-dimensional shape that includes at least 4 different
sides.
26. An article as in claim 1, wherein the nanoparticle comprises a
cross-section in the shape of a rectangle, rod, T, L, branched
structure, diamond, star, square, parallelogram, triangle,
pentagon, or hexagon.
27. An article as in claim 1, wherein the nanoparticle is in the
shape of a polyhedron.
28-29. (canceled)
30. An article as in claim 1, wherein the nanoparticle is an
inorganic nanoparticle.
31. An article as in claim 1, wherein the nanoparticle comprises a
metal.
32. An article as in claim 1, wherein the nanoparticle is an
alloy.
33. An article as in claim 1, wherein the nanoparticle comprises a
semiconductor.
34. An article as in claim 1, wherein the nanoparticle comprises a
polymer.
35. An article as in claim 1, wherein the nanoparticle has at least
one cross-sectional dimension that is less than or equal to 1
micron.
36. (canceled)
37. An article as in claim 1, wherein the nanoparticle has an
aspect ratio of at least 2:1.
38-40. (canceled)
41. An article as in claim 1, wherein the nanoparticle comprises an
isolated nucleic acid strand comprising DNA and/or RNA attached to
a surface of the nanoparticle.
42-43. (canceled)
44. An article as in claim 1, wherein the nucleic acid container
comprises a cavity having a volume, and at least 60% of the volume
is filled with the nanoparticle.
45. (canceled)
46. An article as in claim 1, wherein the nucleic acid container
comprises a cavity in the shape of a polyhedron.
47-50. (canceled)
51. An article as in claim 1, wherein the nucleic acid container
comprises at least one lid that can be open or closed.
52. An article as in claim 1, wherein the nucleic acid container
comprises a cavity, and a cross-sectional dimension of the cavity
is less than or equal to 1 micron.
53-56. (canceled)
57. An article as in claim 1, wherein the nucleic acid container is
formed of a nucleic acid having a molecular weight of at least 640
kDa.
58-77. (canceled)
78. An article as in claim 1, wherein the nanoparticle comprises a
plurality of marker and binding site pairs, wherein each of the
markers are different from one another, and each of the binding
sites are different from one another.
79-97. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is continuation of U.S. application Ser.
No. 14/128,028, filed on Mar. 31, 2014, entitled "METHOD FOR
FORMING NANOPARTICLES HAVING PREDETERMINED SHAPES", which is a
national stage filing under 35 U.S.C. .sctn.371 of International
Application No. PCT/US2012/044846 filed Jun. 29, 2012, which was
published under PCT Article 21(2) in English, and which claims the
benefit of U.S. Provisional Application Ser. No. 61/504,066, filed
on Jul. 1, 2011, entitled "METHOD FOR FORMING NANOPARTICLES HAVING
PREDETERMINED SHAPES", the entire contents of each of which are
incorporated by reference herein.
FIELD OF INVENTION
[0003] The present invention relates generally to articles and
methods for forming nanostructures, and more specifically to
articles and methods for forming nanostructures having unique
and/or predetermined shapes.
BACKGROUND
[0004] Rational synthesis of monodispersed shape-controllable
nanoparticles is the first step towards applications in
bio-detection that use shape-specific properties of nanoparticles
for detection. To direct the growth of nanoparticles (including
inorganic nanoparticles such as gold or silver) with specific
shapes, templates encoded with the designed geometry are often
used. Soft templates, e.g., structures self-assembled from
amphiphilic surfactant molecules, have succeeded in creating
diverse shapes. However, it is generally difficult to predict the
shape of the resultant structure (partially due to the flexible
nature of the template), and hence it is challenging to design
structures with prescribed shapes using this approach and program
the growth of inorganic materials. Hard templates, such as oxides
or viruses, have also been utilized to direct the growth of
nanowires or nanorods, with better predictability of the resultant
structures. However, these approaches may only produce a small
number of shapes, which may limit the programmability of shape
diversity. Improved methods and articles that could address some or
all of these issues, and/or other challenges in the art, would be
beneficial in a number of different fields.
SUMMARY OF THE INVENTION
[0005] The present invention relates generally to articles and
methods for forming nanostructures, and more specifically to
articles and methods for forming nanostructures having unique
and/or predetermined shapes. The subject matter of the present
invention involves, in some cases, interrelated products,
alternative solutions to a particular problem, and/or a plurality
of different uses of one or more articles, compositions and/or
methods.
[0006] In one set of embodiments, a series of articles are
provided. In one embodiment, an article comprises a nanoparticle
positioned inside a nucleic acid container having a predetermined
three-dimensional structure, wherein the nanoparticle comprises at
least one surface portion having a shape that is complementary to a
shape of an inner surface portion of the nucleic acid
container.
[0007] In another embodiment, an article comprises a nanoparticle
comprising at least two opposing surface portions, each of the at
least two opposing surface portions having a shape that is
complementary to a shape of a surface portion of a nucleic acid
nanostructure.
[0008] In another embodiment, an article comprises an inorganic
nanoparticle comprising an isolated nucleic acid strand attached to
a surface of the inorganic nanoparticle, wherein the inorganic
nanoparticle has a non-spherical shape.
[0009] In another embodiment, an article comprises an inorganic
nanoparticle coated with a nucleic acid container, wherein the
nucleic acid container comprises pores; and a nucleic acid strand
attached to a surface of the inorganic nanoparticle, and extending
from the surface of the inorganic nanoparticle, through a pore of
the nucleic acid container.
[0010] In another embodiment, an article comprises an assembly of
nucleic acid-coated nanoparticles, wherein the nucleic acid-coated
nanoparticles are attached to one another by complementary binding
sites.
[0011] In another set of embodiments, a series of methods are
provided. In one embodiment, a method comprises forming a
nanoparticle comprising at least one surface portion having a shape
that is complementary to a shape of an inner surface portion of a
nucleic acid container having a predetermined three-dimensional
structure at the sub-nanometer level.
[0012] In another embodiment, a method comprises forming a
nanoparticle from a nanoparticle precursor positioned inside a
nucleic acid container having a predetermined three-dimensional
structure.
[0013] In another embodiment, a method comprises providing a
nucleic acid container as a template for forming a nanoparticle,
wherein the nucleic acid container comprises a plurality of
components attached to an inner wall of the nucleic acid container
in a predetermined pattern, forming a nanoparticle inside the
nucleic acid container, and attaching the plurality of components
to the nanoparticle.
[0014] In another embodiment, a method comprises attaching an
isolated nucleic acid strand to a surface of an inorganic
nanoparticle having a non-spherical shape.
[0015] In another embodiment, a method comprises providing an
inorganic nanoparticle coated with a nucleic acid container,
wherein the nucleic acid container comprises pores,
[0016] introducing a nucleic strand through a pore of the nucleic
acid container, and attaching a portion of the nucleic strand to a
surface of the inorganic nanoparticle.
[0017] In another embodiment, a method comprises forming an
assembly of nucleic acid-coated nanoparticles, wherein the nucleic
acid-coated nanoparticles are attached to one another by
complementary binding sites.
[0018] In another embodiment, a method comprises using two
nonspherical nanoparticles to detect at least 12 different target
molecules.
[0019] In another set of embodiments, a series of compositions are
provided. In one embodiment, a composition comprises a plurality of
nanoparticles, wherein two of the plurality of nanoparticles can be
used to detect at least 12 different target molecules.
[0020] In another embodiment, a composition comprises a plurality
of nanoparticles, wherein at least 90% of the nanoparticles vary in
a maximum cross-sectional dimension by less than 0.5 standard
deviation of the median maximum cross-sectional dimension of all
the nanoparticles in the composition, and wherein each of the
plurality of nanoparticles includes at least 6 different sides.
[0021] Various configurations of the articles, compositions, and
methods described above and herein are provided. For example, in
some cases, the nanoparticle precursor comprises an inorganic
nanoparticle. In some embodiments, the nanoparticle precursor
comprises a metal, a semiconductor, or a monomer of an organic
polymer. In one embodiment, the nanoparticle precursor comprises
Au, Ag, Cd, Zn, Cu, Pb, Mn, Ni, Mg, Fe, Pd, and/or Pt. The
nanoparticle precursor may have a cross-sectional dimension of, for
example, less than or equal to 50 nm, less than or equal to 25 nm,
less than or equal to 10 nm, less than or equal to 5 nm, less than
or equal to 3 nm, less than or equal to 2 nm, less than or equal to
1 nm, or less than or equal to 0.1 nm.
[0022] In some embodiments, the nanoparticle comprises at least one
surface portion having a shape that is complementary to a shape of
an inner surface portion of the nucleic acid container. The
nanoparticle may have a shape that is complementary to a shape of
the inner surfaces of the nucleic acid container. The nanoparticle
may have a three-dimensional shape that includes at least 4, at
least 5, at least 6, at least 7, at least 8, at least 9, or at
least 10 different sides. The nanoparticle comprises a
cross-section in the shape of a rectangle, rod, T, L, branched
structure, diamond, star, square, parallelogram, triangle,
pentagon, hexagon, ring, or polyhedron. In some embodiments, the
nanoparticle has a non-spherical shape or an asymmetric shape. In
some cases, the nanoparticle is an inorganic nanoparticle. The
nanoparticle may comprises a metal, a semiconductor, or a polymer.
In some cases, the nanoparticle is an alloy. In some embodiments,
the nanoparticle has at least one cross-sectional dimension that is
less than or equal to 1 micron, less than or equal to 500 nm, less
than or equal to 250 nm, less than or equal to 100 nm, less than or
equal to 75 nm, less than or equal to 50 nm, less than or equal to
40 nm, less than or equal to 30 nm, less than or equal to 20 nm,
less than or equal to 10 nm, or less than or equal to 1 nm. In
certain embodiments, the nanoparticle has at least one
cross-sectional dimension that is greater than or equal to 1 nm,
greater than or equal to 5 nm, greater than or equal to 10 nm,
greater than or equal to 50 nm, or greater than or equal to 100 nm.
The nanoparticle may have an aspect ratio of at least 2:1, at least
3:1, at least 5:1, at least 10:1, or at least 20:1. The
nanoparticle may be encapsulated by a nucleic acid nanostructure.
In some embodiments, the nanoparticle comprises an isolated binding
site attached to a surface of the nanoparticle. In some cases, the
nanoparticle comprises at least 2, at least 4, at least 6, at least
8, at least 10, at least 12, at least 14, at least 16, at least 18,
or at least 20 different isolated binding sites attached to a
surface of the nanoparticle. The nanoparticle may comprises an
isolated nucleic acid strand attached to a surface of the
nanoparticle. The isolated binding sites may be positioned at least
2 nm, at least 5 nm, at least 10 nm, at least 15 nm, at least 20
nm, or at least 30 nm apart from one another. In some embodiments,
the nucleic acid strand is a DNA strand or DNA analog, or a RNA
strand or RNA analog.
[0023] In some embodiments, the nanoparticle may have a
cross-sectional shape that includes different numbers of vertexes.
A cross-sectional shape of the nanoparticle may have, for example,
at least 3, at least 4, at least 5, at least 6, at least 7, at
least 8, at least 9, or at least 10 vertexes. In some embodiments,
at least 1, at least 2, at least 3, at least 4, at least 5, at
least 6, at least 7, at least 8, at least 9, or at least 10
vertexes of the cross-sectional shape of the nanoparticle are
rounded. In other embodiments, at least 1, at least 2, at least 3,
at least 4, at least 5, at least 6, at least 7, at least 8, at
least 9, or at least 10 vertexes of the cross-sectional shape of
the nanoparticle are substantially sharp. Combinations of rounded
and sharp vertexes are also possible.
[0024] In some embodiments, the nucleic acid container comprises a
cavity having a volume, and at least 60%, or at least 80% of the
volume is filled with the nanoparticle. In some cases, the nucleic
acid container comprises a cavity having a volume, and
substantially all of the volume is filled with the nanoparticle. In
some embodiments, the nucleic acid container comprises a cavity in
the shape of a polyhedron, has a non-spherical shape, or has an
asymmetric shape. In some cases, the nucleic acid container
comprises at least one open side, or at least two open sides. In
certain cases, the nucleic acid container is substantially closed.
In some embodiments, the nucleic acid container comprises at least
one lid that can be open or closed.
[0025] The nucleic acid container may comprise a cavity, and a
cross-sectional dimension of the cavity may be less than or equal
to 1 micron, less than or equal to 500 nm, less than or equal to
250 nm, less than or equal to 100 nm, less than or equal to 75 nm,
less than or equal to 50 nm, less than or equal to 40 nm, less than
or equal to 30 nm, less than or equal to 20 nm, less than or equal
to 10 nm, or less than or equal to 1 nm. In some embodiments, the
nucleic acid container comprises a cavity, and a cross-sectional
dimension of the cavity is greater than or equal to 1 nm, greater
than or equal to 5 nm, greater than or equal to 10 nm, greater than
or equal to 20 nm, greater than or equal to 30 nm, greater than or
equal to 40 nm, greater than or equal to 50 nm, greater than or
equal to 100 nm, greater than or equal to 500 nm, or greater than
or equal to 1 micron. In some embodiments, the nucleic acid
container comprises walls that surround a cavity, and the average
thickness of the walls is less than or equal to 1 micron, less than
or equal to 500 nm, less than or equal to 250 nm, less than or
equal to 100 nm, less than or equal to 75 nm, less than or equal to
50 nm, less than or equal to 40 nm, less than or equal to 30 nm,
less than or equal to 20 nm, less than or equal to 10 nm, or less
than or equal to 1 nm. In some cases, the nucleic acid container
comprises walls that surround a cavity, and wherein the average
thickness of the walls is greater than or equal to 1 nm, greater
than or equal to 10 nm, greater than or equal to 25 nm, greater
than or equal to 50 nm, greater than or equal to 100 nm, greater
than or equal to 500 nm, or greater than or equal to 1 micron.
[0026] In some cases, the nucleic acid container comprises more
than one layer. The nucleic acid container may be formed of a
nucleic acid having a molecular weight of at least 640 kDa. In some
embodiments, the nucleic acid container is formed of a nucleic acid
having a length of at least 1,000 bases. In some embodiments, a
nucleic acid container comprises an inorganic nanostructure.
[0027] In some embodiments, an assembly is formed by assembling
nucleic acid containers, each of the nucleic acid containers having
a nanoparticle precursor positioned therein, and then synthesizing
the nanoparticle from the nanoparticle precursor inside the nucleic
acid container to form the nucleic acid-coated nanoparticles. The
assembly may be formed by synthesizing a plurality of nucleic
acid-coated nanoparticles, each of the nucleic-acid coated
nanoparticles formed by growing a nanoparticle from a nanoparticle
precursor positioned inside a nucleic acid container, and then
assembling the nucleic acid-coated nanoparticles. The nucleic
acid-coated nanoparticles may be attached to one another by binding
sites that are attached to a nucleic acid portion of the nucleic
acid-coated nanoparticles. In some embodiments, the nucleic
acid-coated nanoparticles are attached to one another by binding
sites that are attached to a nanoparticle portion of the nucleic
acid-coated nanoparticles. In some cases, the nucleic acid-coated
nanoparticles are attached to one another using a thermal process,
a photophysical process, and/or a binding process.
[0028] In some embodiments, a method involves removing a portion of
the nucleic acid from the nucleic acid-coated nanoparticles. In
some cases, a method involves substantially removing the nucleic
acid coating from the nucleic acid-coated nanoparticles. In some
embodiments, a method involves passivating a surface of the
nanoparticle prior to, during, or after the removal step. The
nanoparticles may remain attached to one another in the assembly
after the removal step.
[0029] In embodiments involving assemblies, the assembly may be an
electronic circuit. The assembly may be in the form of a
two-dimensional array, or a three-dimensional array. The assembly
may have at least one length and/or at least one cross-sectional
dimension that is less than or equal to 1 mm, less than or equal to
100 microns, less than or equal to 50 microns, less than or equal
to 10 microns, less than or equal to 1 micron, less than or equal
to 500 nm, less than or equal to 100 nm, less than or equal to 50
nm, less than or equal to 10 nm, or less than or equal to 1 nm. The
assembly may have at least one length and/or at least one
cross-sectional dimension that is greater than or equal to 1 nm,
greater than or equal to 10 nm, greater than or equal to 100 nm,
greater than or equal to 1 micron, greater than or equal to 10
microns, greater than or equal to 50 microns, greater than or equal
to 100 microns, or greater than or equal to 1 mm.
[0030] In some cases, the nanoparticle comprises a marker attached
to a surface of the nanoparticle. In some embodiments, the marker
is isolated on the surface of the nanoparticle. The marker may
comprises a nucleic acid strand, a fluorophore, a nanoparticle, an
antibody, a peptide, or a reporter molecule. In some embodiments,
the marker is a surface-enhanced Raman scattering reporter
molecule. In some cases, the marker is a luminescent probe. In
certain embodiments, each marker is adjacent a binding site
attached to the surface of the nanoparticle. In some embodiments,
the nanoparticle comprises a plurality of marker and binding site
pairs, wherein each of the markers are different from one another,
and each of the binding sites are different from one another. An
article, a composition, or a method may comprise at least 4, at
least 5, at least 6, at least 7, at least 8, at least 9, at least
10, at least 12, at least 15, or at least 20 different markers
positioned on the surface of the nanoparticle. In some cases, each
of the components or markers are isolated from one another and are
positioned at predetermined distances from one another. A method
may involve attaching a predetermined number of components or
markers to the nanoparticle.
[0031] In some embodiments, an article to detect a biomolecule,
e.g., the multiplexed detection of biomolecules. Detection may
comprise introducing a target molecule to a plurality of
nanoparticles, and allowing the target molecule to bind to surfaces
of at least two different nanoparticles. Binding may enhance a
Raman signal from two reporter molecules associated with the
surfaces of the nanoparticles. A method may involve using two
nanoparticles to detect at least 5, at least 10, at least 20, at
least 30, at least 40, at least 50, at least 70, or at least 100
different target molecules in parallel.
[0032] In some embodiments, each of the nucleic acid-coated
nanoparticles is in the form of a nanoparticle positioned inside a
nucleic acid container. A method may involve forming, in parallel,
at least 10, at least 15, at least 20, at least 30, at least 50, or
at least 100 inorganic nanoparticles each having different
shapes.
[0033] A method may comprise synthesizing the nanoparticle from a
seed-mediated growth process. In some cases, the nanoparticle is
synthesized in the absence of a surfactant, or in the absence of an
oxide template. The nucleic acid container may be designed to
include a cavity having a pre-designed three-dimensional structure,
and the shape of the nanoparticle is formed, at least in part, by
molding against the cavity.
[0034] In some embodiments, a method involves controlling ion
diffusion kinetics to control the growth kinetics and/or
composition of the nanoparticle. A method may comprise controlling
the distribution of components in an nanoparticle alloy.
[0035] In some cases, the inorganic nanoparticle is hollow and
comprises a cavity. The inorganic nanoparticle may be used as a
template to fabricate a secondary nanostructure in the cavity of
the nanoparticle. The nanoparticle or nanostructure may have a
complex arbitrary shape.
[0036] The combination of programmable nucleic acid containers with
nanoparticle synthesis enables the programmability of arbitrary
shaped materials by using the containers as molds. Target
structural information may be encoded into the cavity design of
specifically shaped nucleic acid containers. In some embodiments,
growth of certain materials (e.g., inorganic materials) within the
cavities may start using small nanoparticle precursors (e.g.,
nanocrystals) for the nucleation of inorganic materials, on the
interior surface of nucleic acid cavity, and is stopped or
significantly slowed down when the growing lattices encounter the
nucleic acid sidewalls. These approaches may enable a wide variety
of applications that take advantage of the nucleic acid-programmed
synthesis of inorganic materials, such as in multiplexed surface
enhanced Raman scattering (SERS) detection, in DNA-directed
self-assembly of electronic circuits, in surface-specific catalyst
and in structural constrain for electrode materials in
lithium-based fuel cells. Notably, the syntheses described herein
can be executed not only ex-vivo (e.g., in test tubes), but also
under in-vitro/in-vivo conditions, such as in bacteria and
cells.
[0037] Other advantages and novel features of the present invention
will become apparent from the following detailed description of
various non-limiting embodiments of the invention when considered
in conjunction with the accompanying figures. In cases where the
present specification and a document incorporated by reference
include conflicting and/or inconsistent disclosure, the present
specification shall control. If two or more documents incorporated
by reference include conflicting and/or inconsistent disclosure
with respect to each other, then the document having the later
effective date shall control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Non-limiting embodiments of the present invention will be
described by way of example with reference to the accompanying
figures, which are schematic and are not intended to be drawn to
scale. In the figures, each identical or nearly identical component
illustrated is typically represented by a single numeral. For
purposes of clarity, not every component is labeled in every
figure, nor is every component of each embodiment of the invention
shown where illustration is not necessary to allow those of
ordinary skill in the art to understand the invention. In the
figures:
[0039] FIG. 1A shows a method involving the use of a nucleic acid
container as a mold to form a nanoparticle according to one set of
embodiments;
[0040] FIG. 1B shows different shapes of nanoparticles that can be
formed using different shapes of nucleic acid containers according
to one set of embodiments;
[0041] FIGS. 2A-2F show examples of different nucleic acid
containers according to one set of embodiments;
[0042] FIGS. 3A-3D show nucleic acid containers that may include
one or more lids according to another set of embodiments;
[0043] FIGS. 3E-3F show nucleic acid containers that may be used to
form hollow nanoparticles according to another set of
embodiments;
[0044] FIGS. 4A-4F show control of surface addressability of
nanostructures according to one set of embodiments;
[0045] FIG. 5 shows an example of surface-specific self-assembly of
nanostructures according to one set of embodiments;
[0046] FIGS. 6A and 6B shows self-assembly of nanostructures into
an electronic circuit according to one set of embodiments;
[0047] FIG. 7 shows a scheme of target-triggered self-assembly of
nanostructures that can be used for surface enhanced Raman
spectroscopy detection according to one set of embodiments;
[0048] FIG. 8A shows detachment of a nanoparticle precursor into a
cavity of a nucleic acid container according to one set of
embodiments;
[0049] FIG. 8B is a transmission electron microscopy (TEM) image of
nucleic acid containers depicted in FIG. 8A according to one set of
embodiments;
[0050] FIG. 8C is a transmission electron microscopy image of a
gold nanoparticle that has been formed by templated synthesis
inside the nucleic acid containers shown in FIG. 8B according to
one set of embodiments;
[0051] FIG. 9A shows a cross-sectional view of a nucleic acid
container according to one set of embodiments;
[0052] FIG. 9B is a transmission electron microscopy image of a
single nanoparticle formed by templated synthesis within the
nucleic acid container shown in FIG. 9A;
[0053] FIGS. 9C and 9D are transmission electron microscopy images
showing two nanoparticles formed by dimerized nucleic acid
containers according to another set of embodiments;
[0054] FIG. 10 is a TEM image of a nanoparticle formed inside a
cavity of a nucleic acid container that includes two quantum dots
associated with the container according to another set of
embodiments;
[0055] FIGS. 11A-11C show a method involving the use of a nucleic
acid container as a mold to form nanoparticles having different
shapes according to one set of embodiments;
[0056] FIGS. 12A-12E are images showing the formation of closed
nucleic acid containers and the use of the containers for forming
nanoparticles having different shapes according to one set of
embodiments;
[0057] FIGS. 13A-13D are images showing the formation of open
nucleic acid containers and the use of the containers for forming
nanoparticles having different shapes according to one set of
embodiments;
[0058] FIG. 14A are images showing the self-assembly of nucleic
acid containers to form a larger container used for growing
nanoparticles according to one set of embodiments; and
[0059] FIG. 14B are images showing the formation of a heterogeneous
quantum dot-silver nanoparticle-quantum dot sandwiched structure
according to one set of embodiments.
DETAILED DESCRIPTION
[0060] Articles and methods for forming nanostructures having
unique and/or predetermined shapes are provided. In some
embodiments, the methods and articles involve the use of nucleic
acid containers as structural molds. For instance, a pre-designed
nucleic acid container including a cavity may be used to control
the shape-specific growth of nanoparticles. Growth of the
nanoparticles within the cavities may be confined by the specific
shape of the nucleic acid container. Using such a method,
nanoparticles having complex and predetermined shapes and sizes can
be formed. The resulting nanoparticle that is coated with a
nucleic-acid container may be used as is, or the nucleic acid
coating may be removed partially or completely if desired.
[0061] Additionally, the methods described herein allow the
material composition of the grown nanoparticles to be controlled by
using different nanoparticle precursors and/or by controlling the
thickness of the walls of the nucleic acid containers. In some
embodiments, controlling such parameters can allow the formation of
nanoparticle alloys having predetermined and controlled ratios of
material components. In some embodiments, the resulting nucleic
acid-nanoparticle structures can be used to control the
orientation, numbers, types, and positioning of components such as
binding sites, markers, and surface ligands on the surface of the
nanoparticles or the surface of the nucleic acid containers.
Advantageously, nanoparticles having a predetermined number and
orientation of unique components attached to the nanoparticle or
nucleic acid container surface may allow addressability of the
structures for applications such as multiplexed detection of target
molecules. Moreover, the addressability of the structures can be
used to form higher ordered assemblies of the structures in some
embodiments.
[0062] The articles and methods provided herein have applications
in a number of different fields, including the areas of
bio-sensing, electronics, environment sciences, and energy. Other
advantages of the articles and methods described herein are
provided in more detail below.
[0063] An example of a method for forming nanoparticles having
unique and/or predetermined shapes in shown in FIG. 1A. As shown
illustratively in FIG. 1A, scheme 10 involves the use of a nucleic
acid container 20 as a mold for the templated synthesis of a
nanoparticle. Nucleic acid container 20 includes an outer surface
24, an inner surface 26, and a wall 25 formed between the outer and
inner surfaces. The nucleic acid container also includes a cavity
30 enclosed by the inner surface portions of the nucleic acid
container. As shown illustratively in FIG. 1A, the nucleic acid
container may also include an end 32 and an end 33, which may be
open in some embodiments, or closed in other embodiments. An
opening into the cavity may allow one or more nanoparticle
precursors 34 to be inserted into the cavity. Alternatively, one or
more nanoparticle precursors can be present inside a cavity that is
completely closed, e.g., by attaching the one or more nanoparticle
precursors to the nucleic acid used to form the container during
formation of the container itself. Once inserted into the cavity,
the one or more nanoparticle precursors may be associated with the
nucleic acid container by, for example, being covalently attached,
physisorbed, chemisorbed, or attached to the nucleic acid container
through ionic interactions, hydrophobic and/or hydrophilic
interactions, electrostatic interactions, van der Waals
interactions, or combinations thereof. In other embodiments, one or
more nanoparticle precursors may be floating inside the cavity.
[0064] The walls of the nucleic acid container may also allow
nanoparticle precursor solutions to flow through it in order to
facilitate the formation of a nanoparticle. Generally, the walls of
the nucleic acid container are porous and may allow penetration
and/or transport of certain molecules and components into or out of
the container, but may prevent penetration and/or transport of
other molecules and components into or out of the container. The
ability of certain molecules to penetrate and/or be transported
into and/or across a wall of the container may depend on, for
example, the packing density of the nucleic acids forming the wall,
the thickness of the walls, and the chemical and physical
properties of the wall, as described in more detail below.
Accordingly, a nucleic acid container need not be open, and in some
embodiments may be substantially closed, while still allowing
certain nanoparticle precursors to enter into the cavity via the
pores and facilitating the formation of a nanoparticle in the
container.
[0065] Once one or more nanoparticle precursor are positioned in
the cavity, they can facilitate the formation of a nanoparticle 38
that may fill all portions of the cavity. The shape of nanoparticle
38 may be determined, at least in part, by the shape of the cavity
of the nucleic acid container. The shape of the cavity of the
nucleic acid container, in turn, may be varied by controlling the
configuration and orientation of the nucleic acid strands that form
the inner surfaces of the container, as described in more detail
below. FIG. 1B shows examples of nucleic acid containers having
cavities with different shapes that can be used to form
nanoparticles 38 having different shapes. Advantageously, a wide
variety of unique and/or predetermined shapes of nanoparticles can
be formed using the methods described herein.
[0066] In certain embodiments, growth of a nanoparticle in a cavity
of the container continues until the nanoparticle encounters an
inner surface or wall of the container. Chemical and/or physical
interactions between the growing nanoparticle and the inner surface
of the container may stop or significantly slow down the growth of
nanoparticle. In certain embodiments involving nucleic acid
containers that are substantially closed, the entire size and/or
shape of nanoparticle 38 may be controlled by the size and/or shape
of the cavity of the container, as described in more detail below.
In other embodiments, the growth of all or portions of a
nanoparticle stops before it is in contact with an inner surface
portion of the container.
[0067] Growing the nanoparticle inside a cavity of a nucleic acid
container generally involves more than simply the addition of
surface ligands to the surface of the nanoparticle precursor. For
example, in some embodiments in which a nanoparticle precursor is
used to grow a nanoparticle, the nanoparticle precursor may have a
shape that is substantially different from the shape of the
resulting nanoparticle that is grown from the nanoparticle
precursor. In some instances, the resulting nanoparticle has a more
complex shape than that of the nanoparticle precursor. In addition,
as described in more detail below, the volume of the resulting
nanoparticle may be substantially different (e.g., substantially
greater) than that of the nanoparticle precursor.
[0068] The combined nucleic acid container 20 and nanoparticle 38
shown in FIG. 1A may form a composite nanostructure 40 that may be
used in a variety of different applications, as described in more
detail below.
[0069] Although FIG. 1A shows the introduction of nanoparticle
precursor 34 into the cavity of the nucleic acid container after
the nucleic acid container has been formed, in other embodiments, a
nanoparticle precursor can be associated with a nucleic acid
container while the container is being formed. For example, design
of the nucleic acid container may involve including a binding site
on a portion of a nucleic acid that will form an inner surface
portion of the container. During annealing of the nucleic acids to
form the shape of the container, a nanoparticle precursor having a
binding site complementary to the binding site attached to nucleic
acid can be introduced to allow binding between the interior
surface of the container and the nanoparticle precursor.
[0070] As shown illustratively in FIG. 1A, nanoparticle 38 may be
formed by a seed-mediated growth process involving the use of
nanoparticle precursor 34. That is, the nanoparticle precursor,
which may itself be in the form of a nanoparticle, may be used as a
seed to grow a larger nanoparticle in the presence of other
precursors (e.g., nanoparticle precursor solutions) that may
determine the material composition of nanoparticle 38. Any suitable
combinations of nanoparticle precursors and nanoparticle precursor
solutions can be used. For example, to form a nanoparticle formed
of gold, a gold nanoparticle precursor and precursor solutions of
HAuCl.sub.4 and ascorbic acid may be used. To form a nanoparticle
of silver, a gold nanoparticle and precursor solutions of
AgNO.sub.3 and ascorbic acid may be used. Accordingly, the material
composition of the resulting nanoparticle can be controlled by
varying the types of precursors used. Examples of additional types
of nanoparticle precursors are provided in more detail below.
[0071] In some embodiments, nanoparticles can be formed by a method
other than a seed-mediated process. For example, one or more
nanoparticle precursors may fill all or portions of the cavity of a
nucleic acid container and optionally an external force such as
heat, light, pressure, electrical potential, magnetic force, and/or
electromagnetic force can be applied to facilitate the growth or
formation of the nanoparticle. In some cases, a chemical component
can be added to facilitate the growth of the nanoparticle. In one
particular embodiment, a nanoparticle precursor such as a monomer
(e.g., a monomer of an organic polymer, such as a synthetic organic
polymer), and optionally one or more catalysts to trigger the
growth of the monomer, can be introduced in solution form into the
cavity of a nucleic acid container. Polymerization of the monomers
can take place in the cavity of the nucleic acid container by, for
example, applying heat, light or other stimulus to allow formation
of a polymeric nanoparticle. Monomers such as nucleotides and amino
acids can be used.
[0072] The resulting nanoparticle may have a physical state that is
different from that of the nanoparticle precursor. For example, the
formation of a nanoparticle may involve applying a stimulus to
cause transformation of a nanoparticle precursor into a different
form so as to form the resulting nanoparticle. For example, a
nanoparticle precursor may be in the form of a liquid, and the
resulting nanoparticle may be in the form of a solid or solid-like
substance (e.g., a gel). In other embodiments, the resulting
nanoparticle has the same physical state as that of the
nanoparticle precursor. For example, both the nanoparticle and the
nanoparticle precursor may be in the form of solids.
[0073] As described herein, in some embodiments the formation of a
nanoparticle in a cavity of a nucleic acid container is stopped or
significantly slowed down by confinement of the nanoparticle in the
container. For example, the formation of the nanoparticle may be
stopped by chemical interaction between a surface of the
nanoparticle and an inner surface of the nucleic acid container. It
should be appreciated, however, that in some embodiments, synthesis
of the nanoparticle can be stopped or significantly slowed down
before the nanoparticle fills the entire volume of the nucleic acid
container. For example, where an external force such as those
described herein are used to facilitate formation of the
nanoparticle, application of an external force may be stopped prior
to the nanoparticle filling the entire volume of a nucleic acid
container.
[0074] Accordingly, in some embodiments, the nucleic acid container
comprises a cavity having a volume, and only a portion of the
volume is filled with the nanoparticle. For example, less than
100%, less than 80%, less than 60%, less than 40%, less than 20%,
or less that 10% of the volume of a cavity may be filled with the
nanoparticle. In certain embodiments, at least 5%, at least 10%, at
least 15%, at least 20%, at least 40%, at least 60%, or at least
80%, at least 90%, at least 95%, or at least 99% of the volume of
cavity is filled with a nanoparticle. Combinations of the
above-noted ranges are also possible (e.g., less than 100% but at
least of 20% of the volume of the container may be filled with the
nanoparticle). In yet other embodiments, substantially all of the
volume of a nucleic acid container is filled with a
nanoparticle.
[0075] Methods described herein may be used to form a population of
nanoparticles having relatively high uniformity in size, shape,
and/or mass. For example, in some embodiments, a composition
includes nanoparticles wherein at least 60%, at least 80%, at least
90%, at least 95%, at least 98%, at least 99%, or 100% of the
nanoparticles vary in dimension (e.g., a cross-sectional dimension,
a maximum cross-sectional dimension, a width, a height, or a
length) or mass by less than three standard deviations, less than
two standard deviations, less than one standard deviation, less
than 0.5 standard deviation, or less than 0.2 standard deviation of
the median or average dimension or mass of all the nanoparticles in
the composition. In certain embodiments, a composition may include
nanoparticles that have a distribution of dimensions (e.g.,
cross-sectional dimension, width, height or length) or mass such
that no more than 20%, no more 15%, no more than 10%, no more than
5%, no more than 3%, no more than 2%, or no more than 1% of the
nanoparticles have a dimension or mass that differs by more than
20%, by more than 15%, by more than 10%, by more than 5%, by more
than 3%, by more than 2%, or by more than 1% of the median or
average value of the corresponding dimension or mass of all the
nanoparticles in the composition.
[0076] The methods described herein may also be used to control and
tune the material composition of the nanoparticle at different
regions of the nanoparticle. For example, in a gold/silver
nanoparticle alloy formed by the methods described herein, the
ratio of gold to silver may be 8:1 (wt:wt) at a first region and a
ratio of 1:8 (wt:wt) at a second region. Generally, an alloy
including a first and second component may have a ratio of the
first to the second component of, for example, between 1:20 and
20:1 (wt:wt). In some embodiments, the ratio of the first to the
second component of an nanoparticle alloy may be at least 1:20, at
least 1:15, at least 1:10, at least 1:8, at least 1:6, at least
1:4, at least 1:2, at least 1:1, at least 2:1, at least 4:1, at
least 6:1, at least 8:1, at least 10:1, at least 15:1, or at least
20:1 at a first region of the nanoparticle, and a ratio of the
first to the second component of at least 1:20, at least 1:15, at
least 1:10, at least 1:8, at least 1:6, at least 1:4, at least 1:2,
at least 1:1, at least 2:1, at least 4:1, at least 6:1, at least
8:1, at least 10:1, at least 15:1, or at least 20:1 at a second
region, wherein the ratios are different between the first and
second regions. Alloys including a third component may also be
possible. In some cases, the ratio between the first to third
components, or the second to third components, have one of the
above-noted ratios.
[0077] Methods described herein may also be used to form
nanoparticles having relatively high uniformity in material
composition. For example, in some embodiments, a composition
includes nanoparticles wherein at least 60%, at least 80%, at least
90%, at least 95%, at least 98%, at least 99%, or 100% of the
nanoparticles vary in material composition by less than three
standard deviations, less than two standard deviations, less than
one standard deviation, less than 0.5 standard deviation or less
than 0.2 standard deviation of the median or average material
composition of all the nanoparticles in the composition. In some
embodiments, such compositions having a relatively high uniformity
in material composition include nanoparticle alloys having the
above-noted ratios between first and second components.
[0078] In other embodiments in which a mixture of different
nanoparticles having different shapes is desired, a method may
include using different nucleic acid containers to form a variety
of different nanoparticles in parallel. For example, in some
instances between 2 and 1,000 nanoparticles (e.g., between 2 and
500, between 2 and 200, or between 2 and 100 nanoparticles), each
having different predetermined shapes, may be formed in parallel.
In some embodiments, a method may include using different nucleic
acid containers to form, in parallel, at least 10, at least 15, at
least 20, at least 30, at least 50, or at least 100 nanoparticles
(e.g., inorganic nanoparticles) each having different shapes. A
composition may include such numbers of differently shaped
nanoparticles and/or nanostructures.
[0079] In some embodiments, the methods described herein can be
used to form nanoparticles in the absence of certain materials such
as surfactants (e.g., cetyltrimethylammonium bromide). As such, a
variety of different materials, including materials that are not
compatible with surfactants, may be used in the methods described
herein. In certain embodiments, the nanoparticle may be synthesized
in the absence of an oxide template.
[0080] In certain embodiments, the methods described herein can be
performed ex-vivo (e.g., in test tubes). In other embodiments, the
methods can be performed under in-vitro or in-vivo conditions, such
as in bacteria and cells.
[0081] Other features of nucleic acid containers, nanoparticles,
and combinations thereof are described in more detail below.
[0082] As described herein, nucleic acid containers can be used as
a template to form nanoparticles within one or more cavities of the
container. The nucleic acid container may have a predetermined
three-dimensional shape or structure (e.g., a non-random
three-dimensional shape or structure).
[0083] The nucleic acid containers described herein can be formed
using any suitable method. In some embodiments, a nucleic acid
container may be constructed using a non-random process, such as
process that involves deliberate folding and/or bending of nucleic
acid strands to form the shape of the nucleic acid container. In
some embodiments, a DNA "origami" method may be used. Using such a
method, 3-dimensional (3D) nucleic acid containers with arbitrary
user specified shapes can be formed. In some embodiments, the
nucleic acid container is formed primarily of a single strand of
nucleic acid, with optional multiple shorter strands that may help
define the resulting shape of the container. For example, in some
embodiments involving the use of a DNA "origami" method, a long
"scaffold" DNA strand "rasterizes" a target structure shape, while
many short "staple" strands hybridize to the scaffold and hold it
in the target shape. Other methods of constructing nucleic acid
containers using similar polymers are also possible. For example,
in the field known as structural DNA nanotechnology (e.g., DNA
origami and designs involving single-stranded tiles),
self-assembled nucleic acids (particularly, DNA) have been used to
construct diverse synthetic molecular structures and devices such
as ribbons, tubes, lattices, and arbitrary 2D and 3D shapes.
Moreover, channels can be introduced into hollow DNA
nanostructures, e.g., DNA nanotubes and 3D barrels. These synthetic
molecular structures and hollow DNA nanostructures may include
cavities for nanoparticle growth according to the present invention
and as described herein.
[0084] Nucleic acid containers can be designed with or without
software packages such as caDNAno and other software known in the
art. In some embodiments, the scaffold strand may be naturally
occurring such as that of the M13 virus. In other embodiments, the
scaffold strand may be non-naturally occurring. In either instance,
the sequence of the scaffold strand should be known. Software
packages such as NUPACK can also be used to design dynamic sequence
components. Those of ordinary skill in the art are familiar with
these methods as evidenced by the disclosures in U.S. Pat. Nos.
7,745,594 and 7,842,793; U.S. Patent Publication No. 2010/00696621;
and Goodman et al. Nature Nanotechnology, doi 10.1038/nnano.2008.3,
the entire contents of which including the methods for generating
nucleic acid based structures are incorporated by reference
herein.
[0085] As described herein, in some embodiments, a nucleic acid
container is formed primarily of a single strand of nucleic acid
(i.e., a "scaffold"). For example, in some embodiments, a single
strand of nucleic acid used to form the nucleic acid container
makes up at least 50%, at least 60%, at least 70%, at least 80%, at
least 90%, or at least 95% of the total molecular weight of the
overall nucleic acid container. In certain embodiments, the
molecular weight of a single strand of nucleic acid that forms the
primary structure of the nucleic acid container may have a
molecular weight of, for example, between 100 kDa and 10,000 kDa.
In some embodiments, the molecular weight of a single strand of
nucleic acid that forms the primary structure of the nucleic acid
container may be, for example, at least 300 kDa, at least 600 kDa,
at least 640 kDa, at least 800 kDa, at least 1,000 kDa, at least
2,000 kDa, at least 4,000 kDa, or at least 6,000 kDa. In some
cases, the molecular weight of a single strand of nucleic acid that
forms the primary structure of the nucleic acid container may be,
for example, less than 6,000 kDa, less than 4,000 kDa, less than
2,000 kDa, less than 1,000 kDa, less than 800 kDa, less than 600
kDa, or less than 300 kDa. Other molecular weight values are also
possible. Combinations of the above-referenced ranges are also
possible (e.g., a molecular weight of at least 300 kDa and less
than 1,000 kDa).
[0086] In some cases, a single strand of a nucleic acid used to
form a nucleic acid container has a length of, for example, between
1000 bases (1 kb) and 300 kilobases (300 kb). The single strand of
nucleic acid may have a length of, for example, at least 1,000
bases long (1 kb), at least 2 kb, at least 5 kb, at least 10 kb, at
least 15 kb, at least 20 kb, at least 25 kb, at least 30 kb, at
least 40 kb, at least 50 kb, at least 60 kb, at least 70 kb, at
least 100 kb, at least 150 kb, or at least 200 kb. In certain
embodiments, the single strand of nucleic acid may have a length
of, for example, less than 200 kb, less than 150 kb, less than 100
kb, less than 70 kb, less than 60 kb, less than 50 kb, less than 40
kb, less than 30 kb, less than 20 kb, less than 10 kb, less than 5
kb, or less than 3 kb. Combinations of the above-referenced ranges
are also possible.
[0087] Furthermore, it should be appreciated that the formation of
a nucleic acid container using two or more (e.g., 2, 3, 4, 5, 6,
etc.) single strands of nucleic acids that act as "scaffolds" is
also contemplated. The two or more single strands of nucleic acids
may have molecular weights and/or lengths in the ranges noted
above, or they may have different ranges of molecular weights
and/or lengths.
[0088] A nucleic acid container may include, in some embodiments,
one or more portions that are open relative to other portions of
the structure. For example, in one particular embodiment, a nucleic
acid container includes two opposite ends that are open such that
fluid and certain molecules can flow through its interior. An
example of such a structure is shown in FIGS. 2A and 2B, which show
top and side views of a container, respectively. In other
embodiments, a nucleic acid container may have one open and, as
shown in the embodiment illustrated in FIGS. 2C and 2D which show
top and side views of a container, respectively. In other
embodiments, a nucleic acid container may include more than two
openings. In yet other embodiments, a nucleic acid container may be
substantially closed, as shown illustratively in FIGS. 2E and 2F,
which show top and side views of a container, respectively.
Generally, the nucleic acid container may be made sufficiently
rigid to maintain its shape under thermal and/or other external
forces typical in solution and/or during nanoparticle growth
conditions.
[0089] A nucleic acid container, and a cavity within a nucleic acid
container, may have any suitable shape. Advantageously, nucleic
acid containers can be designed to include a cavity having
particular shapes that may act as a template for forming all the
portions of the nanoparticle. As described herein, the shape of the
cavity of a nucleic acid container may be used, in some
embodiments, for forming a nanoparticle having a complementary
shape. Non-limiting examples of shapes of nucleic acid container
cavities include tubes, boxes, barrels, rectangles, rods, "T" s,
"L" s branched structures, diamonds, stars, squares,
parallelograms, rhomboids, triangles, pentagons, hexagons, and
polyhedrons, including shapes substantially similar thereto.
Portions of the cavity may be linear in some cases, and curved in
other cases. In some instances, one or more channels are present in
the nanostructure. In some cases, the cavity of a nucleic acid
container has a non-spherical shape. In other cases, the cavity of
a nucleic acid container has an arbitrary or irregular shape. In
some embodiments, the cavity of a nucleic acid container has a
symmetric shape. In some embodiments, the cavity of a nucleic acid
container has an asymmetric shape (e.g., no axis of symmetry). It
is to be understood that a nucleic acid container may have a
variety of shapes and forms provided its structure is suitable for
the application contemplated.
[0090] It should be appreciated that the cavity of the nucleic acid
container may have the same shape, or a different shape, compared
to the shape of the outer surface of the nucleic acid container.
For instance, while the cavity of the nucleic acid container may be
designed so that it has no axis of symmetry, the outer surface of
the container may have a shape that does have an axis of
symmetry.
[0091] A cross-section of a cavity of a nucleic acid container may
have any suitable shape. For example, a cross-section may be in the
shape of a rectangle, rod, "T," "L," branched structure, diamond,
star, square, parallelogram, triangle, pentagon, or hexagon,
including shapes substantially similar thereto. Other shapes are
also possible. In some cases, a cross-section of a cavity has a
non-spherical shape. In some embodiments, each cross-section of a
cavity of a nucleic acid container has a non-spherical shape. In
other cases, a cross-section of a cavity has an arbitrary or
irregular shape, a symmetric shape, or an asymmetric shape. In
certain embodiments, each cross-section of a cavity has a symmetric
shape. In other embodiments, each cross-section of a cavity has an
asymmetric shape.
[0092] A nucleic acid container may, in some embodiments, have a
3-dimensional shape that includes various numbers of different
sides. For example, a nucleic acid container may have a cavity that
is in the shape of a prism that includes five sides in its overall
shape, and a cross-section that includes 3 sides. In certain
embodiments, a cavity of a nucleic acid container may include, for
example, between 3 and 10.sup.6 sides (e.g., between 3 and 100,
between 3 and 70, between 3 and 50, or between 3 and 30, between 3
and 25, between 3 and 20, between 3 and 15, between 6 and 15,
between 3 and 10, between 6 and 10, between 3 and 9, between 3 and
5, between 100 and 10.sup.3, between 10.sup.3 and 10.sup.4, between
10.sup.4 and 10.sup.5, or between 10.sup.5 and 10.sup.6 sides) in
its overall shape. In some embodiments, a cavity of a nucleic acid
container may include, for example, at least 4, at least 5, at
least 6, at least 7, at least 8, at least 9, at least 10, at least
11, at least 12, at least 15, at least 20, at least 25, or at least
50 different sides. In certain embodiments, a cavity of a nucleic
acid container may have a cross-section that includes, for example,
between 3 and 10.sup.6 sides (e.g., between 3 and 100, between 3
and 70, between 3 and 50, or between 3 and 30, between 3 and 25,
between 3 and 20, between 3 and 15, between 6 and 15, between 3 and
10, between 6 and 10, between 3 and 9, between 3 and 5, between 100
and 10.sup.3, between 10.sup.3 and 10.sup.4, between 10.sup.4 and
10.sup.5, or between 10.sup.5 and 10.sup.6 sides) in its overall
shape. In some embodiments, a cavity of a nucleic acid container
may have a cross-section that includes at least 4, at least 5, at
least 6, at least 7, at least 8, at least 9, at least 10, at least
11, at least 12, at least 15, at least 20, at least 25, or at least
50 different sides. Combinations of the above-noted ranges are also
possible. In some cases, a side may be non-curved (e.g., linear),
although curved sides or faces may also be possible.
Advantageously, nucleic acid containers including cavities having
complex shapes can be formed using the methods described herein,
and can be used to form nanoparticles having arbitrary complex
shapes.
[0093] The angle between two sides may be, for example, between
1.degree. and 180.degree. (e.g., between 1.degree. and 120.degree.,
between 1.degree. and 90.degree., or between 1.degree. and
45.degree.). In some cases, the angle between two sides may be, for
example, greater than 1.degree., greater than 10.degree., greater
than 30.degree., greater than 45.degree., greater than 60.degree.,
greater than 90.degree., greater than 120.degree., or greater than
150.degree.. In certain cases, the angle between two sides may be,
for example, less than 180.degree., less than 150.degree., less
than 120.degree., less than 90.degree., less than 60.degree., less
than 45.degree., less than 30.degree., or less than 10.degree..
Other angles are also possible. A combination of the above-noted
ranges are also possible.
[0094] A nucleic acid container may comprise any suitable number of
open sides. For example, a nucleic acid container may include at
least one open side, at least two open sides, at least three open
sides, or at least four open sides. In some cases, the nucleic acid
container includes two opposing sides that are open.
[0095] In some embodiments, a nucleic acid container comprises one
or more lids that can be open or closed (e.g., reversibly or
irreversibly). For example, as shown in the embodiments illustrated
in FIGS. 3A and 3B, which show side and perspective views,
respectively, nucleic acid container 20 may include lids 50 and 52
that may be open or closed. FIGS. 3A and 3B show the lids being in
an open configuration, and may be closed by varying the position of
hinge 54. Similarly, the nucleic acid container shown in FIGS. 3C
and 3D include a lid 50 and a hinge 54 that may allow the opening
and the closing of the lids. In some cases, the nucleic acid
container includes a switchable lid. The nucleic acid container may
be designed such that the lid can be switched on or off (e.g., open
or closed) depending on the presence or absence of a specific
nucleic acid strand, producing different dimensional
controllability of the nucleic acid container. For example, in some
embodiments, when the lid is open, the nucleic acid container may
control the diameter of the growing nanoparticle, whereas when the
lid is closed, both the diameter and length of nanoparticles may be
controlled. Examples of articles and methods involving nucleic acid
containers that include lids are described in more detail in U.S.
Provisional Application No. 61/481,542, which is incorporated
herein by reference in its entirety for all purposes.
[0096] In certain embodiments, nucleic acid containers may have
suitable configurations for forming nanoparticles that are at least
partially hollow. For example, as shown illustratively in FIGS. 3E
and 3F, nucleic acid container 20 includes an outer wall 27 and an
inner wall 55 that define the shape of cavity 30. The inner wall
blocks the formation of the nanoparticle at this region, thereby
allowing the formation of a nanoparticle having cross-section in
the shape of a ring. It should be appreciated that other
configurations of inner and outer walls of nucleic acid containers
are possible to make more complex-shaped nanoparticles that are at
least partially hollow.
[0097] Different methods may be used to fabricate the nucleic acid
containers shown in FIGS. 3E and 3F. One exemplary method involves
direct folding of DNA via structural DNA nanotechnology methods,
such as DNA origami or single-stranded tiles as described herein.
Another exemplary method is based on the self-assembly of different
DNA sub-units. For instance, DNA tube (e.g., outer wall 27) and
rods (e.g., inner wall 55) may be prepared separately, and then
assembled together via DNA hybridization or other interactions, to
form a rod-in-tube structure like that shown in FIGS. 3E and
3F.
[0098] In some embodiments, a lid may include at least 1 binding
site for binding to a complementary binding site positioned on at
least a portion (e.g., a surface) of a container. The binding sites
may allow secure attachment of the lid to the container, as
described in more detail herein. In some cases, a lid or a surface
of the container to be closed by a lid comprises at least 2, at
least 4, at least 6, at least 8, at least 10, at least 12, at least
14, at least 16, at least 18, or at least 20 binding sites. The
binding sites may be isolated from one another in some embodiments.
The binding sites may include, for example, nucleic acid strands,
although other binding units may be used.
[0099] Although many of the figures show nucleic acid containers
having a single cavity, it should be appreciated that in some
embodiments a nucleic acid container may include more than one
cavity (and, therefore, multiple positions for the formation of
multiple nanoparticles). For example, a nucleic acid container may
have, in some cases, between 2 and 200 cavities (e.g., between 2
and 100, between 2 and 50, between 2 and 20, between 2 and 10, or
between 2 and 5 cavities). In some embodiments, a nucleic acid
container includes at least 2, at least 5, at least 10, or at least
15 cavities. In certain embodiments, a nucleic acid container
includes less than 20, less than 15, less than 10, less than 5, or
less than 3 cavities. Other numbers of cavities are also possible.
Combinations of the above-noted ranges are also possible.
[0100] The size of a cavity of a nucleic acid container can be
varied as desired. In some embodiments, a cross-sectional dimension
of a cavity (e.g., as measured by the distance between two inner
surface portions of the container that surround a cavity), is
between 2 nm and 1 micron (e.g., between 2 nm and 500 nm, or
between 2 nm and 250 nm). In some embodiments, a cross-sectional
dimension of a cavity may be, for example, less than or equal to 1
micron, less than or equal to 500 nm, less than or equal to 250 nm,
less than or equal to 100 nm, less than or equal to 75 nm, less
than or equal to 50 nm, less than or equal to 40 nm, less than or
equal to 30 nm, less than or equal to 20 nm, less than or equal to
10 nm, or less than or equal to 2 nm. In certain embodiments, a
cross-sectional dimension of a cavity is greater than or equal to 1
nm, greater than or equal to 2 nm, greater than or equal to 5 nm,
greater than or equal to 10 nm, greater than or equal to 20 nm,
greater than or equal to 30 nm, greater than or equal to 40 nm,
greater than or equal to 50 nm, greater than or equal to 100 nm,
greater than or equal to 500 nm, or greater than or equal to 1
micron. Other values are also possible. Combinations of the
above-noted ranges are also possible. In some embodiments, the
above-noted cross-sectional dimension of the cavity is a maximum
cross-sectional dimension of the cavity.
[0101] Although nucleic acid containers and cavities of nucleic
acid containers having sizes generally on the order of nanometers
are primarily described, in some embodiments, a variety of nucleic
acid containers may be used that have significantly different
sizes. For example, in some embodiments, the a plurality of
containers is used with some containers being small enough to be
positioned partially or fully within other containers.
[0102] The thickness of the walls of the nucleic acid container can
also vary as desired. In some embodiments, the nucleic acid
container comprises walls that surround a cavity, and the average
thickness of the walls may be between 1 nm and 1 micron (e.g.,
between 1 nm and 500 nm, or between 1 nm and 250 nm). The average
thickness of the walls may be, for example, less than or equal to 1
micron, less than or equal to 500 nm, less than or equal to 250 nm,
less than or equal to 100 nm, less than or equal to 75 nm, less
than or equal to 50 nm, less than or equal to 40 nm, less than or
equal to 30 nm, less than or equal to 20 nm, less than or equal to
10 nm, or less than or equal to 1 nm. In certain embodiments, the
average thickness of the walls of a nucleic acid container is
greater than or equal to 1 nm, greater than or equal to 10 nm,
greater than or equal to 25 nm, greater than or equal to 50 nm,
greater than or equal to 100 nm, greater than or equal to 500 nm,
or greater than or equal to 1 micron. Other values are also
possible. Combinations of the above-noted ranges are also
possible.
[0103] In some embodiments, the walls of a nucleic acid container
may be formed of more than one layer of nucleic acids. Increasing
the number of layers of nucleic acids may increase the rigidity
(and thickness) of the wall. In some embodiments, increased wall
rigidity may result in the container walls maintaining their shape
during growth of a nanostructure inside the container. For
instance, increased wall rigidity may confine the expansion of a
nanostructure grown inside the container (e.g., by compressing the
nanostructure during growth) such that the nanostructure does not
grow beyond the size of the cavity of the container prior to
expansion. In other embodiments, fewer number of layers may allow a
nanostructure to grow beyond the size of the cavity of the
container prior to expansion, and may cause the walls of the
container to expand (e.g., bend) during nanostructure growth. In
some embodiments, a wall of a nucleic acid container may have at
least 2, at least 3, at least 4, at least 5, at least 6, at least
8, or at least 10 layers. In some cases, a wall of a nucleic acid
container may have less than or equal to 20, less than or equal to
15, less than or equal to 10, less than or equal to 8, or less than
or equal to 5 layers. Other values are also possible. Combinations
of the above-noted ranges are also possible. The layers of the
walls may have any suitable design such as a square-lattice design
or a honeycomb design.
[0104] Advantageously, by controlling the thickness of walls of the
nucleic acid container, diffusion kinetics, such as the diffusion
kinetics of ions or molecules, across the wall of the container can
be controlled. Such control may allow the tuning of the growth
kinetics of the nanoparticle and/or the material composition of
nanoparticle alloys. For example, in some cases, a first portion of
a wall of the nucleic acid container has a first thickness, and a
second portion of the wall has a second thickness, wherein the
first and second thicknesses are defined by one of the above-noted
ranges. A first, thicker wall portion may, for example, impede a
first nanoparticle precursor solution from entering into the cavity
to a greater extent than a second nanoparticle precursor solution,
thereby allowing more of the second nanoparticle precursor solution
to enter into the cavity at the first wall portion. As a result,
the nanoparticle may have a higher amount of a material formed from
the second nanoparticle precursor at the first wall portion.
[0105] Those of ordinary skill in the art are familiar with
techniques to determine sizes of structures and particles. Examples
of suitable techniques include dynamic light scattering (DLS),
transmission electron microscopy (TEM), scanning electron
microscopy, electroresistance counting, and laser diffraction.
Other suitable techniques are known to those or ordinary skill in
the art. Although many methods for determining sizes of
nanostructures are known, the sizes described herein (e.g.,
cross-sectional dimensions, thicknesses) refer to ones measured by
transmission electron microscopy.
[0106] The nucleic acid containers may also include other
components such as those described herein (e.g., markers, binding
sites, quantum dots, nanoparticles, nucleic acids, proteins, etc.).
For example, in some embodiments, a nucleic acid container includes
one or more inorganic structures (e.g., inorganic nanostructures),
such as an inorganic nanoparticle or a quantum dot. The one or more
components may fill a portion of the cavity in some embodiments,
e.g., such that a nanoparticle formed by templated synthesis inside
the cavity forms around the component or is combined with the
component. In some cases, the component is incorporated into the
nanoparticle being formed. In other embodiments, the inorganic
structure is embedded in the nucleic acid container walls and does
not fill a portion of the cavity.
[0107] Any suitable nanoparticle precursor can be used to form a
nanoparticle as described herein. A nanoparticle precursory may be
used to initiate, catalyze and/or grow a nanoparticle. In some
cases, all or portions of the material of the nanoparticle
precursor may be incorporated into the resulting nanoparticle. In
some embodiments, a nanoparticle precursor may be used for forming
a nanoparticle using a seed-mediated growth process. In some such
embodiments, a nanoparticle precursor may be in a solid form. For
example, the nanoparticle precursor may be in the form of a
nanoparticle, such as an inorganic nanoparticle. In some cases, a
nanoparticle precursor comprises a crystal. In certain cases, a
nanoparticle precursor comprises a metal. Non-limiting examples of
metals include Au, Ag, Cd, Cr, Co, Ti, Zn, Cu, Pb, Mn, Ni, Mg, Fe,
Pd, and Pt. In other embodiments, a nanoparticle precursor
comprises a semiconductor (e.g., Rh, Ge, silicon, silicon compounds
and alloys, cadmium selenide, cadmium sulfide, indium arsenide, and
indium phosphide). In some cases, the nanoparticle precursor may
comprise a Group II-VI (e.g., IV-VI) element. In certain
embodiments, the nanoparticle precursor comprises a metal oxide or
a metal fluoride. In some cases, the nanoparticle precursor
comprises an alloy. In some cases, the nanoparticle precursor
comprises a doped compound. Combinations of such and other
materials are also possible.
[0108] Non-limiting examples of nanoparticle precursors include the
following. For the formation of Au, Ag, Pt and Pd nanoparticles,
gold nanoparticles can be used as a precursor. Alternatively, Ag,
Pt and Pd nanoparticles can be used as a precursor. For the
formation of semiconductors and metal oxide nanoparticles (e.g.,
nanoparticles including one or more of Cd, Cr, Co, Ti, Zn, Mn, Ni,
Mg, Fe, or a different material described herein), clusters or
small-sized nanoparticles of corresponding materials can be used as
seeds. In other embodiments, enzymes or peptides can be used as
nucleation sites if they can trigger the growth of nanoparticles
(e.g., organic nanoparticles). Some catalysts can also be used to
trigger the growth of polymers or nanoparticles, such as
Pt(PPh.sub.3).sub.2Cl.sub.2.
[0109] As described herein, nanoparticle precursors can also be in
the form of solutions. The material composition of the resulting
nanoparticle can be controlled by varying the types of precursor
solutions used. Any suitable solution can be used to form a
nanoparticle, and can be chosen using the description provided
herein in combination with general knowledge in the art. For
instance, a HAuCl.sub.4 precursor solution may be used to form gold
nanoparticles and a AgNO.sub.3 precursor solution may be used to
form silver nanoparticles (optionally in combination with other
solutions such as ascorbic acid). Combinations of nanoparticle
precursors and precursor solutions can also be determined by those
of ordinary skill in the art in combination with the description
provided herein.
[0110] In other embodiments, a nanoparticle precursor may be used
for forming a nanoparticle using a non-seed-mediated growth
process. For example, in some embodiments, nanoparticle precursors
may be in the form of a monomer or a polymer, and the formation of
a nanoparticle may comprise polymerizing and/or cross-linking of
monomer and/or polymer units.
[0111] In some cases, a nanoparticle precursor may comprise an
amino acid, a peptide, a nucleotide, or a nucleic acid (e.g., to
form a nanoparticle that is formed substantially of amino acids,
peptides, or nucleic acids).
[0112] In other cases, a nanoparticle precursor may include other
functionalities such as binding sites, or may be imparted with
certain surface functionalities. For example, the nanoparticle
precursor may comprise a self-assembled monolayer to impart a
particular surface chemistry to the precursor. In certain
embodiments, the nanoparticle precursor may include a binding site
or other suitable component to allow it to be attached to a portion
of the nucleic acid container. In other embodiments, the
nanoparticle precursor may be suspended in the cavity of the
container and not attached to a surface of the container.
[0113] A nanoparticle precursor may have any suitable size. In some
embodiments, a nanoparticle precursor has at least one
cross-sectional dimension that is between 0.5 nm and 1 micron
(e.g., between 0.5 nm and 500 nm, or between 0.5 nm and 250 nm). In
some embodiments, a nanoparticle precursor has at least one
cross-sectional dimension that is, for example, less than or equal
to 1 micron, less than or equal to 500 nm, less than or equal to
250 nm, less than or equal to 100 nm, less than or equal to 75 nm,
less than or equal to 50 nm, less than or equal to 40 nm, less than
or equal to 30 nm, less than or equal to 20 nm, less than or equal
to 10 nm, less than or equal to 5 nm, less than or equal to 1 nm,
or less than or equal to 0.5 nm. In some embodiments, a
nanoparticle precursor has at least one cross-sectional dimension
that is greater than or equal to 0.5 nm, greater than or equal to 1
nm, greater than or equal to 2 nm, greater than or equal to 5 nm,
greater than or equal to 10 nm, greater than or equal to 20 nm, or
greater than or equal to 50 nm. Other sizes are also possible.
Combinations of the above-noted ranges are also possible.
[0114] In some cases, the size (e.g., volume) of a nanoparticle
precursor is at least 500 times, at least 300 times, at least 200
times, at least 100 times, at least 50 times, at least 20 times, at
least 10 times, at least 5 times, or at least 2 times smaller than
the nanoparticle that is formed from the precursor or than the
cavity of the container. Other sizes are also possible.
[0115] In certain embodiments, a nanoparticle precursor has an
average molecular weight of, for example, between 20 Da and 10 kDa
(e.g., between 20 Da and 5 kDa, or between 20 Da and 1 kDa). A
nanoparticle precursor has an average molecular weight of, for
example, less than or equal to 10 kDa, less than or equal to 5 kDa,
less than or equal to 1 kDa, less than or equal to 500 Da, less
than or equal to 200 Da, less than or equal to 100 Da, less than or
equal to 50 Da, or less than or equal to 20 Da. In some
embodiments, a nanoparticle precursor has an average molecular
weight of greater than or equal to 10 Da, greater than or equal to
20 Da, greater than or equal to 50 Da, greater than or equal to 100
Da, greater than or equal to 200 Da, greater than or equal to 500
Da, greater than or equal to 1 kDa, or greater than or equal to 10
kDa. Other molecular weights are also possible. Combinations of the
above-noted ranges are also possible.
[0116] As described herein, a nanoparticle having a unique and/or
predetermined shape can be formed using the methods described
herein. For instance, a nanoparticle having a specific shape
(and/or size) can be formed by designing a cavity of a nucleic acid
container to have the complement of the desired shape (and/or size)
of the nanoparticle. The cavity may then act as a template for
forming all the portions of the nanoparticle. Non-limiting examples
of shapes of nanoparticles include tubes, boxes, barrels,
rectangles, rods, "T" s, "L" s branched structures, diamonds,
stars, squares, parallelograms, triangles, pentagons, hexagons,
polyhedrons, and rings, including shapes substantially similar
thereto. In some cases, a nanoparticle has a non-spherical shape.
In other cases, a nanoparticle has an arbitrary or irregular shape.
In some embodiments, a nanoparticle has a symmetric shape. A
symmetric shape may include, in some embodiments, at least 1, at
least 2, at least 3, or at least 4 axes of symmetry. In some
embodiments, a nanoparticle has an asymmetric shape (e.g., no axis
of symmetry).
[0117] In some embodiments, the nanoparticles formed by the methods
described herein are solid or solid-like (e.g., with solid cores),
and are not hollow structures. In other embodiments, portions of
the nanoparticle may be hollow, e.g., as described herein with
respect to FIGS. 3E and 3F. The hollow portion (e.g., cavity) of
the nanoparticle may be completely enclosed by the walls of the
nanoparticle, or partially enclosed (e.g., having one or more ends
that are opened).
[0118] A cross-section of a nanoparticle may have any suitable
shape. For example, a cross-section may be in the shape of a
rectangle, rod, "T," "L," branched structure, diamond, star,
square, parallelogram, triangle, pentagon, hexagon, or ring,
including shapes substantially similar thereto. Other shapes are
also possible. In some cases, a cross-section of a nanoparticle has
a non-spherical shape. In some embodiments, each cross-section of a
nanoparticle has a non-spherical shape. In other cases, a
cross-section of a nanoparticle has an arbitrary or irregular
shape, a symmetric shape, or an asymmetric shape. In certain
embodiments, each cross-section of a nanoparticle has a symmetric
shape. In other embodiments, each cross-section of a nanoparticle
has an asymmetric shape.
[0119] A nanoparticle may, in some embodiments, have a
3-dimensional shape that includes various numbers of different
sides. For example, a nanoparticle may be in the shape of a prism
that includes four sides. In certain embodiments, a nanoparticle
may include, for example, between 3 and 10.sup.6 sides (e.g.,
between 3 and 100, between 3 and 70, between 3 and 50, or between 3
and 30, between 3 and 25, between 3 and 20, between 3 and 15,
between 6 and 15, between 3 and 10, between 6 and 10, between 3 and
9, between 3 and 5, between 3 and 8, between 20 and 50, between 50
and 100, between 100 and 10.sup.3, between 10.sup.3 and 10.sup.4,
between 10.sup.4 and 10.sup.5, or between 10.sup.5 and 10.sup.6
sides). In some cases, a nanoparticle includes at least 4, at least
5, at least 6, at least 7, at least 8, at least 9, at least 10, at
least 11, at least 12, at least 15, at least 20, at least 25, at
least 50, at least 100, at least 10.sup.3, at least 10.sup.4, or at
least 10.sup.5 different sides.
[0120] As described herein, in some embodiments, a nanoparticle
comprises at least one surface portion having a shape that is
complimentary to a shape of an inner surface portion of a nucleic
acid container. A surface portion generally refers to a portion of
a surface that has a surface area that is greater than the surface
area of a single atom. In some instances, a surface portion may
have a surface area of at least 1 nm.sup.2, at least 2 nm.sup.2, at
least 5 nm.sup.2, at least 10 nm.sup.2, at least 15 nm.sup.2, at
least 20 nm.sup.2, at least 25 nm.sup.2, at least 30 nm.sup.2, at
least 50 nm.sup.2, at least 200 nm.sup.2, at least 200 nm.sup.2, at
least 500 nm.sup.2, or at least 1000 nm.sup.2 (where the largest
surface portion is the surface area of the entire
nanoparticle).
[0121] In some cases, a nanoparticle has at least one surface
portion that is complimentary to a shape of an inner surface
portion of a nucleic acid container at the sub-nanometer (e.g., 0.5
nm) level. For example, growth of a nanoparticle inside a container
may stop or significantly slow down upon reaching the confines of
the inner surface portion of the container. In some such
embodiments, the surface chemistry and/or physical interactions
between the nanoparticle and the inner surface of the container
prevents further growth of the nanoparticle. For example,
electrostatic interactions between the negatively charged phosphate
groups of the nucleic acid container and the positively charged
groups of the nanoparticle or nanoparticle precursor may prevent
further growth of the nanoparticle. The particular orientation of
the atoms of the inner surface portion of the nucleic acid
container may determine the final shape of the resulting
nanoparticle, where the inner surface portion and the shape of a
surface portion of the resulting nanoparticle are complementary,
e.g., at the sub-nanometer level.
[0122] In some embodiments, a relatively high percentage of the
surface area of a nanoparticle is complementary to a shape of an
inner surface (e.g., cavity) of a nucleic acid container at the
sub-nanometer (e.g., 0.5 nm) level. For example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%,
at least 97%, at least 98%, or at least 99% of the surface area of
the nanoparticle may be complementary to a shape of an inner
surface of a nucleic acid container at the sub-nanometer level. In
other embodiments, 100% of the surface area of the nanoparticle is
complementary to a shape of an inner surface of the nucleic acid
container at the sub-nanometer level.
[0123] In other embodiments, the resulting nanoparticle has at
least one surface portion that is complementary to a shape of an
inner surface portion of a nucleic acid container at the nanoscale
level (e.g., 1 nm or greater). For example, growth of a
nanoparticle inside a container may stop or significantly slow down
before reaching the confines of the inner surface of the container
such that the resulting nanoparticle has a volume less than the
volume of the cavity. In some such embodiments, the nanoparticle
has a substantially similar shape as that of the cavity, and is
complementary with the inner walls of the container at the
nanoscale level, but is not complementary with the inner walls of
the container at the sub-nanometer level.
[0124] In some embodiments, a relatively high percentage of the
surface area of a nanoparticle is complementary to a shape of an
inner surface portion of a nucleic acid container at the nanoscale
level (e.g., 1 nm or greater) level. For example, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 95%,
at least 97%, at least 98%, or at least 99% of the surface area of
the nanoparticle is complementary to a shape of an inner surface
portion of the a nucleic acid container at the nanoscale level. In
other embodiments, 100% of the surface area of the nanoparticle is
complementary to a shape of an inner surface portion of the nucleic
acid container at the nanoscale level.
[0125] A nanoparticle having complementarity at both the molecular
(e.g., Angstrom) and nanoscale level for different portions of the
nanoparticle is also possible.
[0126] In yet other embodiments, the resulting nanoparticle
includes one or more surface portions that are not complementary to
the inner surface portions of a nucleic acid container. For
example, fabrication of a nanoparticle in a nucleic acid container
that includes two open ends may result in a nanoparticle that has a
middle portion that is complementary (e.g., at the molecular level
or at a nanoscale level) with inner surface portions of the nucleic
acid container, but having ends that are not complementary to any
portions of the nucleic acid container. In some such embodiments,
the nanoparticle may grow outside of the cavity of the nucleic acid
container and may be shaped by other factors.
[0127] In certain embodiments, a nanoparticle comprises at least
two opposing surface portions, each of the at least two opposing
surface portions having a shape that is complementary to a shape of
an inner surface portion of the nucleic acid container. The
complementarity may be at the molecular level or at the nanoscale
level as described above. For example, as shown in the embodiment
illustrated in FIG. 1A, two opposing surface portions of the cavity
of the nucleic acid container are shown as inner surface portions
26A and 26B. The opposing surface portions may be parallel to one
another in some embodiments. Upon formation of nanoparticle 38,
surface portions of the nanoparticle at these positions may be
complementary to inner surface portions 26A and 26B of the nucleic
acid container.
[0128] In other embodiments, the nanoparticle comprises at least
two adjacent surface portions, each of the at least two adjacent
surface portions having a shape that is complementary to a shape of
an inner surface portion of the nucleic acid container. For
example, as shown in the embodiment illustrated in FIG. 1A,
adjacent inner surface portions 26A and 26C of the nucleic acid
cavity may be used to facilitate the formation of nanoparticle 38.
Accordingly, nanoparticle 38 may include adjacent surface portions
that are complementary to the inner surface portions of the cavity
at these positions.
[0129] A nanoparticle can be formed of any suitable material. A
nanoparticle formed by the methods described herein may be an
inorganic nanoparticle in some embodiments. In some cases, a
nanoparticle comprises a metal. Non-limiting examples of metals
include Au, Ag, Cd, Cr, Co, Ti, Zn, Cu, Pb, Mn, Ni, Mg, Fe, Pd, and
Pt. In other embodiments, a nanoparticle comprises a semiconductor
(e.g., Rh, Ge, silicon, silicon compounds and alloys, cadmium
selenide, cadmium sulfide, indium arsenide, and indium phosphide).
In some cases, the nanoparticle may comprise a Group II-VI (e.g.,
IV-VI) element. In certain embodiments, the nanoparticle comprises
a metal oxide or a metal fluoride. In some cases, the nanoparticle
a comprises an alloy. In some cases, the nanoparticle a comprises a
doped compound. Combinations of such and other materials are also
possible. The nanoparticle may be electronically and/or thermally
conductive in some embodiments, or non-electronically and/or
non-thermally conductive in other embodiments.
[0130] In other embodiments, a nanoparticle formed by the methods
described herein may be an organic nanoparticle. In some cases, a
nanoparticle may comprise a polymer, which may be cross-linked or
non-crosslinked. The polymer may be, for example, a synthetic
polymer and/or a natural polymer. Examples of synthetic polymers
include non-degradable polymers such as polymethacrylate and
degradable polymers such as polylactic acid, polyglycolic acid and
copolymers thereof. Examples of natural polymers include hyaluronic
acid, chitosan, and collagen. Conductive polymers may be used in
some embodiments. In certain embodiments, the nanoparticle does not
include a polymeric material (e.g., it is non-polymeric). In some
cases, a nanoparticle may comprise a protein, an enzyme, or a
peptide.
[0131] The surface of the nanoparticle may include the material
used to form the interior portions of nanoparticle, or may
otherwise be imparted with certain surface functionalities such as
binding sites or other components. For example, the nanoparticle
may comprise a self-assembled monolayer to impart a particular
surface chemistry to the nanoparticle. In some cases, a
nanoparticle surface may be passivated by one or more chemicals to
facilitate attachment of components.
[0132] A nanoparticle may have any suitable size. In some
embodiments, a nanoparticle has at least one cross-sectional
dimension (or at least two cross-sectional dimensions) that is/are
between 2 nm and 1 micron (e.g., between 2 nm and 500 nm, or
between 2 nm and 250 nm). In some embodiments, a nanoparticle has
at least one cross-sectional dimension (or at least two
cross-sectional dimensions) that is/are less than or equal to 1
micron, less than or equal to 500 nm, less than or equal to 250 nm,
less than or equal to 100 nm, less than or equal to 75 nm, less
than or equal to 50 nm, less than or equal to 40 nm, less than or
equal to 30 nm, less than or equal to 20 nm, less than or equal to
10 nm, or less than or equal to 5 nm. In certain embodiments, the
nanoparticle has at least one cross-sectional dimension (or at
least two cross-sectional dimensions) that is/are greater than or
equal to 2 nm, greater than or equal to 5 nm, greater than or equal
to 10 nm, greater than or equal to 50 nm, or greater than or equal
to 100 nm. Combinations of the above-noted ranges are also
possible. In some cases, the above-noted cross-sectional dimension
is a maximum cross-sectional dimension.
[0133] In some cases, a nanoparticle has a volume of, for example,
between 8 nm.sup.3 and 1 .mu.m.sup.3 (1*10.sup.9 nm.sup.3). In
certain embodiments, a nanoparticle has a volume of, for example,
at least 20 nm.sup.3, at least 50 nm.sup.3, at least 100 nm.sup.3,
at least 500 nm.sup.3, at least 1.times.10.sup.3 nm.sup.3, at least
5.times.10.sup.3 nm.sup.3, at least 1.times.10.sup.4 nm.sup.3, at
least 5.times.10.sup.4 nm.sup.3, at least 1.times.10.sup.5
nm.sup.4, at least 5.times.10.sup.5 nm.sup.3, at least
1.times.10.sup.6 nm.sup.3, at least 5.times.10.sup.6 nm.sup.3, at
least 1.times.10.sup.7 nm.sup.3, at least 5.times.10.sup.7
nm.sup.3, at least 1.times.10.sup.8 nm.sup.3, or at least
5.times.10.sup.8 nm.sup.3. In some embodiments, a nanoparticle has
a volume of, for example, less than 1.times.10.sup.9 nm.sup.3, less
than 5.times.10.sup.8 nm.sup.3, less than 1.times.10.sup.8
nm.sup.3, less than 5.times.10.sup.7 nm.sup.3, less than
1.times.10.sup.7 nm.sup.3, less than 5.times.10.sup.6 nm.sup.3,
less than 1.times.10.sup.6 nm.sup.3, less than 5.times.10.sup.5
nm.sup.3, less than 1.times.10.sup.5 nm.sup.3, less than
5.times.10.sup.4 nm.sup.3, less than 1.times.10.sup.4 nm.sup.3,
less than 5.times.10.sup.3 nm.sup.3, less than 1.times.10.sup.3
nm.sup.3, less than 500 nm.sup.3, less than 100 nm.sup.3, less than
50 nm.sup.3, or less than 20 nm.sup.3. Other ranges are also
possible. A combination of the above-noted ranges are also
possible. In certain embodiments, such volumes are based on the use
of a single nucleic acid scaffold. Larger volumes may be possible
using multiple nucleic acids scaffolds.
[0134] As described herein, a nanoparticle may have a size,
dimension (e.g., length, width, height), cross-sectional dimension,
and/or volume that is substantially similar to that of a cavity of
a container in which the nanoparticle is grown. In other
embodiments, a nanostructure may have a size, dimension (e.g.,
length, width, height), cross-sectional dimension, and/or volume
that is less than that of a cavity of a container in which the
nanoparticle is grown. For example, growth of the nanoparticle may
be stopped prior to the nanoparticle reaching one or more sides of
the container. In other embodiments, a nanostructure may have a
size, dimension (e.g., length, width, height), cross-sectional
dimension, and/or volume that is greater than that of a cavity of a
container in which the nanoparticle is grown. For example, the
walls of the container may be designed to be flexible such that the
nanoparticle grown within causes the walls of the container to
expand during growth. Other configurations of the nanoparticle
and/or container are also possible.
[0135] In some cases, the volume of the nanoparticle may be at
least two times, at least five times, at least 10 times, at least
20 times, at least 30 times, at least 50 times, at least 100 times,
at least 200 times, at least 500 times, or at least 1,000 times the
volume of a nanoparticle precursor used to form the nanoparticle.
The resulting volume of the nanoparticle may depend, at least in
part, on the size of the cavity of a nucleic acid container, which
may be varied as described herein. It should be appreciated that
nanoparticles having sizes smaller or larger than the volume of the
cavity of the nucleic acid container are also possible. For
example, portions of a nanoparticle may be fabricated inside a
container, and other portions of the nanoparticle may grow outside
of the container.
[0136] In some cases, a nanoparticle has an aspect ratio of at
least 2:1, at least 3:1, at least 5:1, at least 10:1, or at least
20:1. Other values of aspect ratio are also possible. As used
herein, "aspect ratio" refers to the ratio of a length to a width,
where length and width measured perpendicular to one another, and
the length refers to the longest linearly measured dimension.
[0137] As described herein, in some cases all or portions of a
nanoparticle may be encapsulated or coated by a nucleic acid
nanostructure. For example, at least 50%, at least 60%, at least
70%, at least 80%, at least 90%, at least 95%, at least 97%, at
least 98%, or at least 99% of the surface area of a nanoparticle
may be encapsulated or coated by a nucleic acid nanostructure. In
some instances, the entire surface area of a nanoparticle is
encapsulated or coated by a nucleic acid nanostructure. In some
such embodiments, all or portions of the nanoparticle may be
attached to the nucleic acid nanostructure. For example, all or
portions of the nanoparticle may be physisorbed onto the nucleic
acid nanostructure. In other embodiments, all or portions of the
nanoparticle are not attached to the nucleic acid nanostructure but
are merely adjacent to the nucleic nanostructure. The nanoparticle
may be in contact with, or not in contact with, the nucleic acid
nanostructure during encapsulation.
[0138] Compositions including nanoparticles that have one or more
of the features described herein are also provided. For example, in
some cases, a composition includes nanoparticles such that at least
80%, at least 90%, at least 95%, at least 97%, at least 98%, or at
least 99% of the nanoparticles in the composition are
non-spherical, have no axis of symmetry, are complementary to an
inner surface portion of a nucleic acid container (at a molecular
level or at an atomic level), or are encapsulated/coated by a
nucleic acid container.
[0139] In another set of embodiments, one or more surfaces of a
nanoparticle can be patterned (e.g., in two dimensions and/or in
three dimensions) with one or more components. This can be done by,
for example, fabricating a pattern of one or more components onto
the interior surface of the nucleic acid container used to form the
nanoparticle, and then triggering the growth of the nanoparticle
within the container. The growth of the nanoparticle within the
container can result in the incorporation of the pattern of the one
or more components onto the surface of the nanoparticle. With this
method, nano-scaled structures with details at the nanometer or
sub-nanometer level can be fabricated. Examples of such structures
include metallic coins and arbitrary shaped nano-scaled electronic
devices. Other structures such as core-shell type structures in the
form of alloys or polymer/metal hybrid structures can also be
formed. In some such embodiments, the shape of the shell layer may
be different from the general shape of the core. For example, a
hexagonal shell may surround a pentagon core. The methods described
herein are a more rational way of fabricating arbitrary
pre-designed structures compared to certain existing methods.
[0140] In some embodiments, a fabricated nanoparticle may be used
as a template to fabricate other materials that cannot be directly
prepared by DNA-directed synthesis (e.g., those requiring high
temperatures or pressures, and/or those formed in organic
solutions). In some embodiments, nanoparticles formed by the
methods described herein that are at least partially hollow may be
used as a template to form a secondary nanostructure within the
hollow portion (e.g., cavity) of the nanoparticle. In other
embodiments, the outer surface of the nanoparticle can be used as a
template to form a larger secondary nanostructure. The nanoparticle
that is used as a template may be optionally removed after forming
the secondary nanostructure. Nanoparticles formed of or comprising,
for example, gold, silver, or platinum may be suitable for use as
templates. Other materials for use as templates may also be
possible. A secondary nanostructure formed using a nanoparticle as
a template may be made of any suitable material, including those
materials described herein for nanoparticles in general. The
material of the secondary nanostructure may be the same as, or
different from, the material of the nanoparticle used as the
template.
[0141] The hollow portion (e.g., cavity) of a nanoparticle and/or a
secondary nanostructure formed using a nanoparticle may have any
suitable size. In some embodiments, a hollow portion (e.g., cavity)
of a nanoparticle and/or a secondary nanostructure has at least one
cross-sectional dimension (or at least two cross-sectional
dimensions) that is/are between 1 nm and 1 micron (e.g., between 1
nm and 500 nm, or between 1 nm and 250 nm). In some embodiments, a
hollow portion (e.g., cavity) of a nanoparticle and/or a secondary
nanostructure has at least one cross-sectional dimension (or at
least two cross-sectional dimensions) that is/are less than or
equal to 1 micron, less than or equal to 500 nm, less than or equal
to 250 nm, less than or equal to 100 nm, less than or equal to 75
nm, less than or equal to 50 nm, less than or equal to 40 nm, less
than or equal to 30 nm, less than or equal to 20 nm, less than or
equal to 10 nm, or less than or equal to 5 nm. In certain
embodiments, a hollow portion (e.g., cavity) of a nanoparticle
and/or a secondary nanostructure has at least one cross-sectional
dimension (or at least two cross-sectional dimensions) that is/are
greater than or equal to 2 nm, greater than or equal to 5 nm,
greater than or equal to 10 nm, greater than or equal to 50 nm, or
greater than or equal to 100 nm. Combinations of the above-noted
ranges are also possible. In some cases, the above-noted
cross-sectional dimension is a maximum cross-sectional
dimension.
[0142] In some cases, a hollow portion of a nanoparticle and/or a
secondary nanostructure has a volume of, for example, between 1
nm.sup.3 and 1 .mu.m.sup.3 (1*10.sup.9 nm.sup.3). In certain
embodiments, a hollow portion of a nanoparticle and/or a secondary
nanostructure has a volume of, for example, at least 20 nm.sup.3,
at least 50 nm.sup.3, at least 100 nm.sup.3, at least 500 nm.sup.3,
at least 1.times.10.sup.3 nm.sup.3, at least 5.times.10.sup.3
nm.sup.3, at least 1.times.10.sup.4 nm.sup.3, at least
5.times.10.sup.4 nm.sup.3, at least 1.times.10.sup.5 nm.sup.4, at
least 5.times.10.sup.5 nm.sup.3, at least 1.times.10.sup.6
nm.sup.3, at least 5.times.10.sup.6 nm.sup.3, at least
1.times.10.sup.7 nm.sup.3, at least 5.times.10.sup.7 nm.sup.3, at
least 1.times.10.sup.8 nm.sup.3, or at least 5.times.10.sup.8
nm.sup.3. In some embodiments, a hollow portion of a nanoparticle
and/or a secondary nanostructure has a volume of, for example, less
than 1.times.10.sup.9 nm.sup.3, less than 5.times.10.sup.8
nm.sup.3, less than 1.times.10.sup.8 nm.sup.3, less than
5.times.10.sup.7 nm.sup.3, less than 1.times.10.sup.7 nm.sup.3,
less than 5.times.10.sup.6 nm.sup.3, less than 1.times.10.sup.6
nm.sup.3, less than 5.times.10.sup.5 nm.sup.3, less than
1.times.10.sup.5 nm.sup.3, less than 5.times.10.sup.4 nm.sup.3,
less than 1.times.10.sup.4 nm.sup.3, less than 5.times.10.sup.3
nm.sup.3, less than 1.times.10.sup.3 nm.sup.3, less than 500
nm.sup.3, less than 100 nm.sup.3, less than 50 nm.sup.3, or less
than 20 nm.sup.3. Other ranges are also possible. A combination of
the above-noted ranges are also possible.
[0143] As described herein, nanoparticles, which may be optionally
coated or encapsulated by a nucleic acid container or
nanostructure, may have a variety of different shapes. In some
embodiments, the unique shapes of the nanoparticles can be used to
position one or more components (e.g., binding sites, markers,
ligands, etc.) on different portions of a nanoparticle surface
and/or a surface of the nucleic acid container to allow the
nanoparticle or nanostructure to be addressed in different ways.
For example, a nanoparticle having eight different sides may be
functionalized with one or more different components at each of the
eight different sides. In some cases, the components are in the
form of isolated components that can be added to unique positions
on the nanoparticle or nanostructure. For instance, a single,
isolated component may be positioned on a single side of the
nanoparticle, with each of the different sides of the nanoparticle
including a different single, isolated component. These and other
embodiments may be useful for detecting a variety of different
targets as described in more detail below.
[0144] In some embodiments, the positioning of a component on
precise locations of a nanoparticle and/or nano structure may be
controlled by the particular chemistry of the nucleic acid
container. For example, a nanoparticle that has eight different
sides may be surrounded (partially or fully) by a nucleic acid
container that has an inner cavity having eight different sides,
and walls with different chemistry at each of the different sides.
A component may be designed such that it has an affinity for a
certain portion of the nucleic acid container or nanostructure at
one of the sides that coats or encapsulates a side of the
nanoparticle. Thus, by designing the nucleic acid container to have
specific sequences or chemistry at particular positions, the
addition of a specific component to the surface of the nucleic acid
container and/or the surface of the nanoparticle at one of those
positions can be performed. Similarly, additional components can be
specifically added to different positions of the surface of the
nucleic acid container and/or the surface of the nanoparticle using
this method.
[0145] As described herein, the nucleic acid container is typically
porous such that small pores or holes through the thickness of the
walls of the container allow access to the outer surface of the
nanoparticle. The outer surface of the nucleic acid container
and/or the pores of the nucleic acid container can be designed to
include a particular nucleic acid sequence, hydrophilicity,
hydrophobicity, charge and/or size that favors positioning of a
particular component into the pore or onto the surface of the
container. Likewise, the component to be added may be designed to
include a complementary nucleic acid sequence, hydrophilicity,
hydrophobicity, charge, and/or size such that it has an affinity
for a particular portion of the nucleic acid container. In some
cases, upon the component being matched with a particular portion
of the nucleic acid container, the component may be inserted all
the way through the pore such that it contacts a portion of the
surface of the nanoparticle.
[0146] In some such embodiments, the end of the component may have
a particular chemistry that allows it to be attached to the surface
of the nanoparticle. For example, a component may be functionalized
with a thiol that allows it to be physisorbed to a gold
nanoparticle. In some embodiments in which the component is
attached directly to the nanoparticle, all or portions of the
nucleic acid container that surrounds the nanoparticle (partially
or fully) may be optionally removed, while the component remains
attached to the surface of the nanoparticle. In other embodiments,
however, the nucleic acid container may remain surrounding the
nanoparticle even after attachment of the component. In yet other
embodiments, the component is not attached to the surface of the
nanoparticle but is attached to a portion of the nucleic acid
container. Attachment to both the surface of the nanoparticle and a
portion of the nucleic acid container is also possible. Any
suitable method of attachment, e.g., to the surface of the
nanoparticle and/or to a portion of the nucleic acid container, may
be used such as covalent bonding, physisorption, chemisorption, or
attachment through ionic interactions, hydrophilic and/or
hydrophobic interactions, electrostatic interactions, van der Waals
interactions, or combinations thereof.
[0147] A component may have any suitable orientation with respect
to the nanoparticle or nucleic acid container. For instance, a
component may be oriented substantially perpendicular to a wall of
the nanoparticle or nucleic acid container. In some embodiments, a
component may be oriented at a particular angle or range of angles
with respect to a wall of the nanoparticle or nucleic acid
container (e.g., between 0.degree. and 90.degree., between
0.degree. and 15.degree., between 15.degree. and 45.degree.,
between 45.degree. and 60.degree., or between 60.degree. and
90.degree.).
[0148] In certain embodiments, an inorganic nanoparticle comprising
an isolated nucleic acid strand attached to a surface of the
inorganic nanoparticle is provided, wherein the inorganic
nanoparticle has a non-spherical shape. In certain embodiments, an
inorganic nanoparticle coated with a nucleic acid container is
provided, wherein the nucleic acid container comprises pores. A
binding site, such as an isolated nucleic acid strand may be
attached to a surface of the inorganic nanoparticle, and may extend
from the surface of the inorganic nanoparticle, through a pore of
the nucleic acid container. In some cases, the length of the
binding site (e.g., isolated nucleic acid strand) is longer than
the thickness of the nucleic acid container, such that the binding
site extends outwards from the nucleic acid container.
[0149] By using the unique chemistry of the nucleic acid container
that surrounds all or portions of the nanoparticle to direct
positioning of the components (either directly to the surface of
the nanoparticle or to the nucleic acid container), control of many
different parameters can be provided. FIGS. 4A-4D show examples of
different parameters that can be controlled when adding a component
to a nucleic acid nanostructure and/or a nanoparticle.
[0150] As shown in the embodiments illustrated in FIG. 4A, the
number of components 60 may be varied along one or more surface
portions of the nucleic acid container and/or the nanoparticle. For
example, in some embodiments, a single isolated component 60 may be
positioned on a single side of the nucleic acid container and/or
nanoparticle. In other embodiments, two isolated components can be
positioned on a single side of the nucleic acid container and/or
the nanoparticle. In yet other embodiments, three or more
components 60 can be positioned on a single side of a nucleic acid
container and/or a nanoparticle.
[0151] As shown in the embodiments illustrated in FIG. 4B, the
distance between two or more components can also be controlled. For
example, a first component 60 and a second component 62 may be
positioned relatively far apart from each other at a side of a
nucleic acid container and/or nanoparticle. In other embodiments,
two components can be positioned relatively close to one another,
or on opposite sides of the nucleic acid container and/or
nanoparticle.
[0152] As shown in the embodiments illustrated in FIG. 4C,
conformational control can also be provided. For instance, the
positioning of different components at unique positions on one or
more sides of a nucleic acid container and/or a nanoparticle may
allow the components to interact with each other, and/or with
portions of the nucleic acid container, so as to provide a
particular structural configuration of the components and/or to
change the structural configuration of the container.
[0153] Furthermore, as shown in the embodiments illustrated in FIG.
4D, the length of the components and/or the distance of the
component from the surface of the nanoparticle can also be
controlled. For example, if it is desirable to include a component
60 that is positioned a short distance away from the surface of
nanoparticle 38, the nucleic acid container having a relatively
thin wall can be used. If it is desirable to include a component 20
that is relatively further away from the surface of nanoparticle
38, a relatively thick wall can be used. In some embodiments, a
relatively thick wall may be obtained by using a second layer 70 to
coat all or portions of nucleic acid container 20. The second layer
may be formed using a nucleic acid polymer or any other material
described herein that may be suitable for use as a coating.
Additionally or alternatively, the length of component 60 may be
varied to control the distance between the end of the component and
a surface of the nucleic acid container and/or the surface of the
nanoparticle.
[0154] It should be appreciated that the types of parameters that
can be controlled as shown in FIGS. 4A-4D are merely examples, and
that other parameters with respect to the positioning of components
relative to nucleic acid containers and/or nanoparticles may be
possible. Additionally, it should be appreciated that multiple
components (e.g., binding sites), either homogeneous or
heterogeneous, may be positioned on the same surface with
controlled conformation or relative orientation.
[0155] FIGS. 4E and 4F show examples of how the surface
addressability of the structures can be used to form assemblies
(e.g., higher ordered structures) using specific orientations of
the structures. For instance, nucleic acid container 20 having
different sides a-f may include a nanostructure 37 attached to an
inner surface of the container. The nanostructure may fill a
portion of the cavity of the nucleic acid container. Nanoparticle
38 may be formed inside a cavity using the nucleic acid container
and nanostructure 37 as a template as described herein. Components
60 at each side of the structure may be unique and designed to bind
with specific components on the sides of other structures to form
an assembly having a specific configuration, as shown
illustratively in FIG. 4F. Additional examples of assembles using
surface-specific interactions are described in more detail
below.
[0156] In certain embodiments involving the positioning of isolated
components on a nucleic acid container and/or nanoparticle, a
component may be "isolated" in the sense that it is positioned a
certain distance away from another component that is attached to
the same nucleic acid container and/or nanoparticle such that the
isolated component can be uniquely identified and distinguished
from the other components (e.g., at the nanoscale level). In some
cases, an isolated component may facilitate binding or attachment
of other entities to the component, since it is isolated and avoids
or reduces the amount of steric interactions with other nearby
components attached to the same nucleic acid container and/or
nanoparticle.
[0157] In some cases, a first component (e.g., an isolated
component) is positioned at a distance of least 2 nm, at least 5
nm, at least 10 nm, at least 15 nm, at least 20 nm, at least 30 nm,
at least 40 nm, or at least 50 nm apart from the nearest second
component attached to the same nucleic acid container and/or
nanoparticle. In certain cases, a first component (e.g., an
isolated component) is positioned at a distance of less than or
equal to 500 nm, less than or equal to 200 nm, less than or equal
to 100 nm, less than or equal to 50 nm, less than or equal to 40
nm, less than or equal to 30 nm, less than or equal to 20 nm, less
than or equal to 15 nm, less than or equal to 10 nm, or less than
or equal to 50 nm apart from the nearest second component. Other
distances are also possible. Combinations of the above-noted ranges
are also possible. In other embodiments, components may be
positioned directly adjacent to one another (e.g., in the form of a
self-assembled monolayer) such that the individual components are
not isolated and/or not distinguishable from one another (e.g., at
the nanoscale level).
[0158] Any suitable number of components (whether isolated or not
isolated) may be attached to a nucleic acid container and/or
nanoparticle. In some embodiments, the nucleic acid nanostructure
and/or a nanoparticle may include, for example, between 2 and 500
components (e.g., between 2 and 100, between 2 and 50, or between 2
and 20 components). In some embodiments, the nucleic acid
nanostructure and/or a nanoparticle may include at least 2, at
least 4, at least 6, at least 8, at least 10, at least 12, at least
14, at least 20, at least 30, at least 40, at least 50, at least
70, or at least 100 different components. In other embodiments, the
nucleic acid nanostructure and/or a nanoparticle includes less than
100, less than 70, less than 50, less than 40, less than 30, less
than 20, less than 10, less than 7 different components. In some
embodiments, each of the components are isolated from one another
as described herein. Other numbers of components are also possible.
Combinations of the above-noted ranges are also possible.
[0159] As described herein, two or more components positioned on a
surface of a nucleic acid container and/or a nanoparticle may be
positioned at any suitable orientation with respect to one another.
In some embodiments, at least two components are attached to
opposite surface portions of the nucleic acid container and/or
nanoparticle. In other embodiments, at least two components are
positioned on adjacent surface portions of the nucleic acid
container and/or nanoparticle. In some cases, a first component is
positioned on a surface of a nanoparticle and a second component is
attached to the surface of a nucleic acid container. Other
configurations and orientations are also possible.
[0160] The methods described herein may be used to "print"
components such as proteins, organic molecules, and inorganic
nanoparticles, onto surfaces of the nanoparticles that are formed
by the methods described herein. Components may first be decorated
onto the interior surface of nucleic acid container in a
pre-designed pattern, and later transferred onto the exterior
surface of the nanoparticle grown inside the nucleic acid container
through the interactions described herein (e.g., physisorption,
covalent bonding, van der Waal interactions, etc.), while retaining
the pre-designed pattern. For example, nanoparticles that grow
inside the container may reach the inner walls of the container
where they contact the component and allow attachment of the
component to the nanoparticle surface. In some embodiments, even
after the removal of nucleic acid container, the component may
remain on the surface of the nanoparticle. In other embodiments,
components can be attached to staple strands (e.g., nucleic acids)
that are hybridized to the container scaffold and hold the scaffold
in the target shape. Since different staple strands may be used for
holding together different parts of the container, the staple
strands may be targeted individually for attaching different
components.
[0161] A variety of different components can be attached to a
surface of a nucleic acid container and/or a nanoparticle as
described herein. In some embodiments, the component comprises a
binding site. Sometimes, a component can comprise two binding
sites--one for the nanoparticle and one for a target separate from
the nanoparticle. The term "binding" refers to the interaction
between a corresponding pair of molecules that exhibit mutual
affinity or binding capacity, typically specific or non-specific
binding or interaction, including biochemical, physiological,
and/or pharmaceutical interactions. Biological binding defines a
type of interaction that occurs between pairs of biological
molecules including proteins, peptides, nucleic acids,
glycoproteins, carbohydrates, hormones and the like. Specific
examples include antibody/antigen, antibody/hapten,
enzyme/substrate, enzyme/inhibitor, enzyme/cofactor, binding
protein/substrate, carrier protein/substrate, lectin/carbohydrate,
receptor/hormone, receptor/effector, complementary strands of
nucleic acid, protein/nucleic acid repressor/inducer, ligand/cell
surface receptor, virus/ligand, aptamer/protein, etc. Such
molecules are examples of components that can be used with the
nanoparticles and nucleic acid containers described herein.
[0162] In some embodiments, the binding site comprises a nucleic
acid. For example, the nucleic acid may be in the form of DNA, RNA,
other nucleic acids, or combinations thereof, as described in more
detail herein. In some embodiments, the nucleic acid comprises a
single stranded portion. In other embodiments, the nucleic acid
comprises a double stranded portion. Combinations of single and
double stranded portions are also possible. Examples of nucleic
acids are provided in more detail below.
[0163] In certain embodiments, a component comprises a marker.
Examples of markers include readout (or detectable) markers such as
luminescent probes, fluorophores or fluorophore labeled molecules
or compounds, chromophores or chromophore labeled molecules or
compounds, and the like. In some cases, the marker comprises a
nanoparticle (e.g., a quantum dot). In certain embodiments, the
marker comprises a reporter molecule, such as a surface-enhanced
Raman scattering (SERS) reporter molecule.
[0164] In some embodiments, a marker and a corresponding binding
site are attached to the surface of the nanoparticle and/or nucleic
acid container. The marker/binding site pair may be specific and
unique for a target molecule, as described in more detail below. In
some such embodiments, each marker may be used to represent a
specific binding site. In some cases, the nanoparticle and/or
nucleic acid container comprises a plurality of marker/binding site
pairs, wherein each of the marker/binding site pairs is different
from one another, thereby allowing multiplexing. Other combinations
of marker and binding sites are also possible.
[0165] In some embodiments, the positioning of components at
particular orientations with respect to the nucleic acid container
and/or nanoparticle surface allows controlled self-assembly of
multiple nanostructures into higher-ordered assemblies. For
example, as shown in the embodiment illustrated in FIG. 5, assembly
75 includes a plurality of nanostructures 40A-40D that are
positioned relative to one another in specific arrangements. The
specific arrangements of nanostructures relative to one another may
be obtained by using unique components that are placed at specific
positions on the nucleic acid container and/or nanoparticle. For
example, a nanostructure 40A may be assembled with a nanostructure
40B using components 60A and 60B, which are attached to
nanostructures 40A and 40B, respectively. Components 60A and 60B
may, in some embodiments, be binding sites that are complimentary
to one another such that they selectively bind to one another and
not to other components such as components 61A, 61B, 62A, or 62B.
As described herein, the components may be designed to include a
suitable length, to be positioned at a suitable distance from a
surface of the coated nanoparticle, and/or to be positioned on a
particular side of the nucleic acid container and/or nanoparticle.
Similarly, a nanostructure 40B may be assembled with a
nanostructure 40C using a pair of components 61A and 61B, and
nanostructure 40B may be assembled with a nanostructure 40D using a
pair of components 62A and 62B. As shown in this exemplary
embodiment, each surface of the nanostructures can be tagged with
different binding sites, allowing the attachment of different
nanostructures at each distinct surface within a three-dimensional
space.
[0166] As described herein, each nanostructure (including a nucleic
acid container and/or nanoparticle) used in an assembly may have
any suitable shape. For example, as shown illustratively in FIG. 5,
nanostructure 40A may be in the form of a rhomboid, nanostructure
40B may be in the form of a pentagon, nanostructure 40C may be in
the form of a rhomboid having a different orientation with respect
to nanostructure 40A, and nanostructure 40D may be in the form of a
hexagon. Other shapes may be used in other embodiments.
Additionally, it should be appreciated that any suitable materials
may be used in each of the nanostructures. For instance,
nanoparticles 38A-38D may be formed of the same materials, or
different materials, such as those described herein. Additionally,
components 60-62 may vary, and may be components of the same type
or of different types.
[0167] In some embodiments, an assembly such as the one shown in
FIG. 5 can be formed by synthesizing a plurality of nucleic
acid-coated nanoparticles, each of the nucleic-acid coated
nanoparticles formed by growing a nanoparticle from a nanoparticle
precursor positioned inside a nucleic acid container, and then
assembling the nucleic acid-coated nanoparticles. In other
embodiments, a higher order structure can be formed by assembling
nucleic acid containers, each of the nucleic acid containers having
a nanoparticle precursor positioned therein, and then synthesizing
the nanoparticle from the nanoparticle precursor inside the nucleic
acid container to form the nucleic acid-coated nanoparticles. A
combination of such methods is also possible.
[0168] As described herein, any suitable component or binding site
may be used for assembly, and the component or binding site may be
associated with the nanoparticle and/or the nucleic acid portion of
the nanostructure. In some embodiments, the nucleic acid-coated
nanoparticles are attached to one another by components or binding
sites that are attached to a nucleic acid portion of the nucleic
acid-coated nanoparticles. In other embodiments, the nucleic
acid-coated nanoparticles are attached to one another by components
or binding sites that are attached to a nanoparticle portion of the
nucleic acid-coated nanoparticles. In some cases, the nucleic
acid-coated nanoparticles are attached to one another using a
thermal process (e.g., using heat to cause attachment or binding
between two nanoparticles, or two or more components associated
with the nanoparticles). In other cases, the nucleic acid-coated
nanoparticles are attached to one another using a photophysical
process. In certain cases, the nucleic acid-coated nanoparticles
are attached to one another using a binding process.
[0169] After assembly, all or portions of the nucleic acid
container may be optionally removed from the nucleic acid-coated
nanoparticles. In some such embodiments, a surface of the
nanoparticle may be passivated prior to, during, or after the
removal step. The nanoparticles may remain attached to one another
in the assembly after the removal step. In other embodiments, the
nucleic acid container is not removed after assembly of multiple
nano structures.
[0170] Different types of assemblies can be formed. In some cases,
the assembly comprises an electronic circuit. In some embodiments,
the assembly is in the form of a two-dimensional array. In other
embodiments, the assembly is in the form of a three-dimensional
array. In some cases, nanostructures can be assembled
hierarchically based on surface-specific binding.
[0171] An example of a process for forming a circuit is shown in
the embodiments illustrated in FIGS. 6A and 6B. FIG. 6A shows a
process 100 including the assembly of nanostructures 40A, 40B, and
40C with a nanostructure 40E to form an electronic circuit. FIG. 6B
shows a different orientation of structures in the form of
nanorods, which include conductive nanoparticles 38, that can be
used to form an electronic circuit. As shown illustratively in the
figures, a plurality of nanostructures can be used as building
blocks to be placed at designated positions, in some embodiments
due to the specificity of binding between different surfaces, as
described herein. For example, in FIG. 6B, the linkage points
between the structures can be specifically arranged at designed
position on nanorods. Also as described herein, different
strategies can be utilized to form different structures. For
example, in one embodiment an assembly can be formed by assembling
nucleic acid containers, and then triggering the growth of a
conductive material within the container. In another embodiment, a
conductive material can be grown in each of the building blocks
using nucleic acid containers, and then the resulting
nanostructures can be assembled into a larger structure. In some
cases, merging of the ends of the building blocks, e.g., via
thermal/photophysical methods, may produce continuous conductive
networks that can be used as electronic circuits.
[0172] An assembly may have any suitable size and may be on the
nano-, micro-, meso- or macro-scale. In some cases, an assembly has
a length and/or at least one cross-sectional dimension that is less
than or equal to 1 mm, less than or equal to 100 microns, less than
or equal to 50 microns, less than or equal to 10 microns, less than
or equal to 1 micron, less than or equal to 500 nm, less than or
equal to 100 nm, less than or equal to 50 nm, less than or equal to
10 nm, or less than or equal to 1 nm. In other cases, the assembly
has a length and/or at least one cross-sectional dimension that is
greater than or equal to 1 nm, greater than or equal to 10 nm,
greater than or equal to 100 nm, greater than or equal to 1 micron,
greater than or equal to 10 microns, greater than or equal to 50
microns, greater than or equal to 100 microns, or greater than or
equal to 1 mm. Other values are also possible. Combinations of the
above-noted ranges are also possible. A length may of the assembly
may be determined by measuring the distance between two outermost
portions of furthest-spaced apart nanostructures forming the
assembly. Similarly, a cross-sectional dimension of the assembly
may be determined by taking a cross-section between two outermost
portions of nanostructures forming the assembly and measuring the
distance between the outermost portions.
[0173] An assembly of nanostructures may have any suitable
configuration. For example, in some embodiments, an assembly may
have a linear shape (e.g., AAAAAABBBBBBB, where A and B are
different nanostructure building blocks, or an alternative chain
such as ABABABAB). In other cases, an assembly may have a star
shape (e.g., five B around one A). In other embodiments, an
assembly may form a three-dimensional structure such as a tube,
box, barrel, rectangle, rod, "T", "L", branched structure, diamond,
square, parallelogram, rhomboid, triangle, pentagon, hexagon, or
polyhedron, including shapes substantially similar thereto. Other
configurations are also possible.
[0174] In some embodiments, the nanostructures described herein
(e.g., nanoparticles coated or uncoated by a nucleic acid
container) can be used for multiplexed detection. Detection may
involve, in some embodiments, introducing a composition suspected
of comprising a target molecule (e.g., a biomolecule) to a
plurality of nanostructures, and allowing the target molecule, if
present, to bind to surfaces of at least two different
nanostructures. An example of such a method is shown illustratively
in FIG. 7. A process 80 involves the use of nanostructures 40A and
nanostructure 40D, which include different components attached to
different portions/sides of the nanostructure. For example,
nanostructure 40A includes components 81-84 positioned on different
sides of the nanostructure, and nanostructure 40D includes
different components 85-90 positioned on different sides of the
nanostructure. In some cases, each of the components of the
nanostructure are different from one another, although in other
cases, some of the components of the nanostructure may be the same
while others may be different from one another. Upon introduction
of a target molecule 94, binding between a portion of the target
molecule and one of the components of 40A may occur, and binding
between a portion of the target molecule and a component of
structure 40D may occur. Depending on the particular binding site
included in the target molecule, different combinations of binding
between components of nanostructures 40A and 40D may occur. As
shown illustratively in FIG. 7, the target molecule may include a
binding site that is specific to component 82 of nano structure 40A
and a binding site that is specific to component 90 of
nanostructure 40D. Using such a method, every two surfaces (e.g.,
one surface from one nanostructure and another surface from a
different nanostructure) may be used to detect one specific target
molecule.
[0175] In some embodiments, the introduction of a target molecule
can trigger the recognition of two specific surfaces, and a unique
signal as a result of the recognition event may be produced and/or
enhanced. For example, in one set of embodiments, each of the
components shown in FIG. 7 may be reporter molecules for SERS-based
detection. The binding between the target molecule and two specific
surfaces, one on each different nanostructure, can enhance the
Raman signals from the two specific reporter molecules associated
with the binding. Using such a method, a variety of different
target molecules can be detected using relatively few numbers of
nanostructures because the signal from the binding of each target
molecule will be unique and distinguishable from others.
[0176] In some embodiments, the number of different target
molecules that can be recognized by nanostructures described herein
may depend, at least in part, on the number of different components
(e.g., binding sites) positioned on the nanostructure. As described
herein, in some cases the components are in the form of isolated
components. Generally, the number of different target molecules
that can be recognized using the nanostructures described herein
can be determined using the formula (n*x-1)*n*x/2 (assuming
detection of each target molecule involves binding with two
nanostructures), where n is the number of nanostructures and x is
the number of different binding sites (and/or sides) associated
with each of the nanostructures. For example, 2 nanostructures
having 5 binding sites on each nanostructure can result in the
detection of (2*5-1)*2*5/2=45 different target molecules. The
number of different target molecules that can be recognized using
the nanostructures described herein, assuming detection of each
target molecule involves binding with three nanostructures, can be
determined using the formula (n*x-2)*(n*x-1)*n*x/3. In some
embodiments two nanostructures (or 3, 4, 5, 6, etc. nanostructures)
can be used to detect, for example, between 2 and 2,000 different
target molecules (e.g., between 2 and 1,000, between 2 and 500,
between 2 and 200, between 2 and 100, or between 2 and 50 different
target molecules). For instance, in some embodiments two
nanostructures (or 3, 4, 5, 6, etc. nanostructures) can be used to
detect at least 5, at least 10, at least 20, at least 30, at least
40, at least 50, at least 70, at least one 100, at least 500, at
least 1,000, or at least 1,500 different target molecules. In some
cases, such numbers of target molecules can be detected in
parallel. In some embodiments, a composition includes at least 2,
at least 3, at least 5, at least 10, at least 20, at least 30, at
least 50, or at least 100 different nanostructures, each of which
can be used to detect different target molecules.
[0177] It should be appreciated that other methods of detection
other than SERS-based detection can be used using the
nanostructures described herein. For example, other
metal-surface-enhanced luminescent probes can be used, wherein the
luminescent group is utilized to replace Raman signal
reporters.
[0178] Nucleic acids, in the context of the invention, include DNA
and RNA, as well are various modifications thereof. Modifications
include base modifications, sugar modifications, and backbone
modifications. Non-limiting examples of these are provided herein.
Non-limiting examples of DNA variants that may be used include
L-DNA (the backbone enantiomer of DNA, known in the literature),
peptide nucleic acids (PNA) bisPNA clamp, a pseudocomplementary
PNA, a locked nucleic acid (LNA), or co-nucleic acids of the above
such as DNA-LNA co-nucleic acids. It is to be understood that the
nucleic acids used in the embodiments described herein may be
homogeneous or heterogeneous in nature. As an example, they may be
completely DNA in nature or they may be comprised of DNA and
non-DNA (e.g., LNA) monomers or sequences. Thus, any combination of
nucleic acid elements may be used. The nucleic acids described
herein may be referred to as polymers or nucleic acid polymers. The
modification may render the interactions of such polymers more or
less stable under certain conditions.
[0179] The nucleic acids described herein may be obtained from
natural sources, and optionally subsequently modified. They may be
synthesized in vitro, and optionally may mimic a naturally
occurring nucleic acid or may represent a non-naturally occurring
nucleic acid (e.g., due to the present of elements that are not
found in naturally occurring nucleic acids). Methods for harvesting
nucleic acids from in cells, tissues or organisms are known in the
art. Methods for synthesizing nucleic acids, including automated
nucleic acid synthesis, are also known in the art.
[0180] The nucleic acids may have a homogenous backbone (e.g.,
entirely phosphodiester or entirely phosphorothioate) or a
heterogeneous (or chimeric) backbone. Phosphorothioate backbone
modifications render a nucleic acid less susceptible to nucleases
and thus more stable (as compared to a native phosphodiester
backbone nucleic acid) under certain conditions. Other linkages
that may provide more stability to a nucleic acid include without
limitation phosphorodithioate linkages, methylphosphonate linkages,
methylphosphorothioate linkages, boranophosphonate linkages,
peptide linkages, alkyl linkages, dephospho type linkages, and the
like.
[0181] Nucleic acids having modified backbones, such as backbones
comprising phosphorothioate linkages, and including those
comprising chimeric modified backbones may be synthesized using
automated techniques employing either phosphoramidate or
H-phosphonate chemistries. (F. E. Eckstein, "Oligonucleotides and
Analogues--A Practical Approach" IRL Press, Oxford, UK, 1991, and
M. D. Matteucci and M. H. Caruthers, Tetrahedron Lett. 21, 719
(1980)) Aryl- and alkyl-phosphonate linkages can be made, e.g., as
described in U.S. Pat. No. 4,469,863; and alkylphosphotriester
linkages (in which the charged oxygen moiety is alkylated), e.g.,
as described in U.S. Pat. No. 5,023,243 and European Patent No.
092,574, can be prepared by automated solid phase synthesis using
commercially available reagents. Methods for making other DNA
backbone modifications and substitutions have been described.
Uhlmann E et al. (1990) Chem Rev 90:544; Goodchild J (1990)
Bioconjugate Chem 1:165; Crooke S T et al. (1996) Annu Rev
Pharmacol Toxicol 36:107-129; and Hunziker J et al. (1995) Mod
Synth Methods 7:331-417.
[0182] The nucleic acids described herein may additionally or
alternatively comprise modifications in their sugars. For example,
a .beta.-ribose unit or a .beta.-D-2'-deoxyribose unit can be
replaced by a modified sugar unit, wherein the modified sugar unit
is for example selected from .beta.-D-ribose,
.alpha.-D-2'-deoxyribose, L-2'-deoxyribose, 2'-F-2'-deoxyribose,
arabinose, 2'-F-arabinose, 2'-O--(C.sub.1-C.sub.6)alkyl-ribose,
preferably 2'-O--(C.sub.1-C.sub.6)alkyl-ribose is
2'-O-methylribose, 2'-O--(C.sub.2-C.sub.6)alkenyl-ribose,
2'-[O--(C.sub.1-C.sub.6)alkyl-O--(C.sub.1-C.sub.6)alkyl]-ribose,
2'-NH.sub.2-2'-deoxyribose, .beta.-D-xylo-furanose,
.alpha.-arabinofuranose,
2,4-dideoxy-.beta.-D-erythro-hexo-pyranose, and carbocyclic
(described, for example, in Froehler J (1992) Am Chem Soc 114:8320)
and/or open-chain sugar analogs (described, for example, in
Vandendriessche et al. (1993) Tetrahedron 49:7223) and/or
bicyclosugar analogs (described, for example, in Tarkov M et al.
(1993) Helv Chim Acta 76:481).
[0183] The nucleic acids may comprise modifications in their bases.
Modified based include modified cytosines (such as 5-substituted
cytosines (e.g., 5-methyl-cytosine, 5-fluoro-cytosine,
5-chloro-cytosine, 5-bromo-cytosine, 5-iodo-cytosine,
5-hydroxy-cytosine, 5-hydroxymethyl-cytosine,
5-difluoromethyl-cytosine, and unsubstituted or substituted
5-alkynyl-cytosine), 6-substituted cytosines, N4-substituted
cytosines (e.g., N4-ethyl-cytosine), 5-aza-cytosine,
2-mercapto-cytosine, isocytosine, pseudo-isocytosine, cytosine
analogs with condensed ring systems (e.g., N,N'-propylene cytosine
or phenoxazine), and uracil and its derivatives (e.g.,
5-fluoro-uracil, 5-bromo-uracil, 5-bromovinyl-uracil,
4-thio-uracil, 5-hydroxy-uracil, 5-propynyl-uracil), modified
guanines such as 7-deazaguanine, 7-deaza-7-substituted guanine
(such as 7-deaza-7-(C2-C6)alkynylguanine), 7-deaza-8-substituted
guanine, hypoxanthine, N2-substituted guanines (e.g.
N2-methyl-guanine),
5-amino-3-methyl-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione,
2,6-diaminopurine, 2-aminopurine, purine, indole, adenine,
substituted adenines (e.g. N6-methyl-adenine, 8-oxo-adenine)
8-substituted guanine (e.g. 8-hydroxyguanine and 8-bromoguanine),
and 6-thioguanine. The nucleic acids may comprise universal bases
(e.g. 3-nitropyrrole, P-base, 4-methyl-indole, 5-nitro-indole, and
K-base) and/or aromatic ring systems (e.g. fluorobenzene,
difluorobenzene, benzimidazole or dichloro-benzimidazole,
1-methyl-1H-[1,2,4]triazole-3-carboxylic acid amide).
[0184] As used herein, the terms "bind" or "interact" as they
relate to nucleic acids typically refer to hybridization (e.g.,
base-specific binding) between two or more nucleic acid sequences
or strands. The term "annealing" refers to the process of heating
and slowly cooling a mixture of nucleic acids (e.g., in a typical
thermal cycling machine) such that the thermodynamic steady state
(or one relatively near it) of hybridized elements is formed.
Interaction between nucleic acids, according to certain embodiments
described herein, is specific and is typically governed by the
sequence of the interacting strands. These interactions include
Watson-Crick binding in which complementary nucleic acid sequences
hybridize to each other. These interactions may also include other
binding motifs including but not limited to Hoogsteen or quadruplex
binding.
[0185] It should be appreciated that other components such as small
molecules, proteins, and markers may be attached to the nucleic
acids described herein in some embodiments.
[0186] The following examples are intended to illustrate certain
embodiments of the present invention, but are not to be construed
as limiting and do not exemplify the full scope of the
invention.
Example 1
[0187] This example shows a method for forming gold nanoparticles
in nucleic acid containers as described herein.
[0188] Step 1: Formation of nucleic acid containers. A nucleic acid
container was formed by folding DNA using a DNA origami method. The
nucleic acid container was designed using caDNAno software. To fold
the DNA strands into the designed shape, 16.7 uL 200 nM M13 nucleic
acid scaffold (P8064 mutation) (SEQ. ID. NO. 1), 20 uL 500 nM
staple strand (obtained following the procedure described in Dietz
et al., Science, 325:725-730, 7 Aug. 2009 and Douglas et al.,
Nature, 459: 414-418, 21 May 2009), 11 uL folding buffer (50 mM
Tris, 10 mM EDTA, and 120 mM MgCl.sub.2), and 44 uL water were
mixed. The resulting solution was rapidly heat denatured, followed
by slow cooling from 80 to 61 degrees Celsius over 100 min, then
from 60 to 24 degrees Celsius over 72 h. The nucleic acid
containers were purified on a 2% agarose gel (0.5.times.TBE+10 mM
MgCl.sub.2) at 70 V for 3 h in an ice-water bath. The gel was
stained with Sybr Gold and the nucleic acid containers were
extracted from the gel with crash-soak method.
[0189] Step 2: Seed decoration. 5 nm mono-DNA functionalized gold
nanoparticle precursors were used as seeds for the growth of a
templated nanoparticle. To introduce a seed into the cavity of a
nucleic acid container, the purified containers (2 nM in
0.5.times.TBE+10 mM MgCl.sub.2 buffer) were mixed with 50 nM 5 nm
mono-DNA functionalized gold nanoparticles. The solution was
incubated at 37 degree for 16 hours, and then slowly annealed to 24
degree (1 degree/step, 20 min/step). In order to include a single
gold nanoparticle into the cavity of the nucleic acid container,
the mono-DNA from the gold nanoparticle was designed to hybridize
with a single complementary DNA sequence attached to inner surface
of the nucleic acid container.
[0190] Excessive unbound gold nanoparticles were removed via spin
centrifuge. 20 uL of the seed-decorated nucleic acid container
solution was mixed with 180 uL water, and loaded into an Amicon
centrifugal filter (MWCO=100 kDa, from Millipore) to be centrifuged
at 14,000 g for 3 min. This step was repeated twice under the same
conditions. The filter was then reversed, and spun at 1,000 g for 2
min. The residual solution was collected.
[0191] Step 3: Growth of a gold nanoparticle within a nucleic acid
container. To grow a gold nanoparticle having a cross-section in
the shape of a hexagon, steps 1 and 2 above were first performed to
synthesize nucleic acid container 20 including a cavity having a
cross-section in the shape of a hexagon, as shown in FIG. 8A. Then,
a 10 uL purified seed-decorated solution was combined with a 1 uL
14 mM HAuCl.sub.4 precursor solution. 1 uL 20 mM ascorbic acid was
added subsequently. After 2 min, 3.5 uL of the final solution was
dipped onto a copper grid. After 2 min, the solution was wiped away
with filter paper, leaving the nanostructures on the grid. 3.5 uL
of a 2% uranium formation was then added onto the grid to stain the
resulting nanostructures. After 45 sec, the solution was wiped away
with filter paper. The grid was then left for drying in order to
dry the resulting nanostructures.
[0192] FIGS. 8B and 8C show TEM images of the resulting
nanostructures after purification. As shown in FIG. 8C, a
nanoparticle 38 formed of gold had cross-sectional dimensions of 38
nm and 34 nm.
[0193] This example shows that gold nanoparticles can be formed in
nucleic acid containers that act as a template during the growth of
the nanoparticles. The resulting nanoparticles have shapes that are
complementary to the shapes of the cavities of the nucleic acid
containers.
Example 2
[0194] This example shows a method for forming silver nanoparticles
in nucleic acid containers as described herein.
[0195] Steps 1 and 2 described in Example 1 were performed to
synthesize a nucleic acid container having a cross-section in the
shape of a rhomboid, as shown in FIG. 9A. To grow a silver
nanoparticle in the cavity of the nucleic acid container, a 10 uL
purified seed-decorated solution was combined with 1 uL 100 mM
Mg(Ac).sub.2 solution. To this mixture, 1 uL 14 mM AgNO.sub.3 and 1
uL 20 mM ascorbic acid were added subsequently. 3.5 uL of the
resulting solution was dipped onto a copper grid. After 2 min, the
solution was wiped away with filter paper, leaving the
nanostructures on the grid. 3.5 uL 2% uranium formation was then
added onto the grid to stain the resulting nanostructures. After 45
sec, the solution was wiped away with filter paper. The grid was
then left for drying in order to dry the resulting nano
structures.
[0196] FIGS. 9B-9D show TEM images of the resulting nanostructures
after purification. As shown in FIG. 9B, a nanoparticle 38 formed
of silver had cross-sectional dimensions of 17 nm and 18 nm. The
thickness of the walls of nucleic acid container 20 was
approximately 4-6 nm. FIGS. 9C and 9D show two nanostructures that
are dimerized. The dimerized structure was formed using Steps 1 and
2 described in Example 1. Prior to growing the silver nanoparticles
in the cavities of the nucleic acid containers, however, two
nucleic acid containers were attached to one another using
complementary DNA strands.
[0197] This example shows that silver nanoparticles can be formed
in nucleic acid containers that act as a template during the growth
of the nanoparticles. The resulting nanoparticles have shapes that
are complementary to the shapes of the cavities of the nucleic acid
containers. This example also shows that nucleic acid containers
can be attached to one another to form larger nanostructures.
Example 3
[0198] This example shows a method for forming silver nanoparticles
in a nucleic acid container having quantum dots associated with the
inner surface of the container. The quantum dots were used to block
the openings of the nucleic acid container.
[0199] Steps 1 and 2 as described in Example 2 were performed to
synthesize a nucleic acid container having a cross-section in the
shape of a rhomboid, as shown in FIG. 9A. To a solution of 20 uL
seed-decorated origami solution, a 0.5 uL solution of 2 uM quantum
dots (from Invitrogen) was added, and incubated at 35.degree. C.
for 16 hours, and then slowly cooled to room temperature for
another 2 hours. Seed purification and silver growth were performed
following the method described in Example 2.
[0200] FIG. 10 is a TEM image showing the resulting nanostructure
after purification. A nanoparticle 38 made of silver was formed
within nucleic acid container 20. Quantum dots 39 were located at
the two ends of the nucleic acid container, the quantum dots having
dimensions of about 20 nm.
[0201] This example shows that a silver nanoparticle can be formed
in a nucleic acid container that acts as a template during the
growth of the nanoparticle when both ends of the container are
blocked by quantum dots. The resulting nanoparticle had a shape
that was complementary to the shape of the cavity formed by the
nucleic acid container and quantum dots. This example also shows
that nucleic acid containers can be utilized to prepare
heterogeneous structures, which are potentially applicable in solar
cells and hydrogen production from water.
Example 4
[0202] This example shows a method for designing opened nucleic
acid containers and the use of the containers to direct the shape
of silver nanoparticles during their formation. In this example,
the M13 nucleic acid scaffold (P8064 mutation) (SEQ. ID. NO. 1) was
used.
[0203] Hollow DNA containers used as molds were designed using a 3D
DNA origami strategy as described herein (FIG. 11). To ensure the
structural rigidity of nucleic acid (DNA) containers 20, a
multi-layered square-lattice design was used to form walls 25
(e.g., sidewalls). A two-layered design made of 16-helix bundles, a
three-layered design made of 18-helix bundles, and a four-layered
design of 24-helix bundles were tested in different shaped DNA
containers (FIG. 11). The cross-section of cavities 30 of the
containers were designed with distinct shapes of sub-25 nm,
including rectangular, square, triangle, and ring shapes (FIG.
11A); this distinct property imparts programmability, a feature
that existing hard templates lack. The thicknesses of the DNA
containers were also tuned from 10 nm to 30 nm. In order to ensure
metal growth within the central cavity of a single, defined DNA
container, 5-nm gold nanoparticles, used as a seed 34 for silver or
gold growth, were conjugated to the interior surface of the DNA
container via DNA hybridization (FIG. 11B). A 21-nt single-stranded
DNA was immobilized onto a seed surface, and the stoichiometry
ratio between gold seeds and surface DNA was 1:1 in the reaction
buffer. Multiple 21-nt ssDNAs, ranging from 3 to 25, were
immobilized in the interior surface of the DNA containers with
sequences complementary to those on the seed surface. Notably, in
some embodiments, direct reduction of noble metals, such as silver
and gold, without using seeds, may result in the metallization at
the exterior surface of the DNA container. Subsequent reduction of
metal precursors mediated by the seeds produced confined growth of
metal nanostructures 38 within each DNA mold for specific
prescribed shapes and dimensions (FIG. 11C).
[0204] In this experiment, portions of a DNA container such as DNA
barrels and lids were folded by slowly annealing the
staple/scaffold mixtures from 80.degree. C. to 24.degree. C. over
72 h. Then, the crude products were subjected to agarose gel
electrophoresis (1.5% agarose gel) with 0.5.times.TBE/10 mM
MgCl.sub.2 as running buffer. The purified structures were
extracted from the gel and then recovered via centrifugation. Seed
decoration was executed by the incubation of opened DNA containers
(e.g., barrels) with excess of 5-nm gold particles (the
stoichiometry ratio between gold and DNA containers ranged from 2:1
to 5:1) at 35 C for 16 h, and then annealed to 24 C over 3 h.
Excessive gold nanoparticles were removed by using a size-exclusive
spin columns. To form an enclosed cavity, DNA lids were mixed with
the seed-decorated DNA barrels at 35 C for 16 h, and then annealed
to 24 C over 3 h. Metal precursors, such as silver nitrate for
silver nanoparticle and chloroauric acid for the formation of gold
nanoparticles, were then added to the purified gold-DNA barrels
conjugates, followed by a reducing agent, such as ascorbic acid
(AA). After several minutes to hours of growth in the dark at 4 C
or room temperature, the solution was dipped onto a copper grid,
and stained with uranium salt for TEM imaging.
Example 5
[0205] This example shows a method for designing closed nucleic
acid containers (e.g., boxes) and the use of the containers to
direct the shape of silver nanoparticles during their formation. In
this example, the M13 nucleic acid scaffold (P8064 mutation) (SEQ.
ID. NO. 1) was used.
[0206] The three-dimensional confined growth of silver
nanoparticles was examined using a box-shaped DNA container. Each
DNA box container 20 was designed with three independent
components: one barrel 47 and two square shaped lids 50 and 52
(FIG. 12). Cavity 30, surrounded by both the lids and the interior
surface of the barrel, was designed with either a square- or
rectangular-shaped cross-section. The rectangular DNA barrels were
assembled from 88 parallel double helices. The cross-section
dimensions of central cavity were designed as 8 helices by 6
helices. The sidewalls were built from both 16-helix bundles and
18-helix bundles. The lengths of the barrels were set as 6 and 9
double-helix turns, respectively. The square-shaped DNA barrel was
assembled from 108 double helices. Each sidewall was constructed
from 18-helix bundles. The cross-sectional dimensions of the
central cavity were designed as 6 helices by 6 helices, with the
length of 7 double-helix turns. A three-layered DNA lid was
designed with 18 helices in width, 3 helices in thickness and 15
helices turn in length.
[0207] To connect the lids onto the barrels, 6 or 16 15-nt
single-stranded binding sites were introduced at both ends of the
18-helix bundles in the rectangular DNA barrel; while in the square
DNA box, 13 15-nt single-stranded binding sites 49 were immobilized
at each end of the DNA sidewalls (FIG. 12A). The binding sites at
each bundle exhibited the same sequences. On one side of the DNA
lids, 20 15-nt single-stranded DNAs with complement sequences to
those on DNA barrels were introduced. The spacing between two
different sequenced DNA were set to 20 nm, consistent with the
spacing of binding sites at the barrels.
[0208] For both rectangular barrels and lids, the formation yields
were around 10-20%, while for the square barrel, the folding yield
was much lower of 5%, owing to barrel dimerization by sticky-end
stacking. TEM imaging indicated the formation of the designed
shapes. For both rectangular barrels, the cross-sectional
dimensions of the central cavity were 20 nm.times.15 nm, consistent
with 2.5 nm per double helix, and 15 nm.times.15 nm for the square
shaped cavity. However, due to partial dehydration and structural
deformation during TEM sample preparation for imaging, small
deviations of 2 or 3 nm were also observed, as well as corner angle
deviations from 90 degrees and/or or rough inner surfaces. TEM
imaging also revealed the seed decoration yields for different
shaped DNA barrels were approximately 74-91% (N>100).
[0209] The unpurified reaction solution after lid closure was then
imaged with TEM to determine the formation yield (FIGS. 12B-12D
left, N>100). A rectangular DNA barrel with 20-15-30 nm
dimensions was utilized to optimize the formation yield of the
seed-decorated DNA box container. At a lid-to-barrel stoichiometry
ratio of 3:1, including 6 binding sites on each end of the
rectangular barrel, produced less than 10% box formation yield;
whereas increasing the binding sites to 16 promoted the box
formation yield to 31% (FIG. 12B, left). Increasing the
lids-to-barrel stoichiometry ratio from 2:1 to 6:1 resulted in the
slightly increment of box formation yield from 28% to 33%. At a
relatively high stoichiometry ratio, i.e., 6:1, in some cases, each
end of the barrel may connect to two lids, which may prevent the
correct lid closure processes. The yield of defect structures was
also increased from 20% in 2:1 stoichiometry ratio to 50% in 6:1
stoichiometry ratio. The formation of defective structures
prevented further increments of DNA box formation yield at high
stoichiometry ratios. Box closure yields for DNA barrels with
20-15-20 nm dimensions and 15-15-25 dimensions were found to be 13%
and 21%, respectively (FIG. 12C-12D left), at a stoichiometry ratio
of 3:1. Compared with that of the DNA box container, the formation
of seed-decorated DNA box containers was with seed-decorated
barrels was further lowered by a factor of around 20%, which was
consistent with the formation yields of seed-decorated DNA barrels.
Agarose gel electrophoresis was tested to purify the reaction
solution of the seed-decorated DNA box containers. However, after
the extraction of bands corresponding to the seed-decorated DNA box
containers, TEM imaging revealed the presence of both opened and
closed seed-decorated DNA box containers, which resulted from
either small mobility differences or structural deformation during
gel extraction.
[0210] The growth of silver nanoparticles was triggered by the
addition of silver nitrate (1.4 mM) and ascorbic acid (2 mM). After
growth for 4 to 10 min at room temperature, silver nanoparticles 38
grown within the DNA boxes were imaged by TEM (FIG. 12B right). TEM
images indicated the presence of 4-8 nm-thick sidewalls after
silver growth, which suggested that the DNA containers remained
intact after silver growth. In rectangular DNA box containers with
20-15-30 nm dimension cavity, silver nanoparticles were grown into
20-16-30 nm dimension (FIG. 12B, right). Rectangular cross-sections
as well as rounded corners were observed in TEM images. When the
cavity dimensions were reduced to 20-15-20 nm, silver nanoparticles
with similar rectangular cross-shapes and 20-nm thickness were
observed (FIG. 12C, right). Different maximum allowed thicknesses
of silver nanoparticles in DNA box containers confirmed the
confinement of DNA box containers in the thickness direction.
Changing the cross-sectional dimensions of the DNA box containers
from 20 nm.times.15 nm to 15 nm.times.15 nm produced silver
nanoparticles with square-shaped cross-sections (FIG. 12D right).
Each edge was measured to be around 16 nm in TEM image. The bigger
dimensional sizes of the silver nanoparticles, compared with those
of cavities, resulted from the compression of the DNA double
helices by silver nanoparticle growth.
[0211] In some cases, defective DNA structures were also observed
during the silver growth processes. In the growth direction
confined by two two-layered DNA sidewalls made from 16-helix bundle
in a rectangular DNA box container, defective structures were
observed with both sidewall bending and cavity expansion; whereas
in the growth direction confined by two three-layered DNA sidewalls
made from 18-helix bundle, defective structures were mainly
observed with expanded dimension size, e.g., from 20 nm to around
25 nm. TEM images indicated that defect yields for two-layered
sidewalls was 5 times higher than that in the three-layered
sidewalls (N>50). In square shaped DNA box containers with
three-layered DNA sidewalls, defective structures mainly resulted
from the expansion of cavity dimensions, e.g., from 15 nm to around
20 nm. The defect ratio was also dependent on reaction time and
reactant concentration. When the reaction time was 4 min with 0.3
mM AgNO3 and 0.5 mM AA as reactants, the defect ratio at for the
square box was decreased by 2/3.
[0212] Several other designs for lids and barrels were also tested
to fabricate cavities with distinct shapes. 16-helix bundles of DNA
with 10 or 15 nm lengths were introduced onto the top of 30-helix
bundles. After purification, TEM images indicated the well
formation of 30-helix bundles. However, in this particular
experiment, both 16-helix bundles did not connect to the 30-helix
bundles tightly, and could not be utilized for box formation. DNA
barrels with triangular tops was also fabricated. After seed
decoration and lid closure, clear spacing was observed at one
vertex, which was composed of several 10-nm DNA helices. Although a
triangular top was observed in the confined silver nanoparticles,
the spacing between 10-nm DNA helices was expanded. This resulted
from the unstable linkage of 1 or 2 staple crossovers in 10-nm DNA
helices, compared to 4 to 5 staple crossovers linkage in 30-nm DNA
helices. The distorted DNA barrels further evidenced that rigid and
stable DNA sidewalls confined the metal growth.
[0213] As described herein, FIGS. 12A-12D show confined growth of
silver nanoparticles within a DNA box container. FIG. 12B shows
design (top) and TEM images (bottom) for silver nanoparticles grown
within a rectangular-shaped DNA box of 20-15-30 nm dimensions. From
left to right: DNA box, seed-decorated DNA box, and silver growth
within the box. FIG. 12C shows design (top) and TEM images (bottom)
for silver nanoparticle grown within a rectangular-shaped DNA box
of 20-15-20 nm dimensions. From left to right: DNA box,
seed-decorated DNA box, and silver growth within the box. FIG. 12D
shows design (top) and TEM images (bottom) for silver nanoparticle
grown within a square-shaped DNA box of 15-15-25 nm dimensions.
From left to right: DNA box, seed-decorated DNA box, and silver
growth within the box. FIG. 12E shows zoom-out TEM images for
silver nanoparticle grown within the rectangular-shaped DNA box of
20-15-30 nm dimensions.
Example 6
[0214] This example shows a method for designing opened nucleic
acid containers and the use of the containers to direct the shape
of silver and gold nanoparticles during their formation. In this
example, the M13 nucleic acid scaffold (P8064 mutation) (SEQ. ID.
NO. 1) was used.
[0215] The generality of the confined growth of metal
nanostructures in DNA molds was tested in open nucleic acid
containers 20 (e.g., barrels) to demonstrate the cross-section
controllability (FIG. 13). Four-layered DNA helices were connected
to form walls 25 that encircled specific shaped cavities within the
DNA containers. Cavities 30 were designed with three different
cross-sectional shapes, e.g., an equilateral triangle (FIG. 13A), a
right-angled triangle (FIG. 13B), and disk shapes (FIG. 13C). Three
21-nt single stranded binding sites were introduced at the interior
surface of the containers to immobilize seeds 34.
[0216] Gel purification indicated 5-10% folding yields of the DNA
containers. TEM images showed that the equilateral triangle-shaped
DNA channel exhibited an edge length of 25 nm with thickness of 15
nm (FIG. 13A left). In the case of the right-angled triangle, two
different sets of edge dimensions, e.g., 20-24-31 nm (FIG. 13B
left) and 15-29-33 nm (not shown) have been observed. Shape
diversity was ascribed to the presence of 16-base single-stranded
regions at the edges of the DNA containers (not shown). At
different stretching statuses, single-stranded regions may exhibit
distinct lengths, which in turn may produce variable container
dimensions. For the DNA ring, the inner diameter of the disk-shaped
container was determined to be 25 nm and the thickness was 10 nm
(FIG. 13C left). After gold seed decoration, TEM imaging revealed
60-75% decoration yields for each container (N>100). Although
multiple binding sites were present at the interior surface of the
containers, most DNA containers were conjugated with one seed (FIG.
13A-C middle), which was ascribed to spatial repulsion among
nanoparticles within the containers.
[0217] The silver nanoparticles grown within the DNA containers
(for 4 to 8 min at room temperature) were imaged by TEM.
Approximately 5-10% of the silver nanoparticles formed were found
to have the shape of the designed cross-sections of the container
in which the nanoparticles were grown (the remaining nanoparticles
having the shape of spheres). In the equilateral triangle-shaped
container, a fully confined silver nanoparticle exhibited an
equilateral triangle-shaped cross-section, with each edge having a
length of 25 nm and three round vertexes (FIG. 13A right). The DNA
container remained intact after silver growth, and was found to be
fully wrapped around the side surface of the grown silver
nanoparticle. No obvious bending or curvature of DNA sidewalls was
observed. In the center of the silver nanoparticle, a round shade
with 5-nm diameter was assigned to the decorated gold seed in the
DNA container. In the right-angled triangle-shaped channel with
20-24-31 nm dimensions, a right-angled silver nanoparticle was
grown with 19-24-29 nm dimensions (FIG. 13B right). Similar with
that in the equilateral triangle-shaped channel, round vertexes
were also observed in the right-angled silver nanoparticle. In the
disk-shaped channel, silver sphere was observed with cross-section
diameter of 25 nm (FIG. 13C right).
[0218] In the open containers, only particles grown laterally
rather than vertically were confined to the shape of the container,
which resulted in the 5-10% confinement yields. Most of the
unconfined particles exhibited sphere shapes.
[0219] It was observed that four-layered DNA helices increased
sidewall rigidity. In the case of the equilateral triangle-shaped
DNA containers, the number of defective structures having bent
sidewalls was lower than those in two- and three-layered
rectangular DNA containers. It was also observed that right-angled
triangular containers with 15-29-33 nm dimensions also confine the
silver growth within. However, the nanoparticle grown in a
container having a sharp vertex including a 30-degree angle was not
well formed, compared with that formed in a container having angles
of 60 degrees in an equilateral triangle, or in a container having
an angle of 50 degree in a right-angled triangle.
[0220] Several other open containers were also tested. Equilateral
triangle-shaped DNA containers with 15-nm edge exhibited less rigid
sidewalls compared with that having a 25-nm edge, owing to less
staple crossovers to interconnect DNA double helices. The silver
nanoparticle grown within produced triangle shaped silver
nanoparticles (1%, N>100), but most of the nanostructures were
sphere-like. A honeycomb lattice was employed to build hexagonal
DNA containers; however, after silver growth, orientation
transformation of double helices in the DNA sidewalls were
observed, indicating less rigid structure of the honeycomb lattice
compared with that of a square lattice.
[0221] Gold nanoparticles were also grown within open rectangular
DNA containers. Compared with that of silver nanoparticles, the
growth kinetics of gold in 0.5.times.TBE/10 mM Mg(NO.sub.3).sub.2
buffer was much slower. After reaction for thirty minutes, no
obvious size increment had been observed for the nanoparticle
within, which was ascribed to the chelating effect of EDTA to the
gold precursor. Removing EDTA from the reaction buffer
significantly promoted the growth kinetics. Thirty minutes reaction
produced a 15 nm.times.20 nm rectangular cross-shaped gold
nanoparticle within the rectangular barrel (FIG. 13D right).
Example 7
[0222] This example describes a method of performing directed
self-assembly of nucleic acid (e.g., DNA) containers as described
herein. In this example, the M13 nucleic acid scaffold (P8064
mutation) (SEQ. ID. NO. 1) was used.
[0223] DNA containers, including DNA containers containing
inorganic nanoparticles, provide not only structural confinement
but also surface addressability information. For example, for DNA
containers containing nanoparticles therein, due to the sequence
specificity of 3D DNA origami, each staple strand (e.g., a DNA
strand) that is located near a silver nanoparticle surface can be
independently addressed and modulated, which enables a surface
addressability resolution down to 2.5 nm.times.3.4 nm on the
nanoparticle surface. Each staple strand can be further modified
with distinct binding features, including biotin or multiple
different sequenced single-stranded regions, controlled
orientations and stoichiometry ratio. Different from previous
post-assembly strategies known in the art, surface addressability
of DNA nanostructures enabled metal growth within a pre-assembled
network of containers. Based on this feature, branched metallic
trimers and quantum dot (QD)-silver heterogeneous structures were
fabricated, as shown in FIG. 14.
[0224] To assemble a Y-shaped DNA container 51 from three
individual DNA barrel containers 20, six single-stranded connectors
were arranged into two parallel rows at one end of each rectangular
barrel by the extension of specific staple strands at both 3 and 5
positions. Each row was composed of three different sequenced 15-nt
single stranded DNAs, and hybridized to their complementary strands
in another partner barrel. Incubation of separately prepared and
purified DNA barrels (3 nM) in the presence of 10 nM gold seeds 34
produced seed-decorated Y-shaped barrels. TEM imaging indicated the
5% formation yield of seed-decorated Y-shaped barrels (FIG. 14A,
left and middle). Multimers, such as pentamers and hexamers, were
also observed in the unpurified solution. Silver growth within the
Y-shaped barrel produced individual nanoparticles within each
barrel, and a Y-shaped orientation for the trimer, as confined by
the orientation of DNA barrel containers (FIG. 14A, right). The
widths of silver nanoparticles 38 within each barrel were
determined to be 20, 22, and 23 nm. The slightly increased width
than the width of the rectangular barrel cavity (20 nm) was
attributed to the spacing expansion among DNA double helices by
metal growth. In the center of the Y-shape barrel where the growth
frontier of silver nanoparticles encountered, three clear particle
interfaces were observed, which confirmed that the as-formed silver
nanoparticles were originated and assembled from three independent
silver fragments. The presence of particle interfaces also
indicated the low growth kinetics of different oriented
crystallographic facets at the center, owing to the absence of
seeds in the center of Y-shaped barrel.
[0225] To build a heterogeneous quantum dot (QD)-silver
nanoparticle-quantum dot sandwiched structure 53 as shown in FIG.
14B, 5 or 6 biotin groups were introduced at each ends of an open
rectangular barrel container 20 with 20-15-30 nm dimensions and
having a cavity. The quantum dots were used as lids for the
containers to confine the growth of silver nanoparticles within the
containers. Biotinylation of DNA container barrels was achieved by
the extension of selected staples strands at 5 position via a TT
spacer. The biotinylated rectangular barrel container (5 nM) was
firstly incubated with gold seeds 34 (10 nM) for 17 hours, and then
incubated in the presence of excessive quantum dots 53
(streptavidin-coated QDs (50 nM)) for another 17 hours. Excessive
quantum dots 57 and gold seeds were removed via spin column
purification. TEM imaging revealed the formation of the designed
sandwiched structure between QDs and seed-decorated barrels ((FIG.
14B, left and middle)). After staining, the white spheres with
15-20 nm diameter was attributed to the PEG and streptavidin shell
around QD cores. 70% seed-decorated barrels were found conjugated
with two QDs at both ends (N>100). Notably, no QD was found
attached to the side surface of DNA barrels. Growth mediated by the
decorated gold seeds produced silver nanoparticles between two QDs,
with designed QD-Ag-QD heterogeneous structures (FIG. 14B, right).
The dimension sizes of Ag nanoparticle within DNA barrels were
determined to be 21 nm by 30 nm, which was in consistent with the
size of cavity of the container.
TABLE-US-00001 NUCLEIC ACID SEQUENCES M13 nucleic acid scaffold
(P8064 mutation) (SEQ ID NO: 1)
GAATTCGAGCTCGGTACCCGGGGATCCTCAACTGTGAGGAGGCTCACG
GACGCGAAGAACAGGCACGCGTGCTGGCAGAAACCCCCGGTATGACCG
TGAAAACGGCCCGCCGCATTCTGGCCGCAGCACCACAGAGTGCACAGG
CGCGCAGTGACACTGCGCTGGATCGTCTGATGCAGGGGGCACCGGCAC
CGCTGGCTGCAGGTAACCCGGCATCTGATGCCGTTAACGATTTGCTGA
ACACACCAGTGTAAGGGATGTTTATGACGAGCAAAGAAACCTTTACCC
ATTACCAGCCGCAGGGCAACAGTGACCCGGCTCATACCGCAACCGCGC
CCGGCGGATTGAGTGCGAAAGCGCCTGCAATGACCCCGCTGATGCTGG
ACACCTCCAGCCGTAAGCTGGTTGCGTGGGATGGCACCACCGACGGTG
CTGCCGTTGGCATTCTTGCGGTTGCTGCTGACCAGACCAGCACCACGC
TGACGTTCTACAAGTCCGGCACGTTCCGTTATGAGGATGTGCTCTGGC
CGGAGGCTGCCAGCGACGAGACGAAAAAACGGACCGCGTTTGCCGGAA
CGGCAATCAGCATCGTTTAACTTTACCCTTCATCACTAAAGGCCGCCT
GTGCGGCTTTTTTTACGGGATTTTTTTATGTCGATGTACACAACCGCC
CAACTGCTGGCGGCAAATGAGCAGAAATTTAAGTTTGATCCGCTGTTT
CTGCGTCTCTTTTTCCGTGAGAGCTATCCCTTCACCACGGAGAAAGTC
TATCTCTCACAAATTCCGGGACTGGTAAACATGGCGCTGTACGTTTCG
CCGATTGTTTCCGGTGAGGTTATCCGTTCCCGTGGCGGCTCCACCTCT
GAAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAAC
CCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCC
AGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAG
TTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCA
GAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGAGGCCGAT
ACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCC
ATCTACACCAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTT
CCCACGGAGAATCCGACGGGTTGTTACTCGCTCACATTTAATGTTGAT
GAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTT
CCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAATGCGAATT
TTAACAAAATATTAACGTTTACAATTTAAATATTTGCTTATACAATCT
TCCTGTTTTTGGGGCTTTTCTGATTATCAACCGGGGTACATATGATTG
ACATGCTAGTTTTACGATTACCGTTCATCGATTCTCTTGTTTGCTCCA
GACTCTCAGGCAATGACCTGATAGCCTTTGTAGATCTCTCAAAAATAG
CTACCCTCTCCGGCATTAATTTATCAGCTAGAACGGTTGAATATCATA
TTGATGGTGATTTGACTGTCTCCGGCCTTTCTCACCCTTTTGAATCTT
TACCTACACATTACTCAGGCATTGCATTTAAAATATATGAGGGTTCTA
AAAATTTTTATCCTTGCGTTGAAATAAAGGCTTCTCCCGCAAAAGTAT
TACAGGGTCATAATGTTTTTGGTACAACCGATTTAGCTTTATGCTCTG
AGGCTTTATTGCTTAATTTTGCTAATTCTTTGCCTTGCCTGTATGATT
TATTGGATGTTAATGCTACTACTATTAGTAGAATTGATGCCACCTTTT
CAGCTCGCGCCCCAAATGAAAATATAGCTAAACAGGTTATTGACCATT
TGCGAAATGTATCTAATGGTCAAACTAAATCTACTCGTTCGCAGAATT
GGGAATCAACTGTTATATGGAATGAAACTTCCAGACACCGTACTTTAG
TTGCATATTTAAAACATGTTGAGCTACAGCATTATATTCAGCAATTAA
GCTCTAAGCCATCCGCAAAAATGACCTCTTATCAAAAGGAGCAATTAA
AGGTACTCTCTAATCCTGACCTGTTGGAGTTTGCTTCCGGTCTGGTTC
GCTTTGAAGCTCGAATTAAAACGCGATATTTGAAGTCTTTCGGGCTTC
CTCTTAATCTTTTTGATGCAATCCGCTTTGCTTCTGACTATAATAGTC
AGGGTAAAGACCTGATTTTTGATTTATGGTCATTCTCGTTTTCTGAAC
TGTTTAAAGCATTTGAGGGGGATTCAATGAATATTTATGACGATTCCG
CAGTATTGGACGCTATCCAGTCTAAACATTTTACTATTACCCCCTCTG
GCAAAACTTCTTTTGCAAAAGCCTCTCGCTATTTTGGTTTTTATCGTC
GTCTGGTAAACGAGGGTTATGATAGTGTTGCTCTTACTATGCCTCGTA
ATTCCTTTTGGCGTTATGTATCTGCATTAGTTGAATGTGGTATTCCTA
AATCTCAACTGATGAATCTTTCTACCTGTAATAATGTTGTTCCGTTAG
TTCGTTTTATTAACGTAGATTTTTCTTCCCAACGTCCTGACTGGTATA
ATGAGCCAGTTCTTAAAATCGCATAAGGTAATTCACAATGATTAAAGT
TGAAATTAAACCATCTCAAGCCCAATTTACTACTCGTTCTGGTGTTTC
TCGTCAGGGCAAGCCTTATTCACTGAATGAGCAGCTTTGTTACGTTGA
TTTGGGTAATGAATATCCGGTTCTTGTCAAGATTACTCTTGATGAAGG
TCAGCCAGCCTATGCGCCTGGTCTGTACACCGTTCATCTGTCCTCTTT
CAAAGTTGGTCAGTTCGGTTCCCTTATGATTGACCGTCTGCGCCTCGT
TCCGGCTAAGTAACATGGAGCAGGTCGCGGATTTCGACACAATTTATC
AGGCGATGATACAAATCTCCGTTGTACTTTGTTTCGCGCTTGGTATAA
TCGCTGGGGGTCAAAGATGAGTGTTTTAGTGTATTCTTTTGCCTCTTT
CGTTTTAGGTTGGTGCCTTCGTAGTGGCATTACGTATTTTACCCGTTT
AATGGAAACTTCCTCATGAAAAAGTCTTTAGTCCTCAAAGCCTCTGTA
GCCGTTGCTACCCTCGTTCCGATGCTGTCTTTCGCTGCTGAGGGTGAC
GATCCCGCAAAAGCGGCCTTTAACTCCCTGCAAGCCTCAGCGACCGAA
TATATCGGTTATGCGTGGGCGATGGTTGTTGTCATTGTCGGCGCAACT
ATCGGTATCAAGCTGTTTAAGAAATTCACCTCGAAAGCAAGCTGATAA
ACCGATACAATTAAAGGCTCCTTTTGGAGCCTTTTTTTTGGAGATTTT
CAACGTGAAAAAATTATTATTCGCAATTCCTTTAGTTGTTCCTTTCTA
TTCTCACTCCGCTGAAACTGTTGAAAGTTGTTTAGCAAAATCCCATAC
AGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCG
TTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTAGT
TTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGG
GCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTC
TGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGG
TGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCAC
TTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCT
TGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCAGAATAATAGGTT
CCGAAATAGGCAGGGGGCATTAACTGTTTATACGGGCACTGTTACTCA
AGGCACTGACCCCGTTAAAACTTATTACCAGTACACTCCTGTATCATC
AAAAGCCATGTATGACGCTTACTGGAACGGTAAATTCAGAGACTGCGC
TTTCCATTCTGGCTTTAATGAGGATTTATTTGTTTGTGAATATCAAGG
CCAATCGTCTGACCTGCCTCAACCTCCTGTCAATGCTGGCGGCGGCTC
TGGTGGTGGTTCTGGTGGCGGCTCTGAGGGTGGTGGCTCTGAGGGTGG
CGGTTCTGAGGGTGGCGGCTCTGAGGGAGGCGGTTCCGGTGGTGGCTC
TGGTTCCGGTGATTTTGATTATGAAAAGATGGCAAACGCTAATAAGGG
GGCTATGACCGAAAATGCCGATGAAAACGCGCTACAGTCTGACGCTAA
AGGCAAACTTGATTCTGTCGCTACTGATTACGGTGCTGCTATCGATGG
TTTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGG
TGATTTTGCTGGCTCTAATTCCCAAATGGCTCAAGTCGGTGACGGTGA
TAATTCACCTTTAATGAATAATTTCCGTCAATATTTACCTTCCCTCCC
TCAATCGGTTGAATGTCGCCCTTTTGTCTTTGGCGCTGGTAAACCATA
TGAATTTTCTATTGATTGTGACAAAATAAACTTATTCCGTGGTGTCTT
TGCGTTTCTTTTATATGTTGCCACCTTTATGTATGTATTTTCTACGTT
TGCTAACATACTGCGTAATAAGGAGTCTTAATCATGCCAGTTCTTTTG
GGTATTCCGTTATTATTGCGTTTCCTCGGTTTCCTTCTGGTAACTTTG
TTCGGCTATCTGCTTACTTTTCTTAAAAAGGGCTTCGGTAAGATAGCT
ATTGCTATTTCATTGTTTCTTGCTCTTATTATTGGGCTTAACTCAATT
CTTGTGGGTTATCTCTCTGATATTAGCGCTCAATTACCCTCTGACTTT
GTTCAGGGTGTTCAGTTAATTCTCCCGTCTAATGCGCTTCCCTGTTTT
TATGTTATTCTCTCTGTAAAGGCTGCTATTTTCATTTTTGACGTTAAA
CAAAAAATCGTTTCTTATTTGGATTGGGATAAATAATATGGCTGTTTA
TTTTGTAACTGGCAAATTAGGCTCTGGAAAGACGCTCGTTAGCGTTGG
TAAGATTCAGGATAAAATTGTAGCTGGGTGCAAAATAGCAACTAATCT
TGATTTAAGGCTTCAAAACCTCCCGCAAGTCGGGAGGTTCGCTAAAAC
GCCTCGCGTTCTTAGAATACCGGATAAGCCTTCTATATCTGATTTGCT
TGCTATTGGGCGCGGTAATGATTCCTACGATGAAAATAAAAACGGCTT
GCTTGTTCTCGATGAGTGCGGTACTTGGTTTAATACCCGTTCTTGGAA
TGATAAGGAAAGACAGCCGATTATTGATTGGTTTCTACATGCTCGTAA
ATTAGGATGGGATATTATTTTTCTTGTTCAGGACTTATCTATTGTTGA
TAAACAGGCGCGTTCTGCATTAGCTGAACATGTTGTTTATTGTCGTCG
TCTGGACAGAATTACTTTACCTTTTGTCGGTACTTTATATTCTCTTAT
TACTGGCTCGAAAATGCCTCTGCCTAAATTACATGTTGGCGTTGTTAA
ATATGGCGATTCTCAATTAAGCCCTACTGTTGAGCGTTGGCTTTATAC
TGGTAAGAATTTGTATAACGCATATGATACTAAACAGGCTTTTTCTAG
TAATTATGATTCCGGTGTTTATTCTTATTTAACGCCTTATTTATCACA
CGGTCGGTATTTCAAACCATTAAATTTAGGTCAGAAGATGAAATTAAC
TAAAATATATTTGAAAAAGTTTTCTCGCGTTCTTTGTCTTGCGATTGG
ATTTGCATCAGCATTTACATATAGTTATATAACCCAACCTAAGCCGGA
GGTTAAAAAGGTAGTCTCTCAGACCTATGATTTTGATAAATTCACTAT
TGACTCTTCTCAGCGTCTTAATCTAAGCTATCGCTATGTTTTCAAGGA
TTCTAAGGGAAAATTAATTAATAGCGACGATTTACAGAAGCAAGGTTA
TTCACTCACATATATTGATTTATGTACTGTTTCCATTAAAAAAGGTAA
TTCAAATGAAATTGTTAAATGTAATTAATTTTGTTTTCTTGATGTTTG
TTTCATCATCTTCTTTTGCTCAGGTAATTGAAATGAATAATTCGCCTC
TGCGCGATTTTGTAACTTGGTATTCAAAGCAATCAGGCGAATCCGTTA
TTGTTTCTCCCGATGTAAAAGGTACTGTTACTGTATATTCATCTGACG
TTAAACCTGAAAATCTACGCAATTTCTTTATTTCTGTTTTACGTGCAA
ATAATTTTGATATGGTAGGTTCTAACCCTTCCATTATTCAGAAGTATA
ATCCAAACAATCAGGATTATATTGATGAATTGCCATCATCTGATAATC
AGGAATATGATGATAATTCCGCTCCTTCTGGTGGTTTCTTTGTTCCGC
AAAATGATAATGTTACTCAAACTTTTAAAATTAATAACGTTCGGGCAA
AGGATTTAATACGAGTTGTCGAATTGTTTGTAAAGTCTAATACTTCTA
AATCCTCAAATGTATTATCTATTGACGGCTCTAATCTATTAGTTGTTA
GTGCTCCTAAAGATATTTTAGATAACCTTCCTCAATTCCTTTCAACTG
TTGATTTGCCAACTGACCAGATATTGATTGAGGGTTTGATATTTGAGG
TTCAGCAAGGTGATGCTTTAGATTTTTCATTTGCTGCTGGCTCTCAGC
GTGGCACTGTTGCAGGCGGTGTTAATACTGACCGCCTCACCTCTGTTT
TATCTTCTGCTGGTGGTTCGTTCGGTATTTTTAATGGCGATGTTTTAG
GGCTATCAGTTCGCGCATTAAAGACTAATAGCCATTCAAAAATATTGT
CTGTGCCACGTATTCTTACGCTTTCAGGTCAGAAGGGTTCTATCTCTG
TTGGCCAGAATGTCCCTTTTATTACTGGTCGTGTGACTGGTGAATCTG
CCAATGTAAATAATCCATTTCAGACGATTGAGCGTCAAAATGTAGGTA
TTTCCATGAGCGTTTTTCCTGTTGCAATGGCTGGCGGTAATATTGTTC
TGGATATTACCAGCAAGGCCGATAGTTTGAGTTCTTCTACTCAGGCAA
GTGATGTTATTACTAATCAAAGAAGTATTGCTACAACGGTTAATTTGC
GTGATGGACAGACTCTTTTACTCGGTGGCCTCACTGATTATAAAAACA
CTTCTCAGGATTCTGGCGTACCGTTCCTGTCTAAAATCCCTTTAATCG
GCCTCCTGTTTAGCTCCCGCTCTGATTCTAACGAGGAAAGCACGTTAT
ACGTGCTCGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTA
AGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCC
AGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCC
ACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTA
GGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGAT
TTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTT
CGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTC
CAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTA
TAAGGGATTTTGCCGATTTCGGAACCACCATCAAACAGGATTTTCGCC
TGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCC
AGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAA
AAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGG
CCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCG
GGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCA
CCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATT
GTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTAC Sequences of
staple strands for the DNA container in FIG. 8. oligo1 (SEQ ID NO:
2) TAACCACCCACTACGTGAACCACGTCAAAGGGCGAACCGCCT oligo2 (SEQ ID NO: 3)
CGGGCGCAGGTGCCGTAAAGCCAGTTTGGAACAAGGTTTGCC oligo3 (SEQ ID NO: 4)
CGGCGAACGATTTAGAGCTTGATAAATCAAAAGAAAAATCGG oligo4 (SEQ ID NO: 5)
GTTTTTTTATTAAAGAACGTGTGCAGCAAGCGGTCTGGGCGC oligo5 (SEQ ID NO: 6)
CTAAAGGAGATAGGGTTGAGTTCCTGTTTGATGGTTTAATGA oligo6 (SEQ ID NO: 7)
AAAGTTGTTCACTAAATGAAAGCGTTAGAATCAGAGCGAATCAGT oligo7 (SEQ ID NO: 8)
TTCAGAGAGTGACTCCAATCACCCGGTCACGACTATGG oligo8 (SEQ ID NO: 9)
CAGGGTGAAAGTGTAAAGCCTTTTTCACGGTCATCGTGTGTT oligo9 (SEQ ID NO: 10)
ATCGGCCATTAATTGCGTTGCCCTGTGCACTCTGTCTGCAGC oligo10 (SEQ ID NO: 11)
CGAGCCGTTTCCTGTGTGAAATCATAAACATCCCTGCCCTGC oligo11 (SEQ ID NO: 12)
TGCTGCGTATCACGCTGAGTCCACGGGGTCGTAGGGCGACGTATA oligo12 (SEQ ID NO:
13) GTGAGCTCGGCCAGAATGCGGCGGCATCAGATGCCCAATCCG oligo13 (SEQ ID NO:
14) ACTAGCTGCAGGTTCCGTAGCCCGGAGCCCCCGTGGCGAACAGGA oligo14 (SEQ ID
NO: 15) CAGCAAAGGTATGAGCCGGGTGGTCTGGTCAGCAGGTCTCGT oligo15 (SEQ ID
NO: 16) CAGCGGTCATTGCAGGCGCTTGTCGGTGGTGCCATACGATGC oligo16 (SEQ ID
NO: 17) GGCTGGTGTAGAACGTCAGCGGGCCAGAGCACATCGCGGATCTCAC oligo17 (SEQ
ID NO: 18) CCGGGCGAAGAATGCCAACGGGCAAACGCGGTCCGGGGCGGTATTT oligo18
(SEQ ID NO: 19) ACCTCGCACTGGGTTACGGTGCTGAACTCACAACGCGCGCGA oligo19
(SEQ ID NO: 20) TCCTGGTGCTCACTGTTTACACTGATAGCTGGAAGCATGTTT oligo20
(SEQ ID NO: 21) CGCTGGCGCTCATTTCCGTGGGTTTTCCCTTCGCTTGCC oligo21
(SEQ ID NO: 22) TGATTGCTAAAAAAGCCATGTGCTTTCATCAGGCTTCGC oligo22
(SEQ ID NO: 23) AGCAGTTTTTTTTCCAACCGCCGGTTGCTCGTTAACGGGCCGGGGG
oligo23 (SEQ ID NO: 24)
AAAAAAAAAGTTAACCCACGCGCGGGGTGCCGGTGCTGCGCGGCTC oligo24 (SEQ ID NO:
25) GGAATTAAGTCGAAAGGGGCGCATGGGATAGATTGTAAGAAGATTCAAT oligo25 (SEQ
ID NO: 26) GTGTAAAACGAAGGGCGACAGTATCGTCGGATGTTAAACATATGTAGAG
oligo26 (SEQ ID NO: 27)
TCGACGGGAAAGGCAAAGGCACCGTAGCCAGAAATAATAAGAGAACATT oligo27 (SEQ ID
NO: 28) CGCCAGGTGAAGGGATAGCTCAAACTTAAATTTCTAGCC oligo28 (SEQ ID NO:
29) GTGCCAATTACCAGTCCCGGATGTGTACATCGACACGTTCCGCAGC oligo29 (SEQ ID
NO: 30) AGTAAACGGCTTAAAATTCAGAAATAGCTGAAAAGATTTAAA oligo30 (SEQ ID
NO: 31) ACTAAATGTGAACCAATTCGTAAAGATCTACCCCTCATATTA oligo31 (SEQ ID
NO: 32) GCTTTCCGCGCCATTCGCCATGAGGTGG oligo32 (SEQ ID NO: 33)
CCGTAATCGTAACCGTGCATCATTACGCCAGCTGGTGGGTAA oligo33 (SEQ ID NO: 34)
TGGGAACTTGAGGGGACGACGATCGGTGCGGGCCTCAGTCAC oligo34 (SEQ ID NO: 35)
AACAACCCGGCCTCAGGAAGAGCGCAACTGTTGGGACGGCCA oligo35 (SEQ ID NO: 36)
ACGGTAAAGGAACGCCATCAACTTTCATCAACATTCCAGCCA oligo36 (SEQ ID NO: 37)
ATGCCGGACCCCGGTTGATAATCGCATTAAATTTTTTCTCCG oligo37 (SEQ ID NO: 38)
TAAGCAACAAAAGGGTGAGAAATATTCAACCGTTCAGCCCCA oligo38 (SEQ ID NO: 39)
GCCAAAGGTGGCATCAAACATGTTTTAAATACCTTTAA oligo39 (SEQ ID NO: 40)
CAAAAACATATTTTAAATGCAAGCTATTTTTGAGAACTAGCA oligo40
(SEQ ID NO: 41) TGAAATAACCTGTTTAGTCATTCCATATAACCCAGACC oligo41 (SEQ
ID NO: 42) ATACTTTAAAAATTTTTAGAAAAAGGCTATCAGGTTCGATGA oligo42 (SEQ
ID NO: 43) GTAGCATTGAATATAATGCTGAAGAGGTCATTTTTAGAAAAC oligo43 (SEQ
ID NO: 44) TGATCAGAGCATAAAGCAGTAATGTGTAGGTTAAATTA oligo44 (SEQ ID
NO: 45) GGGGCGCAAAGTACGGTGTCTACAGGTCAGGATTACTGACTA oligo45 (SEQ ID
NO: 46) TTTCGCATCCCAATTCTGCGATAATTCGAGCTTCAATTAAGA oligo46 (SEQ ID
NO: 47) TTGCTCCATCAAAAATCAGGTAATACTGCGGAATCATGCAGA oligo47 (SEQ ID
NO: 48) GGAAGCAAAAGCGGATTGCATCCAGAGGGGGTAATGAGCAAC oligo48 (SEQ ID
NO: 49) TTGCAAAAAGAAGCGAAAGTTGATAATGGTCCCCTGTA oligo49 (SEQ ID NO:
50) ACCACATAACATTATTACAGGCAAATCAACGTAACTCAAGAG oligo50 (SEQ ID NO:
51) CTAGTCATAAACCATAATTTTGATTAGCTCATTCTACTGCAAAAT oligo51 (SEQ ID
NO: 52) TACATAAAATAAAACGAACTACTCATTCAGTGAATGCATAGG oligo52 (SEQ ID
NO: 53) ACGAGGCTTGGGAAGAAAAATGCCCTGACGAGAAATGAACGG oligo53 (SEQ ID
NO: 54) TAAAGTAAAACAGAAGCAACTCCAGGAAGTTCTATATTGTTGTAC oligo54 (SEQ
ID NO: 55) ACTATCACTCATTATACCAGTACGAGTAGTAAATTCCAACTT oligo55 (SEQ
ID NO: 56) CAGACGATTATGCGATTTTAAAGATGGTTTAATTTATCATAA oligo56 (SEQ
ID NO: 57) AAAGCGAGAGCGAAAGACGCGTTTACGAGTAGACCATTGAAGCCT oligo57
(SEQ ID NO: 58) GTTCGCCAAAAGCGTCCCTTTACCGAGAGTATGCAACTGAGC oligo58
(SEQ ID NO: 59) TAATCTTCCCAGCGATTATACAGAGGCAAAAGAATATAACCGCAAT
oligo59 (SEQ ID NO: 60) CTGGCTGGAAACAAAGTACAAAAGGCACCAACCTATGAG
oligo60 (SEQ ID NO: 61)
TGTACAGTTGTATCATCGCCTAAAATACGTAATGCGGCCGCTAGGT oligo61 (SEQ ID NO:
62) TGAAAGATGTGTCGAAATCCGGGAAGTTTCCATTAACCC oligo62 (SEQ ID NO: 63)
GGGAACCCTCCATGTTACTTAGGACTAAAGACTTTCATCGGAAAAA oligo63 (SEQ ID NO:
64) TGACGACCTGGAACTGAGGGCTTGGAACTGGTAACCCTAGTT oligo64 (SEQ ID NO:
65) CGGTCGCAAACGAACAAGCGCACCTTCAAAAGCTGACGGAACTCAA oligo65 (SEQ ID
NO: 66) GCTTGATACCTGCTAAATAGCGTAGTTTAGTGGATAAGTACT oligo66 (SEQ ID
NO: 67) AGTTAAACACTACGCGGAGATACCAGGCAAGGCTTCTAC oligo67 (SEQ ID NO:
68) GATCGTCAACGGGTGATAAATGGACAGACACCAGACAGGACGATAG oligo68 (SEQ ID
NO: 69) TCACGGTTTAAGGAACAAAACTACCACCCTCAGAGAAGGTGC oligo69 (SEQ ID
NO: 70) TAGCAACGCTTTGAGCCGGAAACGGTCACAACTTTAATTACCCGAT oligo70 (SEQ
ID NO: 71) GACTGAATTTTGTCGTCGTGTATCTTGATATGCTTTTGACCGTTCACCA
oligo71 (SEQ ID NO: 72)
GAATTCAGCGTCCACAGAACCGCCGGGTTTTGGGTCAGATCCTCACTCA oligo72 (SEQ ID
NO: 73) GGAGAATAATACTGAGTCATTTTCTTAAGAGGCCCCCTAGGCAGGACCA oligo73
(SEQ ID NO: 74) GAATTGCGCCTTTAATTGTATGCAGCGAAAGACAGTTCA oligo74
(SEQ ID NO: 75) TCATAGTCAACTTTCAACAGTTTTCTTAAACAGCTTGCAGGG oligo75
(SEQ ID NO: 76) GAGAGGGACCGTACTCAGGAGACGATCTAAAGTTTTCTGTAT oligo76
(SEQ ID NO: 77) ATTAGCGACCCTCAGAACCGCAACGCCTGTAGCATGAGTGAG oligo77
(SEQ ID NO: 78) CTCCTCAAGAGCCACCACCCTTTCGTCACCAGTACACTAAAG oligo78
(SEQ ID NO: 79) GAAAGTAAGGGATAGCAAGCCCCATGTACCGTAACAATTTTT oligo79
(SEQ ID NO: 80) GGTGAAAGCGGCCTCCCCCCCCTTCCATTTGGGGAGGG oligo80 (SEQ
ID NO: 81) TTTAACGGCTCAGTACCAGGCACCGCCACCCTCAGACAGCCC oligo81 (SEQ
ID NO: 82) CCGTTGATATGCCACCACGTCAGATACCATTTTACCAG oligo82 (SEQ ID
NO: 83) AGCCGCCTCAGACGATTGGCCTATAAACAGTTAATGCTGAGA oligo83 (SEQ ID
NO: 84) GTCATAGTCAGAGCCGCCACCTTAAAGCCAGAATGAATAAGT oligo84 (SEQ ID
NO: 85) ACTTGAGATTAGCGTTTGCCACCACCACCGGAACCCAGTCTC oligo85 (SEQ ID
NO: 86) GCACCATCTGTAGCGCGTTTTCCGCCACCCTCAGATCACAAA oligo86 (SEQ ID
NO: 87) CCATCGAGTAATCAGTAGCGACCACCACCAGAGCCTTGACAG oligo87 (SEQ ID
NO: 88) TATTCATTAATAACGGAATACCCGAACAAAGTTACTCAAAAA oligo88 (SEQ ID
NO: 89) AAGGTAAAACTGGCATGATTATTAAGAAAAGTAAGTTTACAG oligo89 (SEQ ID
NO: 90) ATTCAACTTATTACGCAGTATGCTATCTTACCGAAAAACAGG oligo90 (SEQ ID
NO: 91) CGCCAAAAACGTAGAAAATACAGAAACAATGAAATGGGAGAA oligo91 (SEQ ID
NO: 92) GGTAGCAAGGCCGGAAAAAGTTTGCCTTTAGCCCTCAG oligo92 (SEQ ID NO:
93) AAAATTCAAGGTGGCAACATATTAAGCCCAATAATTGAACAA oligo93 (SEQ ID NO:
94) TTTAGACTCCCGATTGAGGAATTAGAGCCAGATTTTCG oligo94 (SEQ ID NO: 95)
AGAGAATAAAATAAACAGCCACAAATCAGATATAGAACCAAG oligo95 (SEQ ID NO: 96)
TTAACTGAACGCTAACGAGCGAGGCGTTTTAGCGAATAATCG oligo96 (SEQ ID NO: 97)
CGCCCAAAGAACAAGCAAGCCAGAGAATATAAAGTCATGTAA oligo97 (SEQ ID NO: 98)
AAGTATTATTAGCAGCCCAGATAGCCAAAAGATATTGAGTCACCG oligo98 (SEQ ID NO:
99) ATTCTAATCATTCCAAGAACGAGACGACGACAATACAGTAGG oligo99 (SEQ ID NO:
100) GCGTCTTTCCATTAGACAGCAATAGTTAGCAGACAAAAACCAGTA oligo100 (SEQ ID
NO: 101) GGGAGGTCGAGCATGTAGAAAGCCTGTTTATCAACATGCGTT oligo101 (SEQ
ID NO: 102) AAGCAGCTACGGGTAATAATTGAGTAAAAGAGTCACAAAATGAAA oligo102
(SEQ ID NO: 103) TACCGCAAAAGGTAAAGTAATCGCCATATTTAACATAGTTAA
oligo103 (SEQ ID NO: 104)
GCTGTCTTGTTCAGCTAATGCCAGTATAAAGCCAAACCGACC oligo104 (SEQ ID NO:
105) TCAACCTCCCTCTTACCAACACCCAAGAGCAATACATAATAT oligo105 (SEQ ID
NO: 106) TTTAGGCAAAACTTTTTCAAATGCTGATGCAAATCATTA oligo106 (SEQ ID
NO: 107) AATTCTGTCCGGTATTAAAGGCTTCAGTTACAACATAAGCCC oligo107 (SEQ
ID NO: 108) GCTTAATCTAAATTTAATGGTTTTAACCTCCGGCTGAGTGAAAGCA oligo108
(SEQ ID NO: 109) ATACAAAGCGTTAAATAAGAAAATAGTGAATTTATTTTTCCCTACA
oligo109 (SEQ ID NO: 110) TTTCATCGGTTATATAACTATAGTACATAAACATCTTGC
oligo110 (SEQ ID NO: 111) GTGTGATATAGGTCTGAGAGATAAATCGATTATTCGTTT
oligo111 (SEQ ID NO: 112)
CCTAATTACAAACCTACTACTTCTTAATAGAAAATATCCGAA oligo112 (SEQ ID NO:
113) TGGAAACATGTAAATATATTTACGCCAAACCGACACTCATCGTAGC oligo113 (SEQ
ID NO: 114) GCTTCTGCTACCTTTTGAAATCGCTCAAAACAACATTCCTTAGAAC oligo114
(SEQ ID NO: 115) TAATTAACAAAATCAAATAAGTTCTTACAGAACGCCCAA oligo115
(SEQ ID NO: 116) CCTTGAAAAGAGTCTAAACACGTATCATAATAGATTAATTTATTTG
oligo116 (SEQ ID NO: 117) ATGAAACAAATCAATATATGTTAGGTTGTTCTGACTGAG
oligo117 (SEQ ID NO: 118)
AAAAGAAATTGATGATGAGAAGTATTGGCAAGAACCACCTGA oligo118 (SEQ ID NO:
119) AAACAGTAACCCACCAGATCCTTTGCTGAACTTAACACAGTA oligo119 (SEQ ID
NO: 120) ACGTAATCCTAGATAATGGAATTGTCGCCATACGTGGCTGGT oligo120 (SEQ
ID NO: 121) AACTCATCATCAATTCGCCTCAATACAGAGGGCCAACAGAAA oligo121
(SEQ ID NO: 122) AGATCATTTTAATTTTAAAAAATCCCACGCTAGATTCATCTG
oligo122 (SEQ ID NO: 123)
GGAATTAGTCAGATGAATATATCGCGCAGAGGCGATCGCTAT oligo123 (SEQ ID NO:
124) AGGATTTGCAATTCATCAATATAAAACAGAAATAAGAAGATG oligo124 (SEQ ID
NO: 125) TTATCTATTAGAGCCGTCAATGATTGTTTGGATTACATATCA oligo125 (SEQ
ID NO: 126) TGGTCAGTTAGACTTTACAAAATTCCTGATTATCAGCGTAGA oligo126
(SEQ ID NO: 127) TCACCTTGCCCGAACGTTATTGCGGAACAAAGAAAAGTACCT
oligo127 (SEQ ID NO: 128) CTGTGAATGGAACTCAAATAACATGCGCTTAATGCGCC
oligo128 (SEQ ID NO: 129)
GTCAGTACTCAAATATCAAACACAACTCGTATTAAAAGGAGC oligo129 (SEQ ID NO:
130) TAAGAATTAAAAATACCGAACATCAACAGTTGAAAACATTTG oligo130 (SEQ ID
NO: 131) CCTTACCGCCTCACGCAGACGAGCCTGGCAAGTGTAGCAAATCAA oligo131
(SEQ ID NO: 132) ATACTACATTTTTTTATGGAGCTAAGAAAGGAAGGGAACGGAACC
oligo132 (SEQ ID NO: 133)
GAGGCCAGCTCATGGAAATACAAAGGGACATTCTGTGAGGCG oligo133 (SEQ ID NO:
134) GCTACAGTTCTTTGATTAGTAACTATCGGCCTTGCACAGACA oligo134 (SEQ ID
NO: 135) TTGCTTTAATTAACCGTTGTAATCCAGAACAATATGAAAGCG oligo135 (SEQ
ID NO: 136) ACGTGCTAAAGAGTCTGTCCAAGCCATTGCAACAGGAGATAG oligo136
(SEQ ID NO: 137) GGCCGATAATCCTGAGAAGTGTTGACGCTCAATCGCCAGTCA
oligo137 (SEQ ID NO: 138) CCGAGCTCGAATTCGTAATCA oligo138 (SEQ ID
NO: 139) GGCCCTGTTTTCACCAGTGAGCAACATA oligo139 (SEQ ID NO: 140)
AAAACAGACGTTAATATTTTGGGATTGA oligo140 (SEQ ID NO: 141)
ATGAGGCCGGAGAATTAAATAGTA oligo141 (SEQ ID NO: 142)
GAGAATGATATTCATTGAATCTAGGAAT oligo142 (SEQ ID NO: 143)
GGGATTTGATAGTTGCGCCGAATATATT oligo143 (SEQ ID NO: 144)
TGAATTTATGATACAGGAGTGTGCCGTC oligo144 (SEQ ID NO: 145)
GAGTCTTTTCTATCACCCGGAAAT oligo145 (SEQ ID NO: 146)
TGAAAATTATCCCAATCCAAAATTACCG oligo146 (SEQ ID NO: 147)
AAATTATAAGAAAACAAAATTTTTTTAA oligo147 (SEQ ID NO: 148)
ATATTTTATAGCCCTAAAACAAGGAAGG oligo148 (SEQ ID NO: 149)
AATGCAATACGGCGCGTCTGCGCG oligo149 (SEQ ID NO: 150)
GGCCCTGTTTTCACCAGTGAGCAACATATTCCTCTACCACCTACATCAC oligo150 (SEQ ID
NO: 151) AAAACAGACGTTAATATTTTGGGATTGATTCCTCTACCACCTACATCAC oligo151
(SEQ ID NO: 152) ATGAGGCCGGAGAATTAAATAGTATTCCTCTACCACCTACATCAC
oligo152 (SEQ ID NO: 153)
GAGAATGATATTCATTGAATCTAGGAATTTCCTCTACCACCTACATCAC oligo153 (SEQ ID
NO: 154) GGGATTTGATAGTTGCGCCGAATATATTTTCCTCTACCACCTACATCAC oligo154
(SEQ ID NO: 155) TGAATTTATGATACAGGAGTGTGCCGTCTTCCTCTACCACCTACATCAC
oligo155 (SEQ ID NO: 156)
GAGTCTTTTCTATCACCCGGAAATTTCCTCTACCACCTACATCAC oligo156 (SEQ ID NO:
157) TGAAAATTATCCCAATCCAAAATTACCGTTCCTCTACCACCTACATCAC oligo157
(SEQ ID NO: 158) AAATTATAAGAAAACAAAATTTTTTTAATTCCTCTACCACCTACATCAC
oligo158 (SEQ ID NO: 159)
ATATTTTATAGCCCTAAAACAAGGAAGGTTCCTCTACCACCTACATCAC oligo159 (SEQ ID
NO: 160) AATGCAATACGGCGCGTCTGCGCGTTCCTCTACCACCTACATCAC oligo160
(SEQ ID NO: 161) CAAAATCAAACCTGTCGTGCCGCCCGCT oligo161 (SEQ ID NO:
162) AGCCGCCGCGAAACGTACAGCATCCCGT oligo162 (SEQ ID NO: 163)
GCCCAAGGATTGCGGGAAGATACA oligo163 (SEQ ID NO: 164)
GGAAGCCGCTTTTGCAAAAGACGTTTAC oligo164 (SEQ ID NO: 165)
TCACGTTAAAAAAAAGGCTCCACGAGGG oligo165 (SEQ ID NO: 166)
GAGGTTGGCCTATTTCGGAACGAAACAT
oligo166 (SEQ ID NO: 167) GAACAGAATCCGTCACCTCAATAG oligo167 (SEQ ID
NO: 168) AGTCAGAAATTTTATCCTGAAGACTTGC oligo168 (SEQ ID NO: 169)
TTTACATTTTGAATACCAAGTTTAGAAT oligo169 (SEQ ID NO: 170)
CACGACCCGCCTGCAACAGTGTAAAGCA oligo170 (SEQ ID NO: 171)
AAAACGCCAGTAAAGGGGGAAAGC oligo171 (SEQ ID NO: 172)
CAAAATCAAACCTGTCGTGCCGCCCGCTTATCTTCCTCACACTCCCAAA oligo172 (SEQ ID
NO: 173) AGCCGCCGCGAAACGTACAGCATCCCGTTATCTTCCTCACACTCCCAAA oligo173
(SEQ ID NO: 174) GCCCAAGGATTGCGGGAAGATACATATCTTCCTCACACTCCCAAA
oligo174 (SEQ ID NO: 175)
GGAAGCCGCTTTTGCAAAAGACGTTTACTATCTTCCTCACACTCCCAAA oligo175 (SEQ ID
NO: 176) TCACGTTAAAAAAAAGGCTCCACGAGGGTATCTTCCTCACACTCCCAAA oligo176
(SEQ ID NO: 177) GAGGTTGGCCTATTTCGGAACGAAACATTATCTTCCTCACACTCCCAAA
oligo177 (SEQ ID NO: 178)
GAACAGAATCCGTCACCTCAATAGTATCTTCCTCACACTCCCAAA oligo178 (SEQ ID NO:
179) AGTCAGAAATTTTATCCTGAAGACTTGCTATCTTCCTCACACTCCCAAA oligo179
(SEQ ID NO: 180) TTTACATTTTGAATACCAAGTTTAGAATTATCTTCCTCACACTCCCAAA
oligo180 (SEQ ID NO: 181)
CACGACCCGCCTGCAACAGTGTAAAGCATATCTTCCTCACACTCCCAAA oligo181 (SEQ ID
NO: 182) AAAACGCCAGTAAAGGGGGAAAGCTATCTTCCTCACACTCCCAAA oligo182
(SEQ ID NO: 183) CCAGCAGGGGGAGAGGCGGTTCTAATGA oligo183 (SEQ ID NO:
184) GACGTTGAGAGATAGACTTTCTGCCGCC oligo184 (SEQ ID NO: 185)
TGTCAATTCAGCTCATTTTTTAGCGAGT oligo185 (SEQ ID NO: 186)
GGTATGCCTGTAAATCGTTCATTT oligo186 (SEQ ID NO: 187)
TTATAGTTGTTTAGACTGGATAGGAATT oligo187 (SEQ ID NO: 188)
AATAGAATCAGCTTGCTTTCGTTTGCGG oligo188 (SEQ ID NO: 189)
CAAATAATGCCTTGAGTAACAGATTAGG oligo189 (SEQ ID NO: 190)
GAACATCGGCCAAAATCGGGCGAC oligo190 (SEQ ID NO: 191)
GAAGCGCAGAGCCTAATTTGCATCCGGT oligo191 (SEQ ID NO: 192)
TTTTCAGATTTCAATTACCTGTAACCTT oligo192 (SEQ ID NO: 193)
AACCCTTCAGCAGAAGATAAACAATATC oligo193 (SEQ ID NO: 194)
AACCCGAGTATTCCTCGAAAGGAG oligo194 (SEQ ID NO: 195)
CCAGCAGGGGGAGAGGCGGTTCTAATGATAACATTCCTAACTTCTCATA oligo195 (SEQ ID
NO: 196) GACGTTGAGAGATAGACTTTCTGCCGCCTAACATTCCTAACTTCTCATA oligo196
(SEQ ID NO: 197) TGTCAATTCAGCTCATTTTTTAGCGAGTTAACATTCCTAACTTCTCATA
oligo197 (SEQ ID NO: 198)
GGTATGCCTGTAAATCGTTCATTTTAACATTCCTAACTTCTCATA oligo198 (SEQ ID NO:
199) TTATAGTTGTTTAGACTGGATAGGAATTTAACATTCCTAACTTCTCATA oligo199
(SEQ ID NO: 200) AATAGAATCAGCTTGCTTTCGTTTGCGGTAACATTCCTAACTTCTCATA
oligo200 (SEQ ID NO: 201)
CAAATAATGCCTTGAGTAACAGATTAGGTAACATTCCTAACTTCTCATA oligo201 (SEQ ID
NO: 202) GAACATCGGCCAAAATCGGGCGACTAACATTCCTAACTTCTCATA oligo202
(SEQ ID NO: 203) GAAGCGCAGAGCCTAATTTGCATCCGGTTAACATTCCTAACTTCTCATA
oligo203 (SEQ ID NO: 204)
TTTTCAGATTTCAATTACCTGTAACCTTTAACATTCCTAACTTCTCATA oligo204 (SEQ ID
NO: 205) AACCCTTCAGCAGAAGATAAACAATATCTAACATTCCTAACTTCTCATA oligo205
(SEQ ID NO: 206) AACCCGAGTATTCCTCGAAAGGAGTAACATTCCTAACTTCTCATA
Sequences of staple strands for the DNA container in FIG. 9. oligo1
(SEQ ID NO: 207) ACATCGTGAATACATTAGCGACCAGAG oligo2 (SEQ ID NO:
208) TTAGAAGGTCAATACCGAACACTTTTTA oligo3 (SEQ ID NO: 209)
CCGTACTAGTATAGCCTAAATTATGTAA oligo4 (SEQ ID NO: 210)
CGACGTTTTTTGCAATGTTTAGAAGAGAA oligo5 (SEQ ID NO: 211)
CGAGCATCCCGTCGGGAGTTAGGCGCATA oligo6 (SEQ ID NO: 212)
CCATATGCACTCCAACTAAAAAATTGGGCTTGAG oligo7 (SEQ ID NO: 213)
GCGTGCCATTAAAGGCCGTTCATATTACGGTAATC oligo8 (SEQ ID NO: 214)
AGGTGAGTTAACACTAACGTCATAGCAGCCTTTAC oligo9 (SEQ ID NO: 215)
AGCCAGCAAATCTAAACAGGGGACGGGAGAATTAA oligo10 (SEQ ID NO: 216)
GTTATCTTAGGAGCAATAAGAATGAAATAGCAATA oligo11 (SEQ ID NO: 217)
AAAAGCCTGAGCAATACCTTTCCACCCTCAGAGCC oligo12 (SEQ ID NO: 218)
CGCCATGTTTACCAAACATAGATCAAAAGCGTCAT oligo13 (SEQ ID NO: 219)
AAACGTATGCAAATATTTCATGTTAAATAACACTG oligo14 (SEQ ID NO: 220)
ACGCCGAATAAACAAATTCTTGTAACGAATTTTGC oligo15 (SEQ ID NO: 221)
TTTGAGGGGACGACAACAAGATGCCCTGAACCGAT oligo16 (SEQ ID NO: 222)
TCGGCTAATTCTGTATCAACAGCTTGCTCAACAAC oligo17 (SEQ ID NO: 223)
GCGAGGTTTTTGTTAAATCAGATTGTATCGCCTGT oligo18 (SEQ ID NO: 224)
GACACCACGGAATAACATACAACAAAGATGAGGAT oligo19 (SEQ ID NO: 225)
TCCAACAGGTCTGAAGCCAGTTTTGATCAGAATGA oligo20 (SEQ ID NO: 226)
AAAACCAGGATTAGCGGGGTTAAGTATTATCGGCG oligo21 (SEQ ID NO: 227)
AAAGAGCTCCTGTACGTGGGACACATCCTAATTTA oligo22 (SEQ ID NO: 228)
GCAGTGTTCAATCAAAGGCTAAATTGAGCGATGCCG
oligo23 (SEQ ID NO: 229) AACAATTCTCGTCAAAACCGATCAAAAGGGCTTACC
oligo24 (SEQ ID NO: 230) ATCATAGCATCAGCAGTTTGAACCCTGTGACTCCTT
oligo25 (SEQ ID NO: 231) CAGTAGTGCCGGACAAACAGATCTACTAGGAAGGTA
oligo26 (SEQ ID NO: 232) CCCTTAGACGCAGATGCCGCCGAAGCCCCTTCAAAG
oligo27 (SEQ ID NO: 233) GAGAGATCGGAAAACTGACTAAAGATTAAGCCGTTC
oligo28 (SEQ ID NO: 234) GGTGCGGGCCTCTTAACGCTCAATCTACCAGTTTCA
oligo29 (SEQ ID NO: 235) ATAATCGATCGAGAGGGATCGAGGCTTTGAGTGTAC
oligo30 (SEQ ID NO: 236) GATAGGTACAAACGCCGGATATCATCAAGAGTAATCT
oligo31 (SEQ ID NO: 237) AGTCATAAGTTGCCACATTATTCATCAGTTGAGTTATACC
oligo32 (SEQ ID NO: 238) TCTTCGCCTCCTCTCAAAAACTGGCCTAGACGGTGGAACCG
oligo33 (SEQ ID NO: 239) CCCTCACTTTACCAGAGAATCCTTGAAGTCCCGGCCTCACC
oligo34 (SEQ ID NO: 240) CGCCTGTGCACTCTTGAACCTGAGAGTCCCCTGAACAAAGTC
oligo35 (SEQ ID NO: 241) AATCAACAGTTGAACATCCCTAAGAATTAGAAAGGCCGGAGA
oligo36 (SEQ ID NO: 242) CGGTAGCGCACTCAGCCATCCACCCAACGAATGCACTGGTCT
oligo37 (SEQ ID NO: 243) CTTCTGAGAGGTGTTATGGTTAAAACATTAAAGAAACGCAAA
oligo38 (SEQ ID NO: 244) CGGCCTTTAGTGATTCCGGCAATAAGAGCTGAATATACCCTC
oligo39 (SEQ ID NO: 245) GCTCATTAACAGCGGCTCTCAAGACTTTAGCCGCCGCCAGTG
oligo40 (SEQ ID NO: 246) TGAGAAGGAATAACCTTGCTTTTTTAATCTCATTAAGGCAGG
oligo41 (SEQ ID NO: 247) CCAATCGCAAGACAGGAAACAAAGAGGCTAAACAGTTCAGAA
oligo42 (SEQ ID NO: 248) ATGCTGACCTTTTTATTCTGAGCCCGTATAAACAGAGTGCCT
oligo43 (SEQ ID NO: 249) TGGGAAGTTCGCCAAGTCAGGATTTTAAGAACTGGTGTGAAT
oligo44 (SEQ ID NO: 250) GCAAAGCCACCGCTTACCTTAAATTTCAACTTTAACAAAGCT
oligo45 (SEQ ID NO: 251) CGTGCATTTGGTGTGCTCATTTTACCCAAATCAACACAAGAA
oligo46 (SEQ ID NO: 252) TCATTCCATTAAACGAAAGACCGAGGGTAGCAACGCATGAGG
oligo47 (SEQ ID NO: 253) TTCATCAACCAACCGAAAGAGGACAGATGAACGGGGCCACTA
oligo48 (SEQ ID NO: 254) CTATTTTGCACCATTTGCGGGTGTATCACCCCCAGCGATTAT
oligo49 (SEQ ID NO: 255) AACCCACTACACTGTTCTTTGCGACAACTTTTAAAGGGGTCA
oligo50 (SEQ ID NO: 256) ATCACCATCAATATAATGCCTTAGAACCTTTTACCTTTATTT
oligo51 (SEQ ID NO: 257) GAGTAATGTGTAGGCAGTCAAGAGAGATAGAGGGTTCAGGTC
oligo52 (SEQ ID NO: 258) TAAAGATGGAAACGTGATTAAAATACTTTGTACCATACCAGC
oligo53 (SEQ ID NO: 259) CAAAAGAACTGGCACAATAATTAAAGGTGTGTGTTGTTGGCA
oligo54 (SEQ ID NO: 260) AGAGCATGGGCAAAAATTACGAATAAATATTTTCAGCTGGTC
oligo55 (SEQ ID NO: 261) CAATAGAGACGGAACGACTTGAGCCAATAATAAAGGATTATA
oligo56 (SEQ ID NO: 262) ACTGTAGCGCGTTTTAGCACCCAATAACCGTCAGATGAATAT
oligo57 (SEQ ID NO: 263) CCACCACACCACCCGTAGGATTAGAGAGAAGAAGACAAAATC
oligo58 (SEQ ID NO: 264) AGAACCGAATTGCTAGACCGGTCTCTGAATTTAAGAGCAGTT
oligo59 (SEQ ID NO: 265) GATACAGGAGTGTAATAAATCGGAAACATTTCATTTGAATTA
oligo60 (SEQ ID NO: 266) AACGAGTAACATGATTGCTCATACAGACGACGATATTAGTTA
oligo61 (SEQ ID NO: 267) TTACAGGGAAGAAAAACAGTAGGGCTCAGGCGATCAGGCGAT
oligo62 (SEQ ID NO: 268) GTAGCATTCCACAGTTTTGTCATATGCGGAGGCATTTTCGAG
oligo63 (SEQ ID NO: 269) AAACGGCACCAGTACGCCAACATGTAATAAGGTAATAATTTT
oligo64 (SEQ ID NO: 270) TCGGTTTATAGAACGAGTAGTGGAATTGCTTTCAAGTTAATA
oligo65 (SEQ ID NO: 271) CACTAAAACACTCACGAAGGCACATTAAATGTGAACAAATCA
oligo66 (SEQ ID NO: 272) TCTTTGATCGCCTGATAAATTGCGAACCGATATAGCCGAGCT
oligo67 (SEQ ID NO: 273)
ATCAAAATGGCTTAGATAACTATTAATGGCGACCGTTACAAAC oligo68 (SEQ ID NO:
274) AAGAACGCGAGAAAAACGACGACGGGAAGGATAGCTTGAATCC oligo69 (SEQ ID
NO: 275) TCCCGACTTTGTTAAAATTCGAATTGTACGAACTGAACGAACC oligo70 (SEQ
ID NO: 276) ATTCGCCTGAACAAAATTAACAAGTACATATGTGAGTAGTCAAT oligo71
(SEQ ID NO: 277) ACCAGCTGCTGCGAATAAGAGCAAACAAGAGAATATTGCCTCAAATAT
oligo72 (SEQ ID NO: 278)
GAGAGGTTGAGAGCTAGCATTGTACCCCGGTTGCTTCACGGATCCAGC oligo73 (SEQ ID
NO: 279) GAAGCCAAGTTACCAGTATGGGCAACATATAATGGTAACATCTTTACA oligo74
(SEQ ID NO: 280) ATTACGCAGAAGGTATAGATTAGAGCCTATTAGATATCATTAATTATC
oligo75 (SEQ ID NO: 281)
ATCGGTTTGCGGGTTATTAATCGTATTAAATCCTTAATGGGAACGGAA oligo76 (SEQ ID
NO: 282) AATATTAAATTCACCATTCCTGATTATTTGTTTGAAATTGCACAGTAA oligo77
(SEQ ID NO: 283) CGAACCCCTTTTGAAATTTCAATTACCGCACAGGGGGCGGTTAATTTT
oligo78 (SEQ ID NO: 284)
CAGTAAATCAGGTAATGCTTTGAGACTCCTCACTCGGATAAAATTTGT oligo79 (SEQ ID
NO: 285) GGATTAAAATAGCGCAACACCCACCACCCTCATTTTCAGACGAGGCAT oligo80
(SEQ ID NO: 286) CGGATAACCTATTAACCTCCCATAGGTCTGAGAGAAGACGCTGAGTAA
oligo81 (SEQ ID NO: 287)
GCGGAGTGAGACGACGTTGGTAGAAAGCAGGATAGCAAGCCTGCTGCA oligo82 (SEQ ID
NO: 288) AGACCAAAGGCCGCACGCATACGAGAAACACCCAATAGATACCAATCA oligo83
(SEQ ID NO: 289) CGAATTCTAATGCGAACGTTAGAGCCTAATTTGCCCAATCCAGCCAGAA
oligo84 (SEQ ID NO: 290)
CGTGGCATTTTGAATATCCTGACGCTAACGAGCGTTTTTGTTCGCCTGC oligo85 (SEQ ID
NO: 291) AACAGGGAGAAGATTAGTCTTAAAGCGTTAGCAAGGCAAGCCACGTAAT
oligo86 (SEQ ID NO: 292)
CAAACCCCACTGCGTGCGGCGAATACCGATAGCCCCCGGGTAAAGGCTT oligo87 (SEQ ID
NO: 293) TGCCCTGCGGCATCTTACCTGCAGCCATCTGGTCACAGCAAAAATATCA oligo88
(SEQ ID NO: 294) GGAGCGGTTGCGGATAAAGGTTTAGCAAACGTAGACAGATAGGATAATA
oligo89 (SEQ ID NO: 295)
ACGGCTGATAATGGGCACGTATTGTAGAATCCTCAGCGCAGAGGAAGTT oligo90 (SEQ ID
NO: 296) GTAGATTCCGTCACATTATTCATTAAAGTATTTTGTGGCAATACCAGAA oligo91
(SEQ ID NO: 297) GGCAGCCCGGTCCGTGCAACTGCTGTAGCTCAACATTAATTGGTCATTT
oligo92 (SEQ ID NO: 298)
TAACGGAAACGTCAGTGGCATCATTTGGGAATTAGTTAGCAAGCGTCAG oligo93 (SEQ ID
NO: 299) ACAAACAACAGGAGTCAGAGCCGCCACCCACCGGATTTGCCATTCGGTC oligo94
(SEQ ID NO: 300) TAGGCATTATACACCGGAATATAAGGCCTTCTGACCCGGAAGTACCAGG
oligo95 (SEQ ID NO: 301)
GAGAATACTCCAAACAAAAGGAGCCTTTTGAATTTGAACGCGTTCCTTA oligo96 (SEQ ID
NO: 302) ATTCTCAACAGTTGAGGATCCTAAAACATAAGCAAAAATAAACAGATAA oligo97
(SEQ ID NO: 303) TCAGAAAACAGGAAGCTCATTTAGGAACTCCATGTGAACGAGGCGGCAA
oligo98 (SEQ ID NO: 304)
GTAAAAATCTACAATAGCGGTGCCGGTTCAGACGTCATACCGCCAGCAC oligo99 (SEQ ID
NO: 305) CGATGAATTTATCCAGTTACAATATTTACATTAAACGTTTTAGTGTCGA oligo100
(SEQ ID NO: 306) AGAGAGAAACGATTCTTTCCAATCAGCTACAATTTTGGCTATAAAACAG
oligo101 (SEQ ID NO: 307)
GCCCAATACTAACAACTAAAAAGGAATTACCTTGCGTTGCCACGCTGAG oligo102 (SEQ ID
NO: 308) GCTATTGGAGTTAACTGAACATGGAATAACATAAAAAGCATCGAGGAAG oligo103
(SEQ ID NO: 309) TTTAAATGCGATATTCGCTGATAAATTACTTCGTTAACGGCTGGTTTGA
oligo104 (SEQ ID NO: 310)
ACCGATTGCCAAAGCCAGCTTTTGCAGGCGCTTTCCCGAACGAGAAGCC oligo105 (SEQ ID
NO: 311) GAGGGAGATAGTAGTGAAAAGCCAATGAACAGAATCAATTCTGCGAACG oligo106
(SEQ ID NO: 312) AGCATTATTATTTAAGGGTTAGAACCTCACGCAAACAAAAGAAAGCTAA
oligo107 (SEQ ID NO: 313)
AAAATCAATTATCATTCAGGTCAATATAATCCTGACAGATGATCACAAT oligo108 (SEQ ID
NO: 314) AACCATCAGAGCACACGTCAGCGTGGTGTATCAAAAACATCCACATTCA oligo109
(SEQ ID NO: 315) GCAAAACTTAGTTTGACCATTTTAAATATTTTTTCTTGCCGTGAAGGGT
oligo110 (SEQ ID NO: 316)
TAGCCCCCACAGTTGATTCCCAAGTTTGCCTTTAGGCCGGAACCCTTTT oligo111 (SEQ ID
NO: 317) GGTGTCTGGCGAATTATTCCGTCCGGCCGATAGCATTTGGGGCGCGAGC oligo112
(SEQ ID NO: 318) GCCTCCCTCAGAGCCGCCAATAAAGTACAGTAGATCGTAATCAGTAGCG
oligo113 (SEQ ID NO: 319)
CGGAACCTCATTCCATATATTCAAGTTATGATGAACCAAATCCCGTAAA oligo114 (SEQ ID
NO: 320) TCAAAATAGAACCACGCCGCCATTGGCCCAAACAACTGGTAACGGGGTC oligo115
(SEQ ID NO: 321) ATTCGAGGAAAGACCATCAAATTATAGTATATTCAGTCCAATTAGTAAA
oligo116 (SEQ ID NO: 322)
TCAGACGAGCATTGTCAAGAAATTGCTTGGAGAAAATTACCAAGCCAGC oligo117 (SEQ ID
NO: 323) ACATGGCAATGGAAATCGACATAAAATTCTGTAAATTAGATTACTACAG oligo118
(SEQ ID NO: 324) CCATAACGATAGCTCGTCGCTATTAATTGTGTACAGCGCAGAAGCAAAC
oligo119 (SEQ ID NO: 325)
TTAATGCTTTCGGAAGTGCCGTGATATACAGGAGGCCACCCTCAGAACC oligo120 (SEQ ID
NO: 326) AAAGAAGGAATTACTAATGCAGATACATAGGAATAGGTAACGCAACTGT oligo121
(SEQ ID NO: 327) AGTAAGAGAGAGGCGTAAACTTTTTCAAGGGGATGCAATAGGAACATTA
oligo122 (SEQ ID NO: 328)
TTTACGGTCAGAACCGCCACCGTACCGTAAAGCTGGCGAAAGATATATT oligo123 (SEQ ID
NO: 329) TCACCAGGTGATAACATAATTACTAGAATAGTATCGTCTTTCTAAATGA oligo124
(SEQ ID NO: 330) TCATAGTTAAACTAACGGAACAACCCATCTCAGAGTATCATAACCCTCG
oligo125 (SEQ ID NO: 331)
TAAACAACGAATAAAGCTCAGGAAGATCTTAACAATAAAGCCCGCTATT oligo126 (SEQ ID
NO: 332) ATTTTCTTGAAAATTAAAGTAACGACAATAAACAATAATGCACTTAAAC oligo127
(SEQ ID NO: 333) TTCACGTGTATGGGTCTAAAGACAGCCCAGTTTCGGCCACCCTGTATCA
oligo128 (SEQ ID NO: 334)
AAGGCTAAAATAATATCCCGCTGCCAGTTTCCGGGCTAATTGAGAATCG oligo129 (SEQ ID
NO: 335) ATATTCGGTCGCCACAGTGAAATGGTTTTGATAGAAAGGAACAGCCAGC oligo130
(SEQ ID NO: 336) CATCGCCCTTTTGCACAGCGAGTAACAAGTAGAAAAGTCCTGGACAGTA
oligo131 (SEQ ID NO: 337)
TGCGCCGCAGCAGCCAAGTACTTTTCATATTACCGGAAGCCTTTAGTTG oligo132 (SEQ ID
NO: 338) GGCTGGCAACTTTTAAAACGAAGAATAAATCCGCGACCTGCGCAAGAAC oligo133
(SEQ ID NO: 339) AACTGACCTGACCTTTTGAGGCTTGCAGGATTCTCGCCAGCTATCCGGT
oligo134 (SEQ ID NO: 340)
ACTAAAGACTTTTTGCTACAGTCACCCTACAATGATTCGAGGAATTGTA oligo135 (SEQ ID
NO: 341) GTAAAATGTTTTTACGCACTCGCTGTCTCCTGTTTCCAGACGCCGACAA oligo136
(SEQ ID NO: 342)
TAAACGGACCAAGCGTACAACGGAGATTAGGTTTTCGCCCAAAAGAATA
EQUIVALENTS
[0226] While several embodiments of the present invention have been
described and illustrated herein, those of ordinary skill in the
art will readily envision a variety of other means and/or
structures for performing the functions and/or obtaining the
results and/or one or more of the advantages described herein, and
each of such variations and/or modifications is deemed to be within
the scope of the present invention. More generally, those skilled
in the art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and/or configurations will depend upon the specific application or
applications for which the teachings of the present invention
is/are used. Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. It is, therefore, to be understood that the foregoing
embodiments are presented by way of example only and that, within
the scope of the appended claims and equivalents thereto, the
invention may be practiced otherwise than as specifically described
and claimed. The present invention is directed to each individual
feature, system, article, material, kit, and/or method described
herein. In addition, any combination of two or more such features,
systems, articles, materials, kits, and/or methods, if such
features, systems, articles, materials, kits, and/or methods are
not mutually inconsistent, is included within the scope of the
present invention.
[0227] All definitions, as defined and used herein, should be
understood to control over dictionary definitions, definitions in
documents incorporated by reference, and/or ordinary meanings of
the defined terms.
[0228] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0229] It should also be understood that, unless clearly indicated
to the contrary, in any methods claimed herein that include more
than one step or act, the order of the steps or acts of the method
is not necessarily limited to the order in which the steps or acts
of the method are recited.
[0230] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," "composed of," and
the like are to be understood to be open-ended, i.e., to mean
including but not limited to. Only the transitional phrases
"consisting of" and "consisting essentially of" shall be closed or
semi-closed transitional phrases, respectively, as set forth in the
United States Patent Office Manual of Patent Examining Procedures,
Section 2111.03.
Sequence CWU 1
1
34218064DNAArtificial SequenceSynthetic Polynucleotide 1gaattcgagc
tcggtacccg gggatcctca actgtgagga ggctcacgga cgcgaagaac 60aggcacgcgt
gctggcagaa acccccggta tgaccgtgaa aacggcccgc cgcattctgg
120ccgcagcacc acagagtgca caggcgcgca gtgacactgc gctggatcgt
ctgatgcagg 180gggcaccggc accgctggct gcaggtaacc cggcatctga
tgccgttaac gatttgctga 240acacaccagt gtaagggatg tttatgacga
gcaaagaaac ctttacccat taccagccgc 300agggcaacag tgacccggct
cataccgcaa ccgcgcccgg cggattgagt gcgaaagcgc 360ctgcaatgac
cccgctgatg ctggacacct ccagccgtaa gctggttgcg tgggatggca
420ccaccgacgg tgctgccgtt ggcattcttg cggttgctgc tgaccagacc
agcaccacgc 480tgacgttcta caagtccggc acgttccgtt atgaggatgt
gctctggccg gaggctgcca 540gcgacgagac gaaaaaacgg accgcgtttg
ccggaacggc aatcagcatc gtttaacttt 600acccttcatc actaaaggcc
gcctgtgcgg ctttttttac gggatttttt tatgtcgatg 660tacacaaccg
cccaactgct ggcggcaaat gagcagaaat ttaagtttga tccgctgttt
720ctgcgtctct ttttccgtga gagctatccc ttcaccacgg agaaagtcta
tctctcacaa 780attccgggac tggtaaacat ggcgctgtac gtttcgccga
ttgtttccgg tgaggttatc 840cgttcccgtg gcggctccac ctctgaaagc
ttggcactgg ccgtcgtttt acaacgtcgt 900gactgggaaa accctggcgt
tacccaactt aatcgccttg cagcacatcc ccctttcgcc 960agctggcgta
atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg
1020aatggcgaat ggcgctttgc ctggtttccg gcaccagaag cggtgccgga
aagctggctg 1080gagtgcgatc ttcctgaggc cgatactgtc gtcgtcccct
caaactggca gatgcacggt 1140tacgatgcgc ccatctacac caacgtgacc
tatcccatta cggtcaatcc gccgtttgtt 1200cccacggaga atccgacggg
ttgttactcg ctcacattta atgttgatga aagctggcta 1260caggaaggcc
agacgcgaat tatttttgat ggcgttccta ttggttaaaa aatgagctga
1320tttaacaaaa atttaatgcg aattttaaca aaatattaac gtttacaatt
taaatatttg 1380cttatacaat cttcctgttt ttggggcttt tctgattatc
aaccggggta catatgattg 1440acatgctagt tttacgatta ccgttcatcg
attctcttgt ttgctccaga ctctcaggca 1500atgacctgat agcctttgta
gatctctcaa aaatagctac cctctccggc attaatttat 1560cagctagaac
ggttgaatat catattgatg gtgatttgac tgtctccggc ctttctcacc
1620cttttgaatc tttacctaca cattactcag gcattgcatt taaaatatat
gagggttcta 1680aaaattttta tccttgcgtt gaaataaagg cttctcccgc
aaaagtatta cagggtcata 1740atgtttttgg tacaaccgat ttagctttat
gctctgaggc tttattgctt aattttgcta 1800attctttgcc ttgcctgtat
gatttattgg atgttaatgc tactactatt agtagaattg 1860atgccacctt
ttcagctcgc gccccaaatg aaaatatagc taaacaggtt attgaccatt
1920tgcgaaatgt atctaatggt caaactaaat ctactcgttc gcagaattgg
gaatcaactg 1980ttatatggaa tgaaacttcc agacaccgta ctttagttgc
atatttaaaa catgttgagc 2040tacagcatta tattcagcaa ttaagctcta
agccatccgc aaaaatgacc tcttatcaaa 2100aggagcaatt aaaggtactc
tctaatcctg acctgttgga gtttgcttcc ggtctggttc 2160gctttgaagc
tcgaattaaa acgcgatatt tgaagtcttt cgggcttcct cttaatcttt
2220ttgatgcaat ccgctttgct tctgactata atagtcaggg taaagacctg
atttttgatt 2280tatggtcatt ctcgttttct gaactgttta aagcatttga
gggggattca atgaatattt 2340atgacgattc cgcagtattg gacgctatcc
agtctaaaca ttttactatt accccctctg 2400gcaaaacttc ttttgcaaaa
gcctctcgct attttggttt ttatcgtcgt ctggtaaacg 2460agggttatga
tagtgttgct cttactatgc ctcgtaattc cttttggcgt tatgtatctg
2520cattagttga atgtggtatt cctaaatctc aactgatgaa tctttctacc
tgtaataatg 2580ttgttccgtt agttcgtttt attaacgtag atttttcttc
ccaacgtcct gactggtata 2640atgagccagt tcttaaaatc gcataaggta
attcacaatg attaaagttg aaattaaacc 2700atctcaagcc caatttacta
ctcgttctgg tgtttctcgt cagggcaagc cttattcact 2760gaatgagcag
ctttgttacg ttgatttggg taatgaatat ccggttcttg tcaagattac
2820tcttgatgaa ggtcagccag cctatgcgcc tggtctgtac accgttcatc
tgtcctcttt 2880caaagttggt cagttcggtt cccttatgat tgaccgtctg
cgcctcgttc cggctaagta 2940acatggagca ggtcgcggat ttcgacacaa
tttatcaggc gatgatacaa atctccgttg 3000tactttgttt cgcgcttggt
ataatcgctg ggggtcaaag atgagtgttt tagtgtattc 3060ttttgcctct
ttcgttttag gttggtgcct tcgtagtggc attacgtatt ttacccgttt
3120aatggaaact tcctcatgaa aaagtcttta gtcctcaaag cctctgtagc
cgttgctacc 3180ctcgttccga tgctgtcttt cgctgctgag ggtgacgatc
ccgcaaaagc ggcctttaac 3240tccctgcaag cctcagcgac cgaatatatc
ggttatgcgt gggcgatggt tgttgtcatt 3300gtcggcgcaa ctatcggtat
caagctgttt aagaaattca cctcgaaagc aagctgataa 3360accgatacaa
ttaaaggctc cttttggagc cttttttttg gagattttca acgtgaaaaa
3420attattattc gcaattcctt tagttgttcc tttctattct cactccgctg
aaactgttga 3480aagttgttta gcaaaatccc atacagaaaa ttcatttact
aacgtctgga aagacgacaa 3540aactttagat cgttacgcta actatgaggg
ctgtctgtgg aatgctacag gcgttgtagt 3600ttgtactggt gacgaaactc
agtgttacgg tacatgggtt cctattgggc ttgctatccc 3660tgaaaatgag
ggtggtggct ctgagggtgg cggttctgag ggtggcggtt ctgagggtgg
3720cggtactaaa cctcctgagt acggtgatac acctattccg ggctatactt
atatcaaccc 3780tctcgacggc acttatccgc ctggtactga gcaaaacccc
gctaatccta atccttctct 3840tgaggagtct cagcctctta atactttcat
gtttcagaat aataggttcc gaaataggca 3900gggggcatta actgtttata
cgggcactgt tactcaaggc actgaccccg ttaaaactta 3960ttaccagtac
actcctgtat catcaaaagc catgtatgac gcttactgga acggtaaatt
4020cagagactgc gctttccatt ctggctttaa tgaggattta tttgtttgtg
aatatcaagg 4080ccaatcgtct gacctgcctc aacctcctgt caatgctggc
ggcggctctg gtggtggttc 4140tggtggcggc tctgagggtg gtggctctga
gggtggcggt tctgagggtg gcggctctga 4200gggaggcggt tccggtggtg
gctctggttc cggtgatttt gattatgaaa agatggcaaa 4260cgctaataag
ggggctatga ccgaaaatgc cgatgaaaac gcgctacagt ctgacgctaa
4320aggcaaactt gattctgtcg ctactgatta cggtgctgct atcgatggtt
tcattggtga 4380cgtttccggc cttgctaatg gtaatggtgc tactggtgat
tttgctggct ctaattccca 4440aatggctcaa gtcggtgacg gtgataattc
acctttaatg aataatttcc gtcaatattt 4500accttccctc cctcaatcgg
ttgaatgtcg cccttttgtc tttggcgctg gtaaaccata 4560tgaattttct
attgattgtg acaaaataaa cttattccgt ggtgtctttg cgtttctttt
4620atatgttgcc acctttatgt atgtattttc tacgtttgct aacatactgc
gtaataagga 4680gtcttaatca tgccagttct tttgggtatt ccgttattat
tgcgtttcct cggtttcctt 4740ctggtaactt tgttcggcta tctgcttact
tttcttaaaa agggcttcgg taagatagct 4800attgctattt cattgtttct
tgctcttatt attgggctta actcaattct tgtgggttat 4860ctctctgata
ttagcgctca attaccctct gactttgttc agggtgttca gttaattctc
4920ccgtctaatg cgcttccctg tttttatgtt attctctctg taaaggctgc
tattttcatt 4980tttgacgtta aacaaaaaat cgtttcttat ttggattggg
ataaataata tggctgttta 5040ttttgtaact ggcaaattag gctctggaaa
gacgctcgtt agcgttggta agattcagga 5100taaaattgta gctgggtgca
aaatagcaac taatcttgat ttaaggcttc aaaacctccc 5160gcaagtcggg
aggttcgcta aaacgcctcg cgttcttaga ataccggata agccttctat
5220atctgatttg cttgctattg ggcgcggtaa tgattcctac gatgaaaata
aaaacggctt 5280gcttgttctc gatgagtgcg gtacttggtt taatacccgt
tcttggaatg ataaggaaag 5340acagccgatt attgattggt ttctacatgc
tcgtaaatta ggatgggata ttatttttct 5400tgttcaggac ttatctattg
ttgataaaca ggcgcgttct gcattagctg aacatgttgt 5460ttattgtcgt
cgtctggaca gaattacttt accttttgtc ggtactttat attctcttat
5520tactggctcg aaaatgcctc tgcctaaatt acatgttggc gttgttaaat
atggcgattc 5580tcaattaagc cctactgttg agcgttggct ttatactggt
aagaatttgt ataacgcata 5640tgatactaaa caggcttttt ctagtaatta
tgattccggt gtttattctt atttaacgcc 5700ttatttatca cacggtcggt
atttcaaacc attaaattta ggtcagaaga tgaaattaac 5760taaaatatat
ttgaaaaagt tttctcgcgt tctttgtctt gcgattggat ttgcatcagc
5820atttacatat agttatataa cccaacctaa gccggaggtt aaaaaggtag
tctctcagac 5880ctatgatttt gataaattca ctattgactc ttctcagcgt
cttaatctaa gctatcgcta 5940tgttttcaag gattctaagg gaaaattaat
taatagcgac gatttacaga agcaaggtta 6000ttcactcaca tatattgatt
tatgtactgt ttccattaaa aaaggtaatt caaatgaaat 6060tgttaaatgt
aattaatttt gttttcttga tgtttgtttc atcatcttct tttgctcagg
6120taattgaaat gaataattcg cctctgcgcg attttgtaac ttggtattca
aagcaatcag 6180gcgaatccgt tattgtttct cccgatgtaa aaggtactgt
tactgtatat tcatctgacg 6240ttaaacctga aaatctacgc aatttcttta
tttctgtttt acgtgcaaat aattttgata 6300tggtaggttc taacccttcc
attattcaga agtataatcc aaacaatcag gattatattg 6360atgaattgcc
atcatctgat aatcaggaat atgatgataa ttccgctcct tctggtggtt
6420tctttgttcc gcaaaatgat aatgttactc aaacttttaa aattaataac
gttcgggcaa 6480aggatttaat acgagttgtc gaattgtttg taaagtctaa
tacttctaaa tcctcaaatg 6540tattatctat tgacggctct aatctattag
ttgttagtgc tcctaaagat attttagata 6600accttcctca attcctttca
actgttgatt tgccaactga ccagatattg attgagggtt 6660tgatatttga
ggttcagcaa ggtgatgctt tagatttttc atttgctgct ggctctcagc
6720gtggcactgt tgcaggcggt gttaatactg accgcctcac ctctgtttta
tcttctgctg 6780gtggttcgtt cggtattttt aatggcgatg ttttagggct
atcagttcgc gcattaaaga 6840ctaatagcca ttcaaaaata ttgtctgtgc
cacgtattct tacgctttca ggtcagaagg 6900gttctatctc tgttggccag
aatgtccctt ttattactgg tcgtgtgact ggtgaatctg 6960ccaatgtaaa
taatccattt cagacgattg agcgtcaaaa tgtaggtatt tccatgagcg
7020tttttcctgt tgcaatggct ggcggtaata ttgttctgga tattaccagc
aaggccgata 7080gtttgagttc ttctactcag gcaagtgatg ttattactaa
tcaaagaagt attgctacaa 7140cggttaattt gcgtgatgga cagactcttt
tactcggtgg cctcactgat tataaaaaca 7200cttctcagga ttctggcgta
ccgttcctgt ctaaaatccc tttaatcggc ctcctgttta 7260gctcccgctc
tgattctaac gaggaaagca cgttatacgt gctcgtcaaa gcaaccatag
7320tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
cagcgtgacc 7380gctacacttg ccagcgccct agcgcccgct cctttcgctt
tcttcccttc ctttctcgcc 7440acgttcgccg gctttccccg tcaagctcta
aatcgggggc tccctttagg gttccgattt 7500agtgctttac ggcacctcga
ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg 7560ccatcgccct
gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
7620ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc
ttttgattta 7680taagggattt tgccgatttc ggaaccacca tcaaacagga
ttttcgcctg ctggggcaaa 7740ccagcgtgga ccgcttgctg caactctctc
agggccaggc ggtgaagggc aatcagctgt 7800tgcccgtctc actggtgaaa
agaaaaacca ccctggcgcc caatacgcaa accgcctctc 7860cccgcgcgtt
ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
7920ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
ccaggcttta 7980cactttatgc ttccggctcg tatgttgtgt ggaattgtga
gcggataaca atttcacaca 8040ggaaacagct atgaccatga ttac
8064242DNAArtificial SequenceSynthetic Polynucleotide 2taaccaccca
ctacgtgaac cacgtcaaag ggcgaaccgc ct 42342DNAArtificial
SequenceSynthetic Polynucleotide 3cgggcgcagg tgccgtaaag ccagtttgga
acaaggtttg cc 42442DNAArtificial SequenceSynthetic Polynucleotide
4cggcgaacga tttagagctt gataaatcaa aagaaaaatc gg 42542DNAArtificial
SequenceSynthetic Polynucleotide 5gtttttttat taaagaacgt gtgcagcaag
cggtctgggc gc 42642DNAArtificial SequenceSynthetic Polynucleotide
6ctaaaggaga tagggttgag ttcctgtttg atggtttaat ga 42745DNAArtificial
SequenceSynthetic Polynucleotide 7aaagttgttc actaaatgaa agcgttagaa
tcagagcgaa tcagt 45838DNAArtificial SequenceSynthetic
Polynucleotide 8ttcagagagt gactccaatc acccggtcac gactatgg
38942DNAArtificial SequenceSynthetic Polynucleotide 9cagggtgaaa
gtgtaaagcc tttttcacgg tcatcgtgtg tt 421042DNAArtificial
SequenceSynthetic Polynucleotide 10atcggccatt aattgcgttg ccctgtgcac
tctgtctgca gc 421142DNAArtificial SequenceSynthetic Polynucleotide
11cgagccgttt cctgtgtgaa atcataaaca tccctgccct gc
421245DNAArtificial SequenceSynthetic Polynucleotide 12tgctgcgtat
cacgctgagt ccacggggtc gtagggcgac gtata 451342DNAArtificial
SequenceSynthetic Polynucleotide 13gtgagctcgg ccagaatgcg gcggcatcag
atgcccaatc cg 421445DNAArtificial SequenceSynthetic Polynucleotide
14actagctgca ggttccgtag cccggagccc ccgtggcgaa cagga
451542DNAArtificial SequenceSynthetic Polynucleotide 15cagcaaaggt
atgagccggg tggtctggtc agcaggtctc gt 421642DNAArtificial
SequenceSynthetic Polynucleotide 16cagcggtcat tgcaggcgct tgtcggtggt
gccatacgat gc 421746DNAArtificial SequenceSynthetic Polynucleotide
17ggctggtgta gaacgtcagc gggccagagc acatcgcgga tctcac
461846DNAArtificial SequenceSynthetic Polynucleotide 18ccgggcgaag
aatgccaacg ggcaaacgcg gtccggggcg gtattt 461942DNAArtificial
SequenceSynthetic Polynucleotide 19acctcgcact gggttacggt gctgaactca
caacgcgcgc ga 422042DNAArtificial SequenceSynthetic Polynucleotide
20tcctggtgct cactgtttac actgatagct ggaagcatgt tt
422139DNAArtificial SequenceSynthetic Polynucleotide 21cgctggcgct
catttccgtg ggttttccct tcgcttgcc 392239DNAArtificial
SequenceSynthetic Polynucleotide 22tgattgctaa aaaagccatg tgctttcatc
aggcttcgc 392346DNAArtificial SequenceSynthetic Polynucleotide
23agcagttttt tttccaaccg ccggttgctc gttaacgggc cggggg
462446DNAArtificial SequenceSynthetic Polynucleotide 24aaaaaaaaag
ttaacccacg cgcggggtgc cggtgctgcg cggctc 462549DNAArtificial
SequenceSynthetic Polynucleotide 25ggaattaagt cgaaaggggc gcatgggata
gattgtaaga agattcaat 492649DNAArtificial SequenceSynthetic
Polynucleotide 26gtgtaaaacg aagggcgaca gtatcgtcgg atgttaaaca
tatgtagag 492749DNAArtificial SequenceSynthetic Polynucleotide
27tcgacgggaa aggcaaaggc accgtagcca gaaataataa gagaacatt
492839DNAArtificial SequenceSynthetic Polynucleotide 28cgccaggtga
agggatagct caaacttaaa tttctagcc 392946DNAArtificial
SequenceSynthetic Polynucleotide 29gtgccaatta ccagtcccgg atgtgtacat
cgacacgttc cgcagc 463042DNAArtificial SequenceSynthetic
Polynucleotide 30agtaaacggc ttaaaattca gaaatagctg aaaagattta aa
423142DNAArtificial SequenceSynthetic Polynucleotide 31actaaatgtg
aaccaattcg taaagatcta cccctcatat ta 423228DNAArtificial
SequenceSynthetic Polynucleotide 32gctttccgcg ccattcgcca tgaggtgg
283342DNAArtificial SequenceSynthetic Polynucleotide 33ccgtaatcgt
aaccgtgcat cattacgcca gctggtgggt aa 423442DNAArtificial
SequenceSynthetic Polynucleotide 34tgggaacttg aggggacgac gatcggtgcg
ggcctcagtc ac 423542DNAArtificial SequenceSynthetic Polynucleotide
35aacaacccgg cctcaggaag agcgcaactg ttgggacggc ca
423642DNAArtificial SequenceSynthetic Polynucleotide 36acggtaaagg
aacgccatca actttcatca acattccagc ca 423742DNAArtificial
SequenceSynthetic Polynucleotide 37atgccggacc ccggttgata atcgcattaa
attttttctc cg 423842DNAArtificial SequenceSynthetic Polynucleotide
38taagcaacaa aagggtgaga aatattcaac cgttcagccc ca
423938DNAArtificial SequenceSynthetic Polynucleotide 39gccaaaggtg
gcatcaaaca tgttttaaat acctttaa 384042DNAArtificial
SequenceSynthetic Polynucleotide 40caaaaacata ttttaaatgc aagctatttt
tgagaactag ca 424138DNAArtificial SequenceSynthetic Polynucleotide
41tgaaataacc tgtttagtca ttccatataa cccagacc 384242DNAArtificial
SequenceSynthetic Polynucleotide 42atactttaaa aatttttaga aaaaggctat
caggttcgat ga 424342DNAArtificial SequenceSynthetic Polynucleotide
43gtagcattga atataatgct gaagaggtca tttttagaaa ac
424438DNAArtificial SequenceSynthetic Polynucleotide 44tgatcagagc
ataaagcagt aatgtgtagg ttaaatta 384542DNAArtificial
SequenceSynthetic Polynucleotide 45ggggcgcaaa gtacggtgtc tacaggtcag
gattactgac ta 424642DNAArtificial SequenceSynthetic Polynucleotide
46tttcgcatcc caattctgcg ataattcgag cttcaattaa ga
424742DNAArtificial SequenceSynthetic Polynucleotide 47ttgctccatc
aaaaatcagg taatactgcg gaatcatgca ga 424842DNAArtificial
SequenceSynthetic Polynucleotide 48ggaagcaaaa gcggattgca tccagagggg
gtaatgagca ac 424938DNAArtificial SequenceSynthetic Polynucleotide
49ttgcaaaaag aagcgaaagt tgataatggt cccctgta 385042DNAArtificial
SequenceSynthetic Polynucleotide 50accacataac attattacag gcaaatcaac
gtaactcaag ag 425145DNAArtificial SequenceSynthetic Polynucleotide
51ctagtcataa accataattt tgattagctc attctactgc aaaat
455242DNAArtificial SequenceSynthetic Polynucleotide 52tacataaaat
aaaacgaact actcattcag tgaatgcata gg 425342DNAArtificial
SequenceSynthetic Polynucleotide 53acgaggcttg ggaagaaaaa tgccctgacg
agaaatgaac gg 425445DNAArtificial SequenceSynthetic Polynucleotide
54taaagtaaaa cagaagcaac tccaggaagt tctatattgt tgtac
455542DNAArtificial SequenceSynthetic Polynucleotide 55actatcactc
attataccag tacgagtagt aaattccaac tt 425642DNAArtificial
SequenceSynthetic Polynucleotide 56cagacgatta tgcgatttta aagatggttt
aatttatcat aa 425745DNAArtificial SequenceSynthetic Polynucleotide
57aaagcgagag cgaaagacgc gtttacgagt agaccattga agcct
455842DNAArtificial SequenceSynthetic Polynucleotide 58gttcgccaaa
agcgtccctt taccgagagt atgcaactga gc 425946DNAArtificial
SequenceSynthetic Polynucleotide 59taatcttccc
agcgattata cagaggcaaa agaatataac cgcaat 466039DNAArtificial
SequenceSynthetic Polynucleotide 60ctggctggaa acaaagtaca aaaggcacca
acctatgag 396146DNAArtificial SequenceSynthetic Polynucleotide
61tgtacagttg tatcatcgcc taaaatacgt aatgcggccg ctaggt
466239DNAArtificial SequenceSynthetic Polynucleotide 62tgaaagatgt
gtcgaaatcc gggaagtttc cattaaccc 396346DNAArtificial
SequenceSynthetic Polynucleotide 63gggaaccctc catgttactt aggactaaag
actttcatcg gaaaaa 466442DNAArtificial SequenceSynthetic
Polynucleotide 64tgacgacctg gaactgaggg cttggaactg gtaaccctag tt
426546DNAArtificial SequenceSynthetic Polynucleotide 65cggtcgcaaa
cgaacaagcg caccttcaaa agctgacgga actcaa 466642DNAArtificial
SequenceSynthetic Polynucleotide 66gcttgatacc tgctaaatag cgtagtttag
tggataagta ct 426739DNAArtificial SequenceSynthetic Polynucleotide
67agttaaacac tacgcggaga taccaggcaa ggcttctac 396846DNAArtificial
SequenceSynthetic Polynucleotide 68gatcgtcaac gggtgataaa tggacagaca
ccagacagga cgatag 466942DNAArtificial SequenceSynthetic
Polynucleotide 69tcacggttta aggaacaaaa ctaccaccct cagagaaggt gc
427046DNAArtificial SequenceSynthetic Polynucleotide 70tagcaacgct
ttgagccgga aacggtcaca actttaatta cccgat 467149DNAArtificial
SequenceSynthetic Polynucleotide 71gactgaattt tgtcgtcgtg tatcttgata
tgcttttgac cgttcacca 497249DNAArtificial SequenceSynthetic
Polynucleotide 72gaattcagcg tccacagaac cgccgggttt tgggtcagat
cctcactca 497349DNAArtificial SequenceSynthetic Polynucleotide
73ggagaataat actgagtcat tttcttaaga ggccccctag gcaggacca
497439DNAArtificial SequenceSynthetic Polynucleotide 74gaattgcgcc
tttaattgta tgcagcgaaa gacagttca 397542DNAArtificial
SequenceSynthetic Polynucleotide 75tcatagtcaa ctttcaacag ttttcttaaa
cagcttgcag gg 427642DNAArtificial SequenceSynthetic Polynucleotide
76gagagggacc gtactcagga gacgatctaa agttttctgt at
427742DNAArtificial SequenceSynthetic Polynucleotide 77attagcgacc
ctcagaaccg caacgcctgt agcatgagtg ag 427842DNAArtificial
SequenceSynthetic Polynucleotide 78ctcctcaaga gccaccaccc tttcgtcacc
agtacactaa ag 427942DNAArtificial SequenceSynthetic Polynucleotide
79gaaagtaagg gatagcaagc cccatgtacc gtaacaattt tt
428038DNAArtificial SequenceSynthetic Polynucleotide 80ggtgaaagcg
gcctcccccc ccttccattt ggggaggg 388142DNAArtificial
SequenceSynthetic Polynucleotide 81tttaacggct cagtaccagg caccgccacc
ctcagacagc cc 428238DNAArtificial SequenceSynthetic Polynucleotide
82ccgttgatat gccaccacgt cagataccat tttaccag 388342DNAArtificial
SequenceSynthetic Polynucleotide 83agccgcctca gacgattggc ctataaacag
ttaatgctga ga 428442DNAArtificial SequenceSynthetic Polynucleotide
84gtcatagtca gagccgccac cttaaagcca gaatgaataa gt
428542DNAArtificial SequenceSynthetic Polynucleotide 85acttgagatt
agcgtttgcc accaccaccg gaacccagtc tc 428642DNAArtificial
SequenceSynthetic Polynucleotide 86gcaccatctg tagcgcgttt tccgccaccc
tcagatcaca aa 428742DNAArtificial SequenceSynthetic Polynucleotide
87ccatcgagta atcagtagcg accaccacca gagccttgac ag
428842DNAArtificial SequenceSynthetic Polynucleotide 88tattcattaa
taacggaata cccgaacaaa gttactcaaa aa 428942DNAArtificial
SequenceSynthetic Polynucleotide 89aaggtaaaac tggcatgatt attaagaaaa
gtaagtttac ag 429042DNAArtificial SequenceSynthetic Polynucleotide
90attcaactta ttacgcagta tgctatctta ccgaaaaaca gg
429142DNAArtificial SequenceSynthetic Polynucleotide 91cgccaaaaac
gtagaaaata cagaaacaat gaaatgggag aa 429238DNAArtificial
SequenceSynthetic Polynucleotide 92ggtagcaagg ccggaaaaag tttgccttta
gccctcag 389342DNAArtificial SequenceSynthetic Polynucleotide
93aaaattcaag gtggcaacat attaagccca ataattgaac aa
429438DNAArtificial SequenceSynthetic Polynucleotide 94tttagactcc
cgattgagga attagagcca gattttcg 389542DNAArtificial
SequenceSynthetic Polynucleotide 95agagaataaa ataaacagcc acaaatcaga
tatagaacca ag 429642DNAArtificial SequenceSynthetic Polynucleotide
96ttaactgaac gctaacgagc gaggcgtttt agcgaataat cg
429742DNAArtificial SequenceSynthetic Polynucleotide 97cgcccaaaga
acaagcaagc cagagaatat aaagtcatgt aa 429845DNAArtificial
SequenceSynthetic Polynucleotide 98aagtattatt agcagcccag atagccaaaa
gatattgagt caccg 459942DNAArtificial SequenceSynthetic
Polynucleotide 99attctaatca ttccaagaac gagacgacga caatacagta gg
4210045DNAArtificial SequenceSynthetic Polynucleotide 100gcgtctttcc
attagacagc aatagttagc agacaaaaac cagta 4510142DNAArtificial
SequenceSynthetic Polynucleotide 101gggaggtcga gcatgtagaa
agcctgttta tcaacatgcg tt 4210245DNAArtificial SequenceSynthetic
Polynucleotide 102aagcagctac gggtaataat tgagtaaaag agtcacaaaa tgaaa
4510342DNAArtificial SequenceSynthetic Polynucleotide 103taccgcaaaa
ggtaaagtaa tcgccatatt taacatagtt aa 4210442DNAArtificial
SequenceSynthetic Polynucleotide 104gctgtcttgt tcagctaatg
ccagtataaa gccaaaccga cc 4210542DNAArtificial SequenceSynthetic
Polynucleotide 105tcaacctccc tcttaccaac acccaagagc aatacataat at
4210639DNAArtificial SequenceSynthetic Polynucleotide 106tttaggcaaa
actttttcaa atgctgatgc aaatcatta 3910742DNAArtificial
SequenceSynthetic Polynucleotide 107aattctgtcc ggtattaaag
gcttcagtta caacataagc cc 4210846DNAArtificial SequenceSynthetic
Polynucleotide 108gcttaatcta aatttaatgg ttttaacctc cggctgagtg
aaagca 4610946DNAArtificial SequenceSynthetic Polynucleotide
109atacaaagcg ttaaataaga aaatagtgaa tttatttttc cctaca
4611039DNAArtificial SequenceSynthetic Polynucleotide 110tttcatcggt
tatataacta tagtacataa acatcttgc 3911139DNAArtificial
SequenceSynthetic Polynucleotide 111gtgtgatata ggtctgagag
ataaatcgat tattcgttt 3911242DNAArtificial SequenceSynthetic
Polynucleotide 112cctaattaca aacctactac ttcttaatag aaaatatccg aa
4211346DNAArtificial SequenceSynthetic Polynucleotide 113tggaaacatg
taaatatatt tacgccaaac cgacactcat cgtagc 4611446DNAArtificial
SequenceSynthetic Polynucleotide 114gcttctgcta ccttttgaaa
tcgctcaaaa caacattcct tagaac 4611539DNAArtificial SequenceSynthetic
Polynucleotide 115taattaacaa aatcaaataa gttcttacag aacgcccaa
3911646DNAArtificial SequenceSynthetic Polynucleotide 116ccttgaaaag
agtctaaaca cgtatcataa tagattaatt tatttg 4611739DNAArtificial
SequenceSynthetic Polynucleotide 117atgaaacaaa tcaatatatg
ttaggttgtt ctgactgag 3911842DNAArtificial SequenceSynthetic
Polynucleotide 118aaaagaaatt gatgatgaga agtattggca agaaccacct ga
4211942DNAArtificial SequenceSynthetic Polynucleotide 119aaacagtaac
ccaccagatc ctttgctgaa cttaacacag ta 4212042DNAArtificial
SequenceSynthetic Polynucleotide 120acgtaatcct agataatgga
attgtcgcca tacgtggctg gt 4212142DNAArtificial SequenceSynthetic
Polynucleotide 121aactcatcat caattcgcct caatacagag ggccaacaga aa
4212242DNAArtificial SequenceSynthetic Polynucleotide 122agatcatttt
aattttaaaa aatcccacgc tagattcatc tg 4212342DNAArtificial
SequenceSynthetic Polynucleotide 123ggaattagtc agatgaatat
atcgcgcaga ggcgatcgct at 4212442DNAArtificial SequenceSynthetic
Polynucleotide 124aggatttgca attcatcaat ataaaacaga aataagaaga tg
4212542DNAArtificial SequenceSynthetic Polynucleotide 125ttatctatta
gagccgtcaa tgattgtttg gattacatat ca 4212642DNAArtificial
SequenceSynthetic Polynucleotide 126tggtcagtta gactttacaa
aattcctgat tatcagcgta ga 4212742DNAArtificial SequenceSynthetic
Polynucleotide 127tcaccttgcc cgaacgttat tgcggaacaa agaaaagtac ct
4212838DNAArtificial SequenceSynthetic Polynucleotide 128ctgtgaatgg
aactcaaata acatgcgctt aatgcgcc 3812942DNAArtificial
SequenceSynthetic Polynucleotide 129gtcagtactc aaatatcaaa
cacaactcgt attaaaagga gc 4213042DNAArtificial SequenceSynthetic
Polynucleotide 130taagaattaa aaataccgaa catcaacagt tgaaaacatt tg
4213145DNAArtificial SequenceSynthetic Polynucleotide 131ccttaccgcc
tcacgcagac gagcctggca agtgtagcaa atcaa 4513245DNAArtificial
SequenceSynthetic Polynucleotide 132atactacatt tttttatgga
gctaagaaag gaagggaacg gaacc 4513342DNAArtificial SequenceSynthetic
Polynucleotide 133gaggccagct catggaaata caaagggaca ttctgtgagg cg
4213442DNAArtificial SequenceSynthetic Polynucleotide 134gctacagttc
tttgattagt aactatcggc cttgcacaga ca 4213542DNAArtificial
SequenceSynthetic Polynucleotide 135ttgctttaat taaccgttgt
aatccagaac aatatgaaag cg 4213642DNAArtificial SequenceSynthetic
Polynucleotide 136acgtgctaaa gagtctgtcc aagccattgc aacaggagat ag
4213742DNAArtificial SequenceSynthetic Polynucleotide 137ggccgataat
cctgagaagt gttgacgctc aatcgccagt ca 4213821DNAArtificial
SequenceSynthetic Polynucleotide 138ccgagctcga attcgtaatc a
2113928DNAArtificial SequenceSynthetic Polynucleotide 139ggccctgttt
tcaccagtga gcaacata 2814028DNAArtificial SequenceSynthetic
Polynucleotide 140aaaacagacg ttaatatttt gggattga
2814124DNAArtificial SequenceSynthetic Polynucleotide 141atgaggccgg
agaattaaat agta 2414228DNAArtificial SequenceSynthetic
Polynucleotide 142gagaatgata ttcattgaat ctaggaat
2814328DNAArtificial SequenceSynthetic Polynucleotide 143gggatttgat
agttgcgccg aatatatt 2814428DNAArtificial SequenceSynthetic
Polynucleotide 144tgaatttatg atacaggagt gtgccgtc
2814524DNAArtificial SequenceSynthetic Polynucleotide 145gagtcttttc
tatcacccgg aaat 2414628DNAArtificial SequenceSynthetic
Polynucleotide 146tgaaaattat cccaatccaa aattaccg
2814728DNAArtificial SequenceSynthetic Polynucleotide 147aaattataag
aaaacaaaat ttttttaa 2814828DNAArtificial SequenceSynthetic
Polynucleotide 148atattttata gccctaaaac aaggaagg
2814924DNAArtificial SequenceSynthetic Polynucleotide 149aatgcaatac
ggcgcgtctg cgcg 2415049DNAArtificial SequenceSynthetic
Polynucleotide 150ggccctgttt tcaccagtga gcaacatatt cctctaccac
ctacatcac 4915149DNAArtificial SequenceSynthetic Polynucleotide
151aaaacagacg ttaatatttt gggattgatt cctctaccac ctacatcac
4915245DNAArtificial SequenceSynthetic Polynucleotide 152atgaggccgg
agaattaaat agtattcctc taccacctac atcac 4515349DNAArtificial
SequenceSynthetic Polynucleotide 153gagaatgata ttcattgaat
ctaggaattt cctctaccac ctacatcac 4915449DNAArtificial
SequenceSynthetic Polynucleotide 154gggatttgat agttgcgccg
aatatatttt cctctaccac ctacatcac 4915549DNAArtificial
SequenceSynthetic Polynucleotide 155tgaatttatg atacaggagt
gtgccgtctt cctctaccac ctacatcac 4915645DNAArtificial
SequenceSynthetic Polynucleotide 156gagtcttttc tatcacccgg
aaatttcctc taccacctac atcac 4515749DNAArtificial SequenceSynthetic
Polynucleotide 157tgaaaattat cccaatccaa aattaccgtt cctctaccac
ctacatcac 4915849DNAArtificial SequenceSynthetic Polynucleotide
158aaattataag aaaacaaaat ttttttaatt cctctaccac ctacatcac
4915949DNAArtificial SequenceSynthetic Polynucleotide 159atattttata
gccctaaaac aaggaaggtt cctctaccac ctacatcac 4916045DNAArtificial
SequenceSynthetic Polynucleotide 160aatgcaatac ggcgcgtctg
cgcgttcctc taccacctac atcac 4516128DNAArtificial SequenceSynthetic
Polynucleotide 161caaaatcaaa cctgtcgtgc cgcccgct
2816228DNAArtificial SequenceSynthetic Polynucleotide 162agccgccgcg
aaacgtacag catcccgt 2816324DNAArtificial SequenceSynthetic
Polynucleotide 163gcccaaggat tgcgggaaga taca 2416428DNAArtificial
SequenceSynthetic Polynucleotide 164ggaagccgct tttgcaaaag acgtttac
2816528DNAArtificial SequenceSynthetic Polynucleotide 165tcacgttaaa
aaaaaggctc cacgaggg 2816628DNAArtificial SequenceSynthetic
Polynucleotide 166gaggttggcc tatttcggaa cgaaacat
2816724DNAArtificial SequenceSynthetic Polynucleotide 167gaacagaatc
cgtcacctca atag 2416828DNAArtificial SequenceSynthetic
Polynucleotide 168agtcagaaat tttatcctga agacttgc
2816928DNAArtificial SequenceSynthetic Polynucleotide 169tttacatttt
gaataccaag tttagaat 2817028DNAArtificial SequenceSynthetic
Polynucleotide 170cacgacccgc ctgcaacagt gtaaagca
2817124DNAArtificial SequenceSynthetic Polynucleotide 171aaaacgccag
taaaggggga aagc 2417249DNAArtificial SequenceSynthetic
Polynucleotide 172caaaatcaaa cctgtcgtgc cgcccgctta tcttcctcac
actcccaaa 4917349DNAArtificial SequenceSynthetic Polynucleotide
173agccgccgcg aaacgtacag catcccgtta tcttcctcac actcccaaa
4917445DNAArtificial SequenceSynthetic Polynucleotide 174gcccaaggat
tgcgggaaga tacatatctt cctcacactc ccaaa 4517549DNAArtificial
SequenceSynthetic Polynucleotide 175ggaagccgct tttgcaaaag
acgtttacta tcttcctcac actcccaaa 4917649DNAArtificial
SequenceSynthetic Polynucleotide 176tcacgttaaa aaaaaggctc
cacgagggta tcttcctcac actcccaaa 4917749DNAArtificial
SequenceSynthetic Polynucleotide 177gaggttggcc tatttcggaa
cgaaacatta tcttcctcac actcccaaa 4917845DNAArtificial
SequenceSynthetic Polynucleotide 178gaacagaatc cgtcacctca
atagtatctt cctcacactc ccaaa 4517949DNAArtificial SequenceSynthetic
Polynucleotide 179agtcagaaat tttatcctga agacttgcta tcttcctcac
actcccaaa 4918049DNAArtificial SequenceSynthetic Polynucleotide
180tttacatttt gaataccaag tttagaatta tcttcctcac actcccaaa
4918149DNAArtificial SequenceSynthetic Polynucleotide 181cacgacccgc
ctgcaacagt gtaaagcata tcttcctcac actcccaaa 4918245DNAArtificial
SequenceSynthetic Polynucleotide 182aaaacgccag taaaggggga
aagctatctt cctcacactc ccaaa 4518328DNAArtificial SequenceSynthetic
Polynucleotide 183ccagcagggg gagaggcggt tctaatga
2818428DNAArtificial SequenceSynthetic Polynucleotide 184gacgttgaga
gatagacttt ctgccgcc
2818528DNAArtificial SequenceSynthetic Polynucleotide 185tgtcaattca
gctcattttt tagcgagt 2818624DNAArtificial SequenceSynthetic
Polynucleotide 186ggtatgcctg taaatcgttc attt 2418728DNAArtificial
SequenceSynthetic Polynucleotide 187ttatagttgt ttagactgga taggaatt
2818828DNAArtificial SequenceSynthetic Polynucleotide 188aatagaatca
gcttgctttc gtttgcgg 2818928DNAArtificial SequenceSynthetic
Polynucleotide 189caaataatgc cttgagtaac agattagg
2819024DNAArtificial SequenceSynthetic Polynucleotide 190gaacatcggc
caaaatcggg cgac 2419128DNAArtificial SequenceSynthetic
Polynucleotide 191gaagcgcaga gcctaatttg catccggt
2819228DNAArtificial SequenceSynthetic Polynucleotide 192ttttcagatt
tcaattacct gtaacctt 2819328DNAArtificial SequenceSynthetic
Polynucleotide 193aacccttcag cagaagataa acaatatc
2819424DNAArtificial SequenceSynthetic Polynucleotide 194aacccgagta
ttcctcgaaa ggag 2419549DNAArtificial SequenceSynthetic
Polynucleotide 195ccagcagggg gagaggcggt tctaatgata acattcctaa
cttctcata 4919649DNAArtificial SequenceSynthetic Polynucleotide
196gacgttgaga gatagacttt ctgccgccta acattcctaa cttctcata
4919749DNAArtificial SequenceSynthetic Polynucleotide 197tgtcaattca
gctcattttt tagcgagtta acattcctaa cttctcata 4919845DNAArtificial
SequenceSynthetic Polynucleotide 198ggtatgcctg taaatcgttc
attttaacat tcctaacttc tcata 4519949DNAArtificial SequenceSynthetic
Polynucleotide 199ttatagttgt ttagactgga taggaattta acattcctaa
cttctcata 4920049DNAArtificial SequenceSynthetic Polynucleotide
200aatagaatca gcttgctttc gtttgcggta acattcctaa cttctcata
4920149DNAArtificial SequenceSynthetic Polynucleotide 201caaataatgc
cttgagtaac agattaggta acattcctaa cttctcata 4920245DNAArtificial
SequenceSynthetic Polynucleotide 202gaacatcggc caaaatcggg
cgactaacat tcctaacttc tcata 4520349DNAArtificial SequenceSynthetic
Polynucleotide 203gaagcgcaga gcctaatttg catccggtta acattcctaa
cttctcata 4920449DNAArtificial SequenceSynthetic Polynucleotide
204ttttcagatt tcaattacct gtaaccttta acattcctaa cttctcata
4920549DNAArtificial SequenceSynthetic Polynucleotide 205aacccttcag
cagaagataa acaatatcta acattcctaa cttctcata 4920645DNAArtificial
SequenceSynthetic Polynucleotide 206aacccgagta ttcctcgaaa
ggagtaacat tcctaacttc tcata 4520727DNAArtificial SequenceSynthetic
Polynucleotide 207acatcgtgaa tacattagcg accagag
2720828DNAArtificial SequenceSynthetic Polynucleotide 208ttagaaggtc
aataccgaac acttttta 2820928DNAArtificial SequenceSynthetic
Polynucleotide 209ccgtactagt atagcctaaa ttatgtaa
2821029DNAArtificial SequenceSynthetic Polynucleotide 210cgacgttttt
tgcaatgttt agaagagaa 2921129DNAArtificial SequenceSynthetic
Polynucleotide 211cgagcatccc gtcgggagtt aggcgcata
2921234DNAArtificial SequenceSynthetic Polynucleotide 212ccatatgcac
tccaactaaa aaattgggct tgag 3421335DNAArtificial SequenceSynthetic
Polynucleotide 213gcgtgccatt aaaggccgtt catattacgg taatc
3521435DNAArtificial SequenceSynthetic Polynucleotide 214aggtgagtta
acactaacgt catagcagcc tttac 3521535DNAArtificial SequenceSynthetic
Polynucleotide 215agccagcaaa tctaaacagg ggacgggaga attaa
3521635DNAArtificial SequenceSynthetic Polynucleotide 216gttatcttag
gagcaataag aatgaaatag caata 3521735DNAArtificial SequenceSynthetic
Polynucleotide 217aaaagcctga gcaatacctt tccaccctca gagcc
3521835DNAArtificial SequenceSynthetic Polynucleotide 218cgccatgttt
accaaacata gatcaaaagc gtcat 3521935DNAArtificial SequenceSynthetic
Polynucleotide 219aaacgtatgc aaatatttca tgttaaataa cactg
3522035DNAArtificial SequenceSynthetic Polynucleotide 220acgccgaata
aacaaattct tgtaacgaat tttgc 3522135DNAArtificial SequenceSynthetic
Polynucleotide 221tttgagggga cgacaacaag atgccctgaa ccgat
3522235DNAArtificial SequenceSynthetic Polynucleotide 222tcggctaatt
ctgtatcaac agcttgctca acaac 3522335DNAArtificial SequenceSynthetic
Polynucleotide 223gcgaggtttt tgttaaatca gattgtatcg cctgt
3522435DNAArtificial SequenceSynthetic Polynucleotide 224gacaccacgg
aataacatac aacaaagatg aggat 3522535DNAArtificial SequenceSynthetic
Polynucleotide 225tccaacaggt ctgaagccag ttttgatcag aatga
3522635DNAArtificial SequenceSynthetic Polynucleotide 226aaaaccagga
ttagcggggt taagtattat cggcg 3522735DNAArtificial SequenceSynthetic
Polynucleotide 227aaagagctcc tgtacgtggg acacatccta attta
3522836DNAArtificial SequenceSynthetic Polynucleotide 228gcagtgttca
atcaaaggct aaattgagcg atgccg 3622936DNAArtificial SequenceSynthetic
Polynucleotide 229aacaattctc gtcaaaaccg atcaaaaggg cttacc
3623036DNAArtificial SequenceSynthetic Polynucleotide 230atcatagcat
cagcagtttg aaccctgtga ctcctt 3623136DNAArtificial SequenceSynthetic
Polynucleotide 231cagtagtgcc ggacaaacag atctactagg aaggta
3623236DNAArtificial SequenceSynthetic Polynucleotide 232cccttagacg
cagatgccgc cgaagcccct tcaaag 3623336DNAArtificial SequenceSynthetic
Polynucleotide 233gagagatcgg aaaactgact aaagattaag ccgttc
3623436DNAArtificial SequenceSynthetic Polynucleotide 234ggtgcgggcc
tcttaacgct caatctacca gtttca 3623536DNAArtificial SequenceSynthetic
Polynucleotide 235ataatcgatc gagagggatc gaggctttga gtgtac
3623637DNAArtificial SequenceSynthetic Polynucleotide 236gataggtaca
aacgccggat atcatcaaga gtaatct 3723740DNAArtificial
SequenceSynthetic Polynucleotide 237agtcataagt tgccacatta
ttcatcagtt gagttatacc 4023841DNAArtificial SequenceSynthetic
Polynucleotide 238tcttcgcctc ctctcaaaaa ctggcctaga cggtggaacc g
4123941DNAArtificial SequenceSynthetic Polynucleotide 239ccctcacttt
accagagaat ccttgaagtc ccggcctcac c 4124042DNAArtificial
SequenceSynthetic Polynucleotide 240cgcctgtgca ctcttgaacc
tgagagtccc ctgaacaaag tc 4224142DNAArtificial SequenceSynthetic
Polynucleotide 241aatcaacagt tgaacatccc taagaattag aaaggccgga ga
4224242DNAArtificial SequenceSynthetic Polynucleotide 242cggtagcgca
ctcagccatc cacccaacga atgcactggt ct 4224342DNAArtificial
SequenceSynthetic Polynucleotide 243cttctgagag gtgttatggt
taaaacatta aagaaacgca aa 4224442DNAArtificial SequenceSynthetic
Polynucleotide 244cggcctttag tgattccggc aataagagct gaatataccc tc
4224542DNAArtificial SequenceSynthetic Polynucleotide 245gctcattaac
agcggctctc aagactttag ccgccgccag tg 4224642DNAArtificial
SequenceSynthetic Polynucleotide 246tgagaaggaa taaccttgct
tttttaatct cattaaggca gg 4224742DNAArtificial SequenceSynthetic
Polynucleotide 247ccaatcgcaa gacaggaaac aaagaggcta aacagttcag aa
4224842DNAArtificial SequenceSynthetic Polynucleotide 248atgctgacct
ttttattctg agcccgtata aacagagtgc ct 4224942DNAArtificial
SequenceSynthetic Polynucleotide 249tgggaagttc gccaagtcag
gattttaaga actggtgtga at 4225042DNAArtificial SequenceSynthetic
Polynucleotide 250gcaaagccac cgcttacctt aaatttcaac tttaacaaag ct
4225142DNAArtificial SequenceSynthetic Polynucleotide 251cgtgcatttg
gtgtgctcat tttacccaaa tcaacacaag aa 4225242DNAArtificial
SequenceSynthetic Polynucleotide 252tcattccatt aaacgaaaga
ccgagggtag caacgcatga gg 4225342DNAArtificial SequenceSynthetic
Polynucleotide 253ttcatcaacc aaccgaaaga ggacagatga acggggccac ta
4225442DNAArtificial SequenceSynthetic Polynucleotide 254ctattttgca
ccatttgcgg gtgtatcacc cccagcgatt at 4225542DNAArtificial
SequenceSynthetic Polynucleotide 255aacccactac actgttcttt
gcgacaactt ttaaaggggt ca 4225642DNAArtificial SequenceSynthetic
Polynucleotide 256atcaccatca atataatgcc ttagaacctt ttacctttat tt
4225742DNAArtificial SequenceSynthetic Polynucleotide 257gagtaatgtg
taggcagtca agagagatag agggttcagg tc 4225842DNAArtificial
SequenceSynthetic Polynucleotide 258taaagatgga aacgtgatta
aaatactttg taccatacca gc 4225942DNAArtificial SequenceSynthetic
Polynucleotide 259caaaagaact ggcacaataa ttaaaggtgt gtgttgttgg ca
4226042DNAArtificial SequenceSynthetic Polynucleotide 260agagcatggg
caaaaattac gaataaatat tttcagctgg tc 4226142DNAArtificial
SequenceSynthetic Polynucleotide 261caatagagac ggaacgactt
gagccaataa taaaggatta ta 4226242DNAArtificial SequenceSynthetic
Polynucleotide 262actgtagcgc gttttagcac ccaataaccg tcagatgaat at
4226342DNAArtificial SequenceSynthetic Polynucleotide 263ccaccacacc
acccgtagga ttagagagaa gaagacaaaa tc 4226442DNAArtificial
SequenceSynthetic Polynucleotide 264agaaccgaat tgctagaccg
gtctctgaat ttaagagcag tt 4226542DNAArtificial SequenceSynthetic
Polynucleotide 265gatacaggag tgtaataaat cggaaacatt tcatttgaat ta
4226642DNAArtificial SequenceSynthetic Polynucleotide 266aacgagtaac
atgattgctc atacagacga cgatattagt ta 4226742DNAArtificial
SequenceSynthetic Polynucleotide 267ttacagggaa gaaaaacagt
agggctcagg cgatcaggcg at 4226842DNAArtificial SequenceSynthetic
Polynucleotide 268gtagcattcc acagttttgt catatgcgga ggcattttcg ag
4226942DNAArtificial SequenceSynthetic Polynucleotide 269aaacggcacc
agtacgccaa catgtaataa ggtaataatt tt 4227042DNAArtificial
SequenceSynthetic Polynucleotide 270tcggtttata gaacgagtag
tggaattgct ttcaagttaa ta 4227142DNAArtificial SequenceSynthetic
Polynucleotide 271cactaaaaca ctcacgaagg cacattaaat gtgaacaaat ca
4227242DNAArtificial SequenceSynthetic Polynucleotide 272tctttgatcg
cctgataaat tgcgaaccga tatagccgag ct 4227343DNAArtificial
SequenceSynthetic Polynucleotide 273atcaaaatgg cttagataac
tattaatggc gaccgttaca aac 4327443DNAArtificial SequenceSynthetic
Polynucleotide 274aagaacgcga gaaaaacgac gacgggaagg atagcttgaa tcc
4327543DNAArtificial SequenceSynthetic Polynucleotide 275tcccgacttt
gttaaaattc gaattgtacg aactgaacga acc 4327643DNAArtificial
SequenceSynthetic Polynucleotide 276attcgcctga acaaaattaa
caagtacata tgtgagtagt caa 4327748DNAArtificial SequenceSynthetic
Polynucleotide 277accagctgct gcgaataaga gcaaacaaga gaatattgcc
tcaaatat 4827848DNAArtificial SequenceSynthetic Polynucleotide
278gagaggttga gagctagcat tgtaccccgg ttgcttcacg gatccagc
4827948DNAArtificial SequenceSynthetic Polynucleotide 279gaagccaagt
taccagtatg ggcaacatat aatggtaaca tctttaca 4828048DNAArtificial
SequenceSynthetic Polynucleotide 280attacgcaga aggtatagat
tagagcctat tagatatcat taattatc 4828148DNAArtificial
SequenceSynthetic Polynucleotide 281atcggtttgc gggttattaa
tcgtattaaa tccttaatgg gaacggaa 4828248DNAArtificial
SequenceSynthetic Polynucleotide 282aatattaaat tcaccattcc
tgattatttg tttgaaattg cacagtaa 4828348DNAArtificial
SequenceSynthetic Polynucleotide 283cgaacccctt ttgaaatttc
aattaccgca cagggggcgg ttaatttt 4828448DNAArtificial
SequenceSynthetic Polynucleotide 284cagtaaatca ggtaatgctt
tgagactcct cactcggata aaatttgt 4828548DNAArtificial
SequenceSynthetic Polynucleotide 285ggattaaaat agcgcaacac
ccaccaccct cattttcaga cgaggcat 4828648DNAArtificial
SequenceSynthetic Polynucleotide 286cggataacct attaacctcc
cataggtctg agagaagacg ctgagtaa 4828748DNAArtificial
SequenceSynthetic Polynucleotide 287gcggagtgag acgacgttgg
tagaaagcag gatagcaagc ctgctgca 4828848DNAArtificial
SequenceSynthetic Polynucleotide 288agaccaaagg ccgcacgcat
acgagaaaca cccaatagat accaatca 4828949DNAArtificial
SequenceSynthetic Polynucleotide 289cgaattctaa tgcgaacgtt
agagcctaat ttgcccaatc cagccagaa 4929049DNAArtificial
SequenceSynthetic Polynucleotide 290cgtggcattt tgaatatcct
gacgctaacg agcgtttttg ttcgcctgc 4929149DNAArtificial
SequenceSynthetic Polynucleotide 291aacagggaga agattagtct
taaagcgtta gcaaggcaag ccacgtaat 4929249DNAArtificial
SequenceSynthetic Polynucleotide 292caaaccccac tgcgtgcggc
gaataccgat agcccccggg taaaggctt 4929349DNAArtificial
SequenceSynthetic Polynucleotide 293tgccctgcgg catcttacct
gcagccatct ggtcacagca aaaatatca 4929449DNAArtificial
SequenceSynthetic Polynucleotide 294ggagcggttg cggataaagg
tttagcaaac gtagacagat aggataata 4929549DNAArtificial
SequenceSynthetic Polynucleotide 295acggctgata atgggcacgt
attgtagaat cctcagcgca gaggaagtt 4929649DNAArtificial
SequenceSynthetic Polynucleotide 296gtagattccg tcacattatt
cattaaagta ttttgtggca ataccagaa 4929749DNAArtificial
SequenceSynthetic Polynucleotide 297ggcagcccgg tccgtgcaac
tgctgtagct caacattaat tggtcattt 4929849DNAArtificial
SequenceSynthetic Polynucleotide 298taacggaaac gtcagtggca
tcatttggga attagttagc aagcgtcag 4929949DNAArtificial
SequenceSynthetic Polynucleotide 299acaaacaaca ggagtcagag
ccgccaccca ccggatttgc cattcggtc 4930049DNAArtificial
SequenceSynthetic Polynucleotide 300taggcattat acaccggaat
ataaggcctt ctgacccgga agtaccagg 4930149DNAArtificial
SequenceSynthetic Polynucleotide 301gagaatactc caaacaaaag
gagccttttg aatttgaacg cgttcctta 4930249DNAArtificial
SequenceSynthetic Polynucleotide 302attctcaaca gttgaggatc
ctaaaacata agcaaaaata aacagataa 4930349DNAArtificial
SequenceSynthetic Polynucleotide 303tcagaaaaca ggaagctcat
ttaggaactc catgtgaacg aggcggcaa 4930449DNAArtificial
SequenceSynthetic Polynucleotide 304gtaaaaatct acaatagcgg
tgccggttca gacgtcatac cgccagcac 4930549DNAArtificial
SequenceSynthetic Polynucleotide 305cgatgaattt atccagttac
aatatttaca ttaaacgttt tagtgtcga 4930649DNAArtificial
SequenceSynthetic Polynucleotide 306agagagaaac gattctttcc
aatcagctac aattttggct ataaaacag 4930749DNAArtificial
SequenceSynthetic Polynucleotide 307gcccaatact aacaactaaa
aaggaattac cttgcgttgc cacgctgag 4930849DNAArtificial
SequenceSynthetic Polynucleotide 308gctattggag ttaactgaac
atggaataac ataaaaagca tcgaggaag 4930948DNAArtificial
SequenceSynthetic Polynucleotide 309tttaaatgcg atattcgctg
ataaattact tcgttaacgg ctggtttg 4831049DNAArtificial
SequenceSynthetic Polynucleotide 310accgattgcc
aaagccagct tttgcaggcg ctttcccgaa cgagaagcc 4931149DNAArtificial
SequenceSynthetic Polynucleotide 311gagggagata gtagtgaaaa
gccaatgaac agaatcaatt ctgcgaacg 4931249DNAArtificial
SequenceSynthetic Polynucleotide 312agcattatta tttaagggtt
agaacctcac gcaaacaaaa gaaagctaa 4931349DNAArtificial
SequenceSynthetic Polynucleotide 313aaaatcaatt atcattcagg
tcaatataat cctgacagat gatcacaat 4931449DNAArtificial
SequenceSynthetic Polynucleotide 314aaccatcaga gcacacgtca
gcgtggtgta tcaaaaacat ccacattca 4931549DNAArtificial
SequenceSynthetic Polynucleotide 315gcaaaactta gtttgaccat
tttaaatatt ttttcttgcc gtgaagggt 4931649DNAArtificial
SequenceSynthetic Polynucleotide 316tagcccccac agttgattcc
caagtttgcc tttaggccgg aaccctttt 4931749DNAArtificial
SequenceSynthetic Polynucleotide 317ggtgtctggc gaattattcc
gtccggccga tagcatttgg ggcgcgagc 4931849DNAArtificial
SequenceSynthetic Polynucleotide 318gcctccctca gagccgccaa
taaagtacag tagatcgtaa tcagtagcg 4931949DNAArtificial
SequenceSynthetic Polynucleotide 319cggaacctca ttccatatat
tcaagttatg atgaaccaaa tcccgtaaa 4932049DNAArtificial
SequenceSynthetic Polynucleotide 320tcaaaataga accacgccgc
cattggccca aacaactggt aacggggtc 4932149DNAArtificial
SequenceSynthetic Polynucleotide 321attcgaggaa agaccatcaa
attatagtat attcagtcca attagtaaa 4932249DNAArtificial
SequenceSynthetic Polynucleotide 322tcagacgagc attgtcaaga
aattgcttgg agaaaattac caagccagc 4932349DNAArtificial
SequenceSynthetic Polynucleotide 323acatggcaat ggaaatcgac
ataaaattct gtaaattaga ttactacag 4932449DNAArtificial
SequenceSynthetic Polynucleotide 324ccataacgat agctcgtcgc
tattaattgt gtacagcgca gaagcaaac 4932549DNAArtificial
SequenceSynthetic Polynucleotide 325ttaatgcttt cggaagtgcc
gtgatataca ggaggccacc ctcagaacc 4932649DNAArtificial
SequenceSynthetic Polynucleotide 326aaagaaggaa ttactaatgc
agatacatag gaataggtaa cgcaactgt 4932749DNAArtificial
SequenceSynthetic Polynucleotide 327agtaagagag aggcgtaaac
tttttcaagg ggatgcaata ggaacatta 4932849DNAArtificial
SequenceSynthetic Polynucleotide 328tttacggtca gaaccgccac
cgtaccgtaa agctggcgaa agatatatt 4932949DNAArtificial
SequenceSynthetic Polynucleotide 329tcaccaggtg ataacataat
tactagaata gtatcgtctt tctaaatga 4933049DNAArtificial
SequenceSynthetic Polynucleotide 330tcatagttaa actaacggaa
caacccatct cagagtatca taaccctcg 4933149DNAArtificial
SequenceSynthetic Polynucleotide 331taaacaacga ataaagctca
ggaagatctt aacaataaag cccgctatt 4933249DNAArtificial
SequenceSynthetic Polynucleotide 332attttcttga aaattaaagt
aacgacaata aacaataatg cacttaaac 4933349DNAArtificial
SequenceSynthetic Polynucleotide 333ttcacgtgta tgggtctaaa
gacagcccag tttcggccac cctgtatca 4933449DNAArtificial
SequenceSynthetic Polynucleotide 334aaggctaaaa taatatcccg
ctgccagttt ccgggctaat tgagaatcg 4933549DNAArtificial
SequenceSynthetic Polynucleotide 335atattcggtc gccacagtga
aatggttttg atagaaagga acagccagc 4933649DNAArtificial
SequenceSynthetic Polynucleotide 336catcgccctt ttgcacagcg
agtaacaagt agaaaagtcc tggacagta 4933749DNAArtificial
SequenceSynthetic Polynucleotide 337tgcgccgcag cagccaagta
cttttcatat taccggaagc ctttagttg 4933849DNAArtificial
SequenceSynthetic Polynucleotide 338ggctggcaac ttttaaaacg
aagaataaat ccgcgacctg cgcaagaac 4933949DNAArtificial
SequenceSynthetic Polynucleotide 339aactgacctg accttttgag
gcttgcagga ttctcgccag ctatccggt 4934049DNAArtificial
SequenceSynthetic Polynucleotide 340actaaagact ttttgctaca
gtcaccctac aatgattcga ggaattgta 4934149DNAArtificial
SequenceSynthetic Polynucleotide 341gtaaaatgtt tttacgcact
cgctgtctcc tgtttccaga cgccgacaa 4934249DNAArtificial
SequenceSynthetic Polynucleotide 342taaacggacc aagcgtacaa
cggagattag gttttcgccc aaaagaata 49
* * * * *