HK1196961A

HK1196961A - Method for forming nanoparticles having predetermined shapes

Info

Publication number: HK1196961A
Application number: HK14110340.0A
Authority: HK
Inventors: Wei Sun; Peng Yin
Original assignee: 哈佛学院院长等
Priority date: 2011-07-01
Filing date: 2012-06-29
Publication date: 2014-12-24

Description

method for forming nanoparticles having a predetermined shape

Technical Field

The present invention relates generally to articles and methods for forming nanostructures, and more particularly to articles and methods for forming nanostructures having unique and/or predetermined shapes.

Background

The proper synthesis of monodisperse shape-controllable nanoparticles is the first step for applications in biological assays that use the shape-specific properties of nanoparticles for detection. To direct the growth of nanoparticles having a particular shape, including inorganic nanoparticles such as gold or silver, templates encoded by designed geometries are typically used. Soft templates have successfully created different shapes, for example, from the self-assembled structure of amphiphilic surfactant molecules. However, it is generally difficult to predict the shape of the resulting structure (due in part to the elastic nature of the template), and therefore it is challenging to design structures with predetermined shapes and program the growth of inorganic materials using this approach. Hard templates such as oxides or viruses have also been used to direct the growth of nanowires or nanorods, with better predictability of the resulting structure. However, these methods are only capable of generating a few shapes, which may limit the programmability of shape diversity. Improved methods and articles that can address some or all of these issues and/or other challenges in the art would be advantageous in many different fields.

Summary of The Invention

The present invention relates generally to articles and methods for forming nanostructures, and more particularly to articles and methods for forming nanostructures having unique and/or predetermined shapes. In some cases, the inventive subject matter relates to related products, alternative solutions to specific problems, and/or a variety of different uses of one or more articles, compositions, and/or methods.

In one set of embodiments, a series of articles is provided. In one embodiment, an article comprises a nanoparticle positioned within a nucleic acid container having a predetermined three-dimensional structure, wherein the nanoparticle comprises at least one surface portion having a shape complementary to a shape of an interior surface portion of the nucleic acid container.

In another embodiment, an article includes a nanoparticle comprising at least two opposing surface portions each having a shape complementary to a shape of a surface portion of a nucleic acid nanostructure.

In another embodiment, an article includes 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.

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 the surface of the inorganic nanoparticle and extending from the surface of the inorganic nanoparticle through the pore of the nucleic acid container.

In another embodiment, an article comprises an assembly of nucleic acid-coated nanoparticles, wherein the nucleic acid-coated nanoparticles are attached to each other by complementary binding sites.

In another set of embodiments, a series of methods are provided. In one embodiment, a method includes forming a nanoparticle comprising at least one surface portion having a shape complementary to a shape of an interior surface portion of a nucleic acid container having a predetermined three-dimensional structure on a sub-nanometer level.

In another embodiment, a method includes forming nanoparticles from nanoparticle precursors positioned within a nucleic acid container having a predetermined three-dimensional structure.

In another embodiment, a method includes 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 the nanoparticle within the nucleic acid container, and attaching the plurality of components to the nanoparticle.

In another embodiment, a method comprises attaching an isolated nucleic acid strand to the surface of an inorganic nanoparticle having a non-spherical shape.

In another embodiment, a method comprises providing an inorganic nanoparticle coated with a nucleic acid container, wherein the nucleic acid container comprises a pore, introducing a nucleic acid strand through the pore of the nucleic acid container, and attaching a portion of the nucleic acid strand to a surface of the inorganic nanoparticle.

In another embodiment, a method comprises forming an assembly of nucleic acid-coated nanoparticles, wherein the nucleic acid-coated nanoparticles are attached to each other by complementary binding sites.

In another embodiment, the method comprises using two non-spherical nanoparticles to detect at least 12 different target molecules.

In another set of embodiments, a series of compositions is 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.

In another embodiment, a composition comprises a plurality of nanoparticles, wherein at least 90% of the nanoparticles vary in maximum cross-sectional dimension by less than 0.5 standard deviation from the median maximum cross-sectional dimension of all nanoparticles in the composition, and wherein each of the plurality of nanoparticles comprises at least 6 distinct sides.

Various configurations of the articles, compositions, and methods described above and herein are provided. For example, in some cases, the nanoparticle precursor comprises inorganic nanoparticles. In some embodiments, the nanoparticle precursor comprises a monomer of a metal, semiconductor, or 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 can have a cross-sectional dimension of, for example, less than or equal to 50nm, less than or equal to 25nm, less than or equal to 10nm, less than or equal to 5nm, less than or equal to 3nm, less than or equal to 2nm, less than or equal to 1nm, or less than or equal to 0.1 nm.

In some embodiments, the nanoparticle comprises at least one surface portion having a shape complementary to a shape of an interior surface portion of the nucleic acid container. The nanoparticle may have a shape complementary to a shape of an inner surface of the nucleic acid container. The nanoparticle can 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 nanoparticles comprise a cross-section in the shape of a rectangle, rod, T, L, branch, rhombus, star, square, parallelogram, triangle, pentagon, hexagon, toroid, or polyhedron. In some embodiments, the nanoparticles have a non-spherical shape or an asymmetric shape. In some cases, the nanoparticle is an inorganic nanoparticle. The nanoparticles may comprise a metal, a semiconductor, or a polymer. In some cases, the nanoparticles are alloys. In some embodiments, the nanoparticles have at least one cross-sectional dimension that is less than or equal to 1 micron, less than or equal to 500nm, less than or equal to 250nm, less than or equal to 100nm, less than or equal to 75nm, less than or equal to 50nm, less than or equal to 40nm, less than or equal to 30nm, less than or equal to 20nm, less than or equal to 10nm, or less than or equal to 1 nm. In particular embodiments, the nanoparticles have at least one cross-sectional dimension that is greater than or equal to 1nm, greater than or equal to 5nm, greater than or equal to 10nm, greater than or equal to 50nm, or greater than or equal to 100 nm. The nanoparticles 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 nanoparticles may be encapsulated by nucleic acid nanostructures. In some embodiments, the nanoparticle comprises an isolated binding site attached to the 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 the surface of the nanoparticle. The nanoparticle may comprise an isolated nucleic acid strand attached to the surface of the nanoparticle. The isolated binding sites may be located at least 2nm, at least 5nm, at least 10nm, at least 15nm, at least 20nm, or at least 30nm away from each other. In some embodiments, the nucleic acid strand is a DNA strand or DNA analog, or an RNA strand or RNA analog.

In some embodiments, the nanoparticles may have a cross-sectional shape that includes a different number of vertices. The cross-sectional shape of the nanoparticle can 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 vertices. 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 vertices 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 vertices of the cross-sectional shape of the nanoparticle are substantially sharp. A combination of rounded and sharp vertices is also possible.

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 nanoparticles. In some cases, the nucleic acid container comprises a cavity having a volume, and substantially all of the volume is filled with nanoparticles. 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 container is substantially closed. In some embodiments, the nucleic acid container comprises at least one lid that can be opened or closed.

The nucleic acid container can comprise a cavity, and the cross-sectional dimension of the cavity can be less than or equal to 1 micron, less than or equal to 500nm, less than or equal to 250nm, less than or equal to 100nm, less than or equal to 75nm, less than or equal to 50nm, less than or equal to 40nm, less than or equal to 30nm, less than or equal to 20nm, less than or equal to 10nm, or less than or equal to 1 nm. In some embodiments, the nucleic acid container comprises a cavity, and the cavity has a cross-sectional dimension of greater than or equal to 1nm, greater than or equal to 5nm, greater than or equal to 10nm, greater than or equal to 20nm, greater than or equal to 30nm, greater than or equal to 40nm, greater than or equal to 50nm, greater than or equal to 100nm, greater than or equal to 500nm, or greater than or equal to 1 micron. In some embodiments, the nucleic acid container comprises a wall surrounding the cavity, and the average thickness of the wall is less than or equal to 1 micron, less than or equal to 500nm, less than or equal to 250nm, less than or equal to 100nm, less than or equal to 75nm, less than or equal to 50nm, less than or equal to 40nm, less than or equal to 30nm, less than or equal to 20nm, less than or equal to 10nm, or less than or equal to 1 nm. In some cases, the nucleic acid container comprises a wall surrounding the cavity, and wherein the average thickness of the wall is greater than or equal to 1nm, greater than or equal to 10nm, greater than or equal to 25nm, greater than or equal to 50nm, greater than or equal to 100nm, greater than or equal to 500nm, or greater than or equal to 1 micron.

In some cases, the nucleic acid container comprises more than one layer. The nucleic acid container may be formed from nucleic acids having a molecular weight of at least 640 kDa. In some embodiments, the nucleic acid container is formed from nucleic acids having a length of at least 1,000 bases. In some embodiments, the nucleic acid container comprises an inorganic nanostructure.

In some embodiments, the assembly is formed by: nucleic acid containers each having a nanoparticle precursor positioned therein are assembled, and nanoparticles are subsequently synthesized from the nanoparticle precursors within the nucleic acid containers to form nucleic acid-coated nanoparticles. The assembly may be formed by: synthesizing a plurality of nucleic acid-coated nanoparticles, each formed by growing a nanoparticle from a nanoparticle precursor positioned within a nucleic acid container, and subsequently assembling the nucleic acid-coated nanoparticles. The nucleic acid-coated nanoparticles may be attached to each other through binding sites that are attached to the nucleic acid portion of the nucleic acid-coated nanoparticles. In some embodiments, the nucleic acid-coated nanoparticles are attached to each other by binding sites that are attached to the nanoparticle portion of the nucleic acid-coated nanoparticles. In some cases, the nucleic acid-coated nanoparticles are attached to each other using thermal, photophysical, and/or binding processes.

In some embodiments, the method involves removing a portion of the nucleic acid from the nucleic acid-coated nanoparticle. In some cases, the method involves substantially removing the nucleic acid coating from the nucleic acid-coated nanoparticle. In some embodiments, the method involves passivating the surface of the nanoparticle before, during, or after the removing step. The nanoparticles may remain attached to each other in the assembly after the removing step.

In embodiments involving components, the components may be electronic circuitry. The components may be in the form of a two-dimensional array or a three-dimensional array. The component can have at least one length and/or at least one cross-sectional dimension of less than or equal to 1mm, 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 500nm, less than or equal to 100nm, less than or equal to 50nm, less than or equal to 10nm, or less than or equal to 1 nm. The component can have at least one length and/or at least one cross-sectional dimension greater than or equal to 1nm, greater than or equal to 10nm, greater than or equal to 100nm, 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.

In some cases, the nanoparticle comprises a label attached to the surface of the nanoparticle. In some embodiments, the labels are separated on the surface of the nanoparticle. The label may comprise a nucleic acid strand, a fluorophore, a nanoparticle, an antibody, a peptide, or a reporter. In some embodiments, the label is a surface-enhanced raman scattering reporter. In some cases, the label is a luminescent probe. In particular embodiments, each label is adjacent to a binding site attached to the surface of the nanoparticle. In some embodiments, the nanoparticle comprises a plurality of pairs of labels and binding sites, wherein each label is different from each other and each binding site is different from each other. The article, composition, or method can include 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 labels positioned on the surface of the nanoparticle. In some cases, the components or markers are each separated from one another and positioned at a predetermined distance from one another. The method may involve attaching a predetermined number of components or labels to the nanoparticles.

In some embodiments, the article detects biomolecules, e.g., multiplex detection of biomolecules. The detecting can include introducing the target molecule to a plurality of nanoparticles and allowing the target molecule to bind to the surface of at least two different nanoparticles. Binding may enhance the raman signal from two reporter molecules bound to the nanoparticle surface. The method may involve the use of 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.

In some embodiments, the nucleic acid-coated nanoparticles are each in the form of a nanoparticle positioned within the nucleic acid container. The method may involve forming at least 10, at least 15, at least 20, at least 30, at least 50, or at least 100 inorganic nanoparticles each having a different shape in parallel.

The method may include synthesizing nanoparticles from a seed-mediated growth process. In some cases, the nanoparticles are 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 predetermined three-dimensional structure, and the shape of the nanoparticle is formed at least in part by contouring the cavity.

In some embodiments, the methods involve controlling ion diffusion kinetics to control the growth kinetics and/or composition of the nanoparticles. The method may include controlling a composition distribution in the nanoparticle alloy.

In some cases, the inorganic nanoparticles are hollow and comprise a cavity. The inorganic nanoparticles may be used as templates to build secondary nanostructures in the cavities of the nanoparticles. The nanoparticles or nanostructures may have complex arbitrary shapes.

The combination of a programmable nucleic acid container with nanoparticle synthesis allows for programmability of arbitrarily shaped materials by using the container as a mold. Target structure information can be encoded into the cavity design of a specifically shaped nucleic acid container. In some embodiments, growth of a particular material (e.g., an inorganic material) within a cavity may be initiated using a small nanoparticle precursor (e.g., a nanocrystal) for nucleation of the inorganic material on the interior surfaces of the nucleic acid cavity, and stopped or slowed significantly when the growing lattice encounters the nucleic acid sidewalls. These methods can allow a wide variety of applications of inorganic material synthesis using nucleic acid programming, for example in multiplexed Surface Enhanced Raman Scattering (SERS) detection, DNA-guided electronic circuit self-assembly, surface-specific catalysts, and structural constraints on electrode materials in lithium-based fuel cells. Notably, the syntheses described herein may be performed not only ex vivo (e.g., in vitro), but also under in vitro/in vivo conditions, e.g., in bacteria and cells.

Other advantages and novel features of the invention may become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the drawings. In the event that the present specification and the documents incorporated by reference include conflicting and/or inconsistent disclosure, the present specification controls. If two or more documents incorporated by reference include conflicting and/or inconsistent disclosure with respect to each other, then the document with the later effective period controls.

Brief Description of Drawings

Non-limiting embodiments of the invention will be described with reference to the accompanying drawings, which are schematic and are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every drawing, not every component of every embodiment of the invention is shown, where illustration does not necessarily allow those of ordinary skill in the art to understand the invention. In the drawings:

FIG. 1A shows a method involving the use of a nucleic acid container as a mold to form nanoparticles, according to one set of embodiments;

FIG. 1B shows differently shaped nanoparticles that can be formed using differently shaped nucleic acid containers, according to one set of embodiments;

FIGS. 2A-2F show examples of different nucleic acid containers according to one set of embodiments;

FIGS. 3A-3D show a nucleic acid container that can include one or more lids, according to another set of embodiments;

FIGS. 3E-3F show nucleic acid containers that can be used to form hollow nanoparticles, according to another set of embodiments;

FIGS. 4A-4F illustrate control of surface addressability of nanoparticles, according to one set of embodiments;

FIG. 5 shows an example of surface-specific self-assembly of nanoparticles, according to one set of embodiments;

FIGS. 6A and 6B show the self-assembly of nanostructures into electronic circuits, according to one set of embodiments;

FIG. 7 shows a schematic of target-triggered self-assembly of nanostructures that can be used to form surface-enhanced Raman spectroscopy assays, according to one set of embodiments;

fig. 8A shows the dissociation of a nanoparticle precursor into the cavities of a nucleic acid container, according to one set of embodiments;

FIG. 8B is a Transmission Electron Microscopy (TEM) image of the nucleic acid container depicted in FIG. 8A, according to one set of embodiments;

FIG. 8C is a transmission electron microscopy image of gold nanoparticles that have been formed by template synthesis within the nucleic acid container shown in FIG. 8B, according to one set of embodiments;

FIG. 9A shows a cross-sectional view of a nucleic acid container according to one set of embodiments.

FIG. 9B is a transmission electron microscopy image of a single nanoparticle formed by template synthesis within the nucleic acid container shown in FIG. 9A;

FIGS. 9C and 9D are transmission electron microscopy images showing two types of nanoparticles formed by dimerized nucleic acid container, according to another set of embodiments;

fig. 10 is a TEM image of a nanoparticle formed within a cavity of a nucleic acid container including two quantum dots bound to the container, according to another set of embodiments;

FIGS. 11A-11C show a method involving using a nucleic acid container as a mold to form nanoparticles having different shapes, according to one set of embodiments;

12A-12E are images showing the formation of closed nucleic acid containers and the use of the containers to form nanoparticles having different shapes, according to one set of embodiments;

FIGS. 13A-13D are images showing the formation of open nucleic acid containers and the use of the containers to form nanoparticles having different shapes, according to one set of embodiments;

FIG. 14A is an image showing self-assembly of a nucleic acid container to form a larger container for growing nanoparticles, according to one set of embodiments; and

fig. 14B is an image showing the formation of a heterogeneous quantum dot-silver nanoparticle-quantum dot sandwich structure, according to one set of embodiments.

Detailed Description

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 a nucleic acid container as a structural mold. For example, a pre-designed nucleic acid container comprising a cavity may be used to control shape-specific growth of nanoparticles. The growth of nanoparticles within the cavity may be constrained by the particular shape of the nucleic acid container. Using such methods, nanoparticles having complex and predetermined shapes and sizes can be formed. The resulting nanoparticles coated from the nucleic acid container can be used as such, or the nucleic acid coating can be partially or completely removed, if desired.

In addition, the methods described herein allow for control of the material composition of the growing nanoparticles through the use of different nanoparticle precursors and/or by controlling the wall thickness of the nucleic acid container. In some embodiments, controlling such parameters may allow for the formation of nanoparticle alloys having predetermined and controlled proportions of material components. In some embodiments, the resulting nucleic acid nanoparticle structures can be used to control the orientation, number, type, and location of components, such as binding sites, labels, and surface ligands, on the surface of a nanoparticle or nucleic acid container, advantageously, a predetermined number and orientation of nanoparticles with unique components attached to the surface of a nanoparticle or nucleic acid container can allow addressability of the structure for applications such as multiplex detection of targeting molecules. In addition, in some embodiments, the addressability of the structures may be used to form higher level assembly of the structures.

The articles and methods provided herein have application in many different fields, including biosensing, electronics, environmental science, and energy. Other advantages of the articles and methods described herein are provided in more detail below.

An example of a method for forming nanoparticles having unique and/or predetermined shapes is shown in fig. 1A. As shown by way of example in fig. 1A, scheme 10 involves using nucleic acid container 20 as a mold for template synthesis of nanoparticles. The 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 further comprises a cavity 30 which is partially closed by an inner surface of the nucleic acid container. As illustratively shown in fig. 1A, the nucleic acid container may also include a tip 32 and a tip 33, which may be open in some embodiments, or closed in other embodiments. The opening into the cavity may allow one or more nanoparticle precursors 34 to be inserted into the cavity. Alternatively, one or more nanoparticle precursors may be present within a fully closed chamber, for example 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, one or more nanoparticle precursors can be associated with the nucleic acid container, attached to the nucleic acid container by, for example, covalent attachment, physisorption, chemisorption, or by 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 float within the cavity.

The walls of the nucleic acid container may also allow a nanoparticle precursor solution to flow therethrough in order to facilitate nanoparticle formation. Typically, the walls of the nucleic acid container are porous and may allow certain molecules and components to penetrate and/or be transported into or out of the container, but may prevent other molecules and components from penetrating and/or being transported into or out of the container. The ability of a particular molecule to penetrate and/or transport into and/or across a wall of a container can depend on, for example, the packing density of the nucleic acids forming the wall, the wall thickness, and the chemical and physical properties of the wall, as described in more detail below. Accordingly, the nucleic acid container need not be open, and in some embodiments, can be substantially closed, while still allowing the particular nanoparticle precursor to enter the cavity via the pores and facilitating formation of the nanoparticles in the container.

Once the one or more nanoparticle precursors are positioned within the cavity, they can facilitate the formation of nanoparticles 38 that can fill all portions of the cavity. The shape of the 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 can, in turn, be varied by controlling the configuration and orientation of the nucleic acid strands forming the inner surface of the container, as described in more detail below. FIG. 1B shows an example of a nucleic acid container having a differently shaped cavity that may be used to form nanoparticles 38 having a different shape. Advantageously, a wide variety of unique and/or predetermined shapes of nanoparticles can be formed using the methods described herein.

In particular embodiments, the growth of nanoparticles in the chamber of the container continues until the nanoparticles encounter the inner surface or wall of the container. Chemical and/or physical interactions between the growing nanoparticles and the inner surface of the container can stop or significantly slow the growth of the nanoparticles. In particular embodiments involving a substantially closed nucleic acid container, the overall size and/or shape of the nanoparticles 38 may be controlled by the size and/or shape of the container cavity, as described in more detail below. In other embodiments, the growth of all or a portion of the nanoparticles is stopped before it comes into contact with the portion of the interior surface of the container.

Growing nanoparticles within a nucleic acid container cavity typically involves more than just adding surface ligands to the surface of the nanoparticle precursor. For example, in some embodiments in which nanoparticle precursors are used to grow nanoparticles, the nanoparticle precursors may have a shape that is substantially different from the shape of the resulting nanoparticles grown from the nanoparticle precursors. In some cases, the resulting nanoparticles have a more complex shape than the shape of the nanoparticle precursor. In addition, as described in more detail below, the volume of the resulting nanoparticles may be substantially different from (e.g., substantially greater than) the volume of the nanoparticle precursor.

The combined nucleic acid container 20 and nanoparticles 38 shown in fig. 1A may form a composite nanostructure 40, which may be used in a variety of different applications, as described in more detail below.

Although fig. 1A shows the nanoparticle precursor 30 being introduced into the cavity of the nucleic acid container after the nucleic acid container has been formed, in other embodiments, the nanoparticle precursor can be combined with the nucleic acid container as the container is formed. For example, the design of a nucleic acid container may involve the inclusion of binding sites on the nucleic acid portion, forming part of the interior surface of the container. During annealing of the nucleic acid to form the container shape, nanoparticle precursors having binding sites complementary to the binding sites attached to the nucleic acid can be introduced to allow binding between the interior surface portions of the container and the nanoparticle precursors.

As shown by way of example in fig. 1A, the nanoparticles 38 may be formed by a seed-mediated growth process involving the use of the nanoparticle precursors 34. That is, nanoparticle precursors, which themselves may be in the form of nanoparticles, may be used as seeds to grow larger nanoparticles in the presence of other precursors (e.g., nanoparticle precursor solutions), which may determine the material composition of the nanoparticles 38. Any suitable combination of nanoparticle precursors and nanoparticle precursor solutions may be used. For example, to form nanoparticles formed of gold, gold nanoparticle precursors and HAuCl may be used₄And a precursor solution of ascorbic acid. For the formation of silver nanoparticles, gold nanoparticles and AgNO may be used₃And a precursor solution of ascorbic acid. Accordingly, the material composition of the resulting nanoparticles can be controlled by varying the type of precursor used. Examples of additional types of nanoparticle precursors are provided in more detail below.

In some embodiments, the nanoparticles may be formed by methods other than seed-mediated processes. For example, one or more nanoparticle precursors can fill all or a portion of a nucleic acid container cavity, and an external force, such as heat, light, pressure, an electrical potential, a magnetic force, and/or an electromagnetic force, can optionally be applied to promote growth or formation of nanoparticles. In some cases, chemical components may be added to promote the growth of the nanoparticles. In a particular embodiment, nanoparticle precursors, such as monomers (e.g., organic polymers such as monomers to synthesize organic polymers) and optionally one or more catalysts to trigger monomer growth, can be introduced into the lumen of a nucleic acid container in solution. Polymerization of the monomer may occur in the lumen of the nucleic acid container by, for example, application of heat, light, or other stimulus to allow formation of the polymeric nanoparticles. Monomers such as nucleotides and amino acids may be used.

The resulting nanoparticles may have a different physical state than the nanoparticle precursor. For example, formation of nanoparticles may involve applying a stimulus to cause conversion of a nanoparticle precursor into a different form in order to form the resulting nanoparticles. For example, the nanoparticle precursor may be in the form of a liquid, and the resulting nanoparticles may be in the form of a solid or solid-like substance (e.g., a gel). In other embodiments, the resulting nanoparticles have the same physical state as the nanoparticle precursor. For example, both the nanoparticles and nanoparticle precursors can be in solid form.

As described herein, in some embodiments, nanoparticle formation in a nucleic acid container cavity is stopped or significantly slowed by the confinement of the nanoparticle in the container. For example, nanoparticle formation can be stopped by chemical interaction between the nanoparticle surface and the inner surface of the nucleic acid container. However, it is understood that in some embodiments, synthesis of nanoparticles may be stopped or significantly slowed before the nanoparticles fill the entire volume of the nucleic acid container. For example, when an external force such as those described herein is used to promote nanoparticle formation, the application of the external force may be stopped before the nanoparticles fill the entire volume of the nanoparticle precursor.

Accordingly, in some embodiments, the nucleic acid container comprises a cavity having a volume, or only a portion of the volume is filled with nanoparticles. For example, less than 100%, less than 80%, less than 60%, less than 40%, less than 20%, or less than 10% of the cavity volume may be filled with nanoparticles. In particular 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 cavity volume is filled with nanoparticles. Combinations of the above ranges are also possible (e.g., less than 100% but at least 20% of the cavity volume may be filled with nanoparticles). In yet other embodiments, substantially all of the cavity volume is filled with nanoparticles.

The methods described herein may be used to form populations of nanoparticles having relatively high uniformity in size, shape, and/or mass. For example, in some embodiments, the composition comprises 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 size (e.g., cross-sectional size, maximum cross-sectional size, width, height, or length) or mass by less than the median or average size or mass of all nanoparticles in the composition by 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. In particular embodiments, the composition may include nanoparticles having a size (e.g., cross-sectional size, width, height, or length) or mass distribution such that no more than 20%, no more than 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 size or mass that differs by more than 20%, more than 15%, more than 10%, more than 5%, more than 3%, more than 2%, or more than 1% of the median or average of the respective sizes or masses of all the nanoparticles in the composition.

The methods described herein may also be used to control and fine tune the material composition of the nanoparticles at different regions of the nanoparticles. 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 the first region and 1:8 (wt: wt) at the second region. In general, an alloy comprising the first and second components may have a ratio of the first to second components, for example, between 1:20 and 20:1 (wt: wt). In some embodiments, the ratio of the first to second components of the 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 the first region of the nanoparticle, and the ratio of the first to second components 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 the second region, wherein the ratio is different between the first and second regions. Alloys comprising a third component may also be possible. In some cases, the ratio between the first and third components or between the second and third components has one of the ratios described above.

The methods described herein may also be used to form nanoparticles having relatively high uniformity in material composition. For example, in some embodiments, the composition comprises 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 from the median or average material composition of all nanoparticles in the composition. In some embodiments, such compositions having relatively high consistency in material composition include nanoparticle alloys having the above-described ratios between the first and second components.

In other embodiments where a mixture of different nanoparticles having different shapes is desired, the method may include using different nucleic acid containers to form a plurality of different nanoparticles in parallel. For example, in some cases, 2 to 1,000 kinds of nanoparticles (e.g., 2 to 500, 2 to 200, or 2 to 100 kinds of nanoparticles) each having a different predetermined shape may be formed in parallel. In some embodiments, a method can include using different nucleic acid containers to form at least 10, at least 15, at least 20, at least 30, at least 50, or at least 100 nanoparticles (e.g., inorganic nanoparticles) in parallel, each having a different predetermined shape. The composition may include such a number of differently shaped nanoparticles and/or nanostructures.

In some embodiments, the methods described herein can be used to form nanoparticles in the absence of specific materials such as surfactants (e.g., cetyltrimethylammonium bromide). As such, a variety of different materials, including materials that are incompatible with surfactants, can be used in the methods described herein. In particular embodiments, the nanoparticles may be synthesized in the absence of an oxide template.

In certain embodiments, the methods described herein can be performed ex vivo (e.g., in a test tube). In other embodiments, the methods may be performed under in vitro or in vivo conditions, such as in bacteria and cells.

Other features of the nucleic acid container, nanoparticles, and combinations thereof are described in more detail below.

As described herein, a nucleic acid container can be used as a template to form nanoparticles within one or more cavities of the container. The nucleic acid container can have a predetermined three-dimensional shape or structure (e.g., a non-random three-dimensional shape or structure).

The nucleic acid containers described herein can be formed using any suitable method. In some embodiments, the nucleic acid container can be constructed using non-random processes, such as processes involving intentional folding and/or bending of a nucleic acid strand to form a nucleic acid container shape. In some embodiments, a DNA "origami" approach may be used. Using such methods, three-dimensional (3D) nucleic acid containers having any user-specified shape can be formed. In some embodiments, the nucleic acid container is formed primarily of single strands of nucleic acids, with optional multiple shorter strands that may help define the resulting container shape. For example, in some embodiments involving the use of the DNA "origami" method, a long "scaffold" DNA strand "grids the target structure shape (rasterizes)" 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 area of what are known as structured DNA nanotechnology (e.g., DNA origami and design involving single-stranded sheets), self-assembled nucleic acids (particularly DNA) have been used to construct different synthetic molecular structures and devices such as ribbons, tubes, grids, and arbitrary 2D and 3D shapes. In addition, channels can be introduced into hollow DNA nanostructures such as DNA nanotubes and 3D buckets. These synthetic molecular structures and hollow DNA nanostructures may comprise a cavity for nanoparticle growth according to the invention and as described herein.

The nucleic acid container may be designed with or without the use of software packages such as cadano and other software known in the art. In some embodiments, the scaffold strand may be that of a naturally occurring, e.g., M13 virus. In other embodiments, the scaffold strands may be non-naturally occurring. In either case, the sequence of the scaffold strand should be known. Software packages such as NUPACK may also be used to design dynamic sequence components. Those of ordinary skill in the art are familiar with such methods, as exemplified by U.S. Pat. nos. 7,745,594 and 7,842,793; U.S. patent publication numbers 2010/00696621; and the disclosure in Goodman et al Nature Nanotechnology, doi10.1038/nnano.2008.3, the entire contents of which are incorporated herein by reference, including methods for generating nucleic acid-based structures.

As described herein, in some embodiments, the nucleic acid container is formed primarily of a single strand of nucleic acid (i.e., a scaffold). For example, in some embodiments, the single strands of nucleic acids used to form the nucleic acid containers constitute 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 particular embodiments, the molecular weight of the single strands of nucleic acid forming the primary structure of the nucleic acid container may have a molecular weight of, for example, 100kDa to 10,000 kDa. In some embodiments, the molecular weight of the single strands of nucleic acid forming the primary structure of the nucleic acid container may be, for example, at least 300kDa, at least 600kDa, at least 640kDa, at least 800kDa, at least 1,000kDa, at least 2,000kDa, at least 4,000kDa, or at least 6,000 kDa. In some cases, the molecular weight of the single strands of nucleic acids forming the primary structure of the nucleic acid container can be, for example, less than 6,000kDa, less than 4,000kDa, less than 2,000kDa, less than 1,000kDa, less than 800kDa, less than 600kDa, or less than 300 kDa. Other molecular weight values are also possible. Combinations of the above ranges are also possible (e.g., molecular weights of at least 300kDa and less than 1,000 kDa).

In some cases, the single strand of nucleic acid used to form the nucleic acid container has a length of, for example, 1000 bases (1 kb) to 300 kilobases (300 kb). The single nucleic acid strand may have a length of, for example, at least 1,000 bases (1 kb), at least 2kb, at least 5kb, at least 10kb, at least 15kb, at least 20kb, at least 25kb, at least 30kb, at least 40kb, at least 50kb, at least 60kb, at least 70kb, at least 100kb, at least 150kb, or at least 200kb in length. In particular embodiments, the nucleic acid single strand may have a length of, for example, less than 200kb, less than 150kb, less than 100kb, less than 70kb, less than 60kb, less than 50kb, less than 40kb, less than 30kb, less than 20kb, less than 10kb, less than 5kb, or less than 3 kb. Combinations of the above ranges are also possible.

Furthermore, it is to be understood that formation of nucleic acid containers using two or more (e.g., 2, 3, 4,5, 6, etc.) nucleic acid single strands that serve as "scaffolds" is also contemplated. The two or more nucleic acid single strands may have molecular weights and/or lengths within the above-described ranges, or they may have different ranges of molecular weights and/or lengths.

In some embodiments, the nucleic acid container may include one or more portions that are open relative to other portions of the structure. For example, in one particular embodiment, the nucleic acid container includes two opposing ends that are open, thereby allowing fluid and particular molecules to flow through the interior thereof. Examples of such structures are shown in fig. 2A and 2B, which fig. 2A and 2B show top and side views, respectively, of a container. In other embodiments, the nucleic acid container may have one open end, and as shown in the embodiments illustrated in fig. 2C and 2D, fig. 2C and 2D show top and side views, respectively, of the container. In other embodiments, the nucleic acid container may comprise more than two openings. In yet other embodiments, the nucleic acid container may be substantially closed, as illustratively shown in fig. 2E and 2F, which show top and side views of the container, respectively, in fig. 2E and 2F. In general, the nucleic acid container can be made sufficiently rigid to maintain its shape in solution and/or under the typical heat and/or other external forces during nanoparticle growth conditions.

The nucleic acid container and the cavity within the nucleic acid container may have any suitable shape. Advantageously, the nucleic acid container may be designed to comprise a cavity with a specific shape, which may serve as a template for forming all parts of the nanoparticle. As described herein, in some embodiments, the shape of the nucleic acid container cavity can be used to form a nanoparticle having a complementary shape. Non-limiting examples of the shape of the nucleic acid container cavity include a tube, a cassette, a barrel, a rectangle, a rod, a "T" shape, an "L" branched structure, a diamond, a star, a square, a parallelogram, a rhomboid, a triangle, a pentagon, a hexagon, and a polyhedron, including shapes substantially similar thereto. Portions of the cavity may be linear in some cases and curved in other cases. In some cases, one or more channels are present in the nanostructure. In some cases, the cavity of the nucleic acid container has a non-spherical shape. In other cases, the cavity of the nucleic acid container has an arbitrary or irregular shape. In some embodiments, the cavity of the nucleic acid container has a symmetrical shape. In some embodiments, the cavity of the nucleic acid container has an asymmetric shape (e.g., no axis of symmetry). It will be appreciated that the nucleic acid container may have a variety of shapes and forms, provided that its structure is suitable for the application in question.

It is understood that the cavities of the nucleic acid container may have the same shape or different shapes compared to the shape of the outer surface of the nucleic acid container. For example, although the cavity of the nucleic acid container may be designed such that it does not have an axis of symmetry, the outer surface of the container may have a shape that does have an axis of symmetry.

The cross-section of the nucleic acid container cavity may have any suitable shape. For example, the cross-section may be in the shape of a rectangle, a bar, a "T," an "L," a branched structure, a diamond, a star, a square, a parallelogram, a triangle, a pentagon, or a hexagon, including substantially similar shapes thereto. Other shapes are also possible. In some cases, the cross-section of the cavity has a non-spherical shape. In some embodiments, each cross-section of the nucleic acid container cavity has a non-spherical shape. In other cases, the cross-section of the cavity has an arbitrary or irregular shape, a symmetric shape, or an asymmetric shape. In a particular embodiment, each cross-section of the cavity has a symmetrical shape. In other embodiments, each cross-section of the cavity has an asymmetric shape.

In some embodiments, a nucleic acid container can have a 3-dimensional shape that includes a plurality of numbers of different sides. For example, the nucleic acid container may have a cavity in the shape of a prism including five sides and a cross section including three sides in its overall shape. In particular embodiments, the cavity of the nucleic acid container may comprise, for example, 3-10 in its overall shape⁶Side (e.g., 3-100, 3-70, 3-50, or 3-30, 3-25, 3-20, 3-15, 6-15, 3-10, 6-10, 3-9, 3-5, 100-10)³、10³-10⁴、10⁴-10⁵Or 10⁵-10⁶One side). In some embodiments, the cavity of the nucleic acid container can 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 particular embodiments, the cavity of the nucleic acid container may have a cross-section that includes, for example, 3-10 in its overall shape⁶Side (e.g., 3-100, 3-70, 3-50, or 3-30, 3-25, 3-20, 3-15, 6-15, 3-10, 6-10, 3-9, 3-5, 100-10)³、10³-10⁴、10⁴-10⁵Or 10⁵-10⁶One side). In some embodimentsThe cavity of the nucleic acid container can 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 ranges are also possible. In some cases, the sides may be non-curved (e.g., linear), although curved sides or faces may also be possible. Advantageously, nucleic acid containers comprising cavities having complex shapes can be formed using the methods described herein and can be used to form nanoparticles having any complex shape.

The angle between the two sides may be, for example, 1 ° -180 ° (e.g., 1 ° -120 °, 1 ° -90 °, or 1 ° -45 °). In some cases, the angle between two sides may be, for example, greater than 1 °, greater than 10 °, greater than 30 °, greater than 45 °, greater than 60 °, greater than 90 °, greater than 120 °, or greater than 150 °. In particular instances, the angle between two sides can be, for example, less than 180 °, less than 150 °, less than 120 °, less than 90 °, less than 60 °, less than 45 °, less than 30 °, or less than 10 °. Other angles are also possible. Combinations of the above ranges are also possible.

The nucleic acid container can comprise any suitable number of open sides. For example, the nucleic acid container can 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.

In some embodiments, the nucleic acid container comprises at least one lid that can be opened or closed (e.g., reversibly or irreversibly). For example, as shown in the embodiments illustrated in fig. 3A and 3B, which show side and perspective views, respectively, the nucleic acid container 20 may include lids 50 and 52 that can be opened or closed. Fig. 3A and 3B show the lid in an open configuration and may be closed by changing the position of the hinge 54. Similarly, the nucleic acid container shown in FIGS. 3C and 3D includes a lid 50 and a hinge 54, the hinge 54 may allow opening and closing of the lid. In some cases, the nucleic acid container includes an openable and closable lid. The nucleic acid container can be designed such that the lid can be switched on or off (e.g., opened or closed) depending on the presence or absence of a particular nucleic acid strand, resulting in different size controllability of the nucleic acid container. For example, in some embodiments, the nucleic acid container can control the diameter of the growing nanoparticle when the lid is open, and the diameter and length of the nanoparticle can be controlled when the lid is closed. Examples of articles and methods relating to nucleic acid containers including lids are described in more detail in U.S. provisional application No. 61/481,542, which is incorporated herein by reference for all purposes.

In particular embodiments, the nucleic acid container may have a suitable configuration for forming nanoparticles that are at least partially hollow. For example, as illustratively shown in fig. 3E and 3F, the nucleic acid container 20 includes an outer wall 27 and an inner wall 55 that define the shape of the cavity 30. The inner wall blocks nanoparticle formation at this region, allowing nanoparticle formation with a cross section in the shape of a ring. It will be appreciated that other configurations of the inner and outer walls of the nucleic acid container are possible to produce more complex shaped nanoparticles that are at least partially hollow.

Different methods can be used to construct the nucleic acid containers shown in FIGS. 3E and 3F. One exemplary method involves direct folding of DNA via a structured DNA nanotechnology method, such as DNA origami or single-stranded sheets as described herein. Another exemplary method is based on self-assembly of different DNA subunits. For example, a DNA tube (e.g., outer wall 27) and a rod (e.g., inner wall 55) may be prepared separately and then assembled together via DNA hybridization or other interaction to form a rod-in-tube (rod) structure, such as that shown in fig. 3E and 3F.

In some embodiments, the lid may comprise at least 1 binding site for binding to a complementary binding site located on at least a portion (e.g., a surface) of the container. The binding sites may allow for secure attachment of the lid to the container, as described in more detail herein. In some cases, the lid or the surface to be closed by the lid of the container 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. In some embodiments, the binding sites may be separated from each other. The binding site may comprise, for example, a nucleic acid strand, although other binding elements may be used.

Although many of the figures show a nucleic acid container having a single cavity, it should be understood that in some embodiments, a nucleic acid container may include more than one cavity (and thus multiple locations for forming multiple nanoparticles). For example, in some cases, a nucleic acid container can have 2-200 chambers (e.g., 2-100, 2-50, 2-20, 2-10, or 2-5 chambers). In some embodiments, the nucleic acid container comprises at least 2, at least 5, at least 10, or at least 15 chambers. In particular embodiments, the nucleic acid container comprises less than 20, less than 15, less than 10, less than 5, or less than 3 chambers. Other numbers of cavities are also possible. Combinations of the above ranges are also possible.

The size of the nucleic acid container cavity may be varied as desired. In some embodiments, the cross-sectional dimension of the cavity (e.g., as measured by the distance between two interior surface portions of the vessel surrounding the cavity) is 2nm to 1 micron (e.g., 2nm to 500nm, or 2nm to 250 nm). In some embodiments, the cross-sectional dimension of the cavity can be, for example, less than or equal to 1 micron, less than or equal to 500nm, less than or equal to 250nm, less than or equal to 100nm, less than or equal to 75nm, less than or equal to 50nm, less than or equal to 40nm, less than or equal to 30nm, less than or equal to 20nm, less than or equal to 10nm, or less than or equal to 2 nm. In particular embodiments, the cavity has a cross-sectional dimension of greater than or equal to 1nm, greater than or equal to 2nm, greater than or equal to 5nm, greater than or equal to 10nm, greater than or equal to 20nm, greater than or equal to 30nm, greater than or equal to 40nm, greater than or equal to 50nm, greater than or equal to 100nm, greater than or equal to 500nm, or greater than or equal to 1 micron. Other values are also possible. Combinations of the above ranges are also possible. In some embodiments, the above-described cross-sectional dimension of the lumen is the largest cross-sectional dimension of the lumen.

Although primarily described with nucleic acid containers and chambers of nucleic acid containers generally sized on the nanometer scale, in some embodiments, a variety of nucleic acid containers having significantly different sizes may be used. For example, in some embodiments, multiple containers are used, some of which are small enough to be positioned partially or fully within other containers.

The wall thickness of the nucleic acid container can also be varied as desired. In some embodiments, the nucleic acid container comprises walls surrounding the cavity, and the average thickness of the walls can be from 1nm to 1 micron (e.g., from 1nm to 500nm, or from 1nm to 250 nm). The average thickness of the walls can be, for example, less than or equal to 1 micron, less than or equal to 500nm, less than or equal to 250nm, less than or equal to 100nm, less than or equal to 75nm, less than or equal to 50nm, less than or equal to 40nm, less than or equal to 30nm, less than or equal to 20nm, less than or equal to 10nm, or less than or equal to 1 nm. In particular embodiments, the average thickness of the walls is greater than or equal to 1nm, greater than or equal to 10nm, greater than or equal to 25nm, greater than or equal to 50nm, greater than or equal to 100nm, greater than or equal to 500nm, or greater than or equal to 1 micron. Other values are also possible. Combinations of the above ranges are also possible.

In some embodiments, the walls of the nucleic acid container can be formed from more than one layer of nucleic acid. Increasing the number of nucleic acid layers can increase the rigidity (and thickness) of the wall. In some embodiments, the increased wall rigidity may result in a wall of the container that maintains its shape during growth of the nanostructures within the container. For example, the increased wall rigidity may constrain the enlargement of the nanostructures grown within the container (e.g., by compressing the nanostructures during growth), such that the nanostructures do not grow beyond the size of the container cavity prior to enlargement. In other embodiments, a smaller number of layers may allow the nanostructures to grow beyond the size of the container cavity prior to expansion, and may cause the container walls to expand (e.g., bend) during nanostructure growth. In some embodiments, the walls of the nucleic acid container can 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 the nucleic acid container can 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 ranges are also possible. The layers of the walls may have any suitable design, such as a square grid design or a honeycomb design.

Advantageously, by controlling the wall thickness of the nucleic acid container, diffusion kinetics, such as diffusion kinetics of ions or molecules across the container wall, can be controlled. Such control may allow fine tuning of the growth kinetics and/or material composition of the nanoparticles of the nanoparticle alloy. For example, in some cases, a first portion of a wall of a 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 ranges above. The first thicker wall portion may, for example, block the first nanoparticle precursor solution from entering the chamber to a greater extent than the second nanoparticle precursor solution, thereby allowing more of the second nanoparticle precursor solution to enter the chamber at the first wall portion. Thus, the nanoparticles may have a higher amount of material formed by the second nanoparticle precursor at the first wall portion.

Those of ordinary skill in the art are familiar with techniques for determining structure and particle size. Examples of suitable techniques include Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM), scanning electron microscopy, resistive (electroluminescence) counting, and laser diffraction. Other suitable techniques are known to those of ordinary skill in the art. Although many methods for determining the size of nanostructures are known, the sizes (e.g., cross-sectional dimensions, thickness) described herein refer to sizes measured by transmission electron microscopy.

The nucleic acid container can also include other components such as those described herein (e.g., labels, binding sites, quantum dots, nanoparticles, nucleic acids, proteins, etc.). For example, in some embodiments, the nucleic acid container includes one or more inorganic structures (e.g., inorganic nanostructures), such as inorganic nanoparticles or quantum dots. In some embodiments, one or more components may fill portions of the cavity, e.g., such that nanoparticles formed within the cavity by template synthesis form around or combine with the components. In some cases, the component is incorporated into the nanoparticle to be formed. In other embodiments, the inorganic structure is embedded in the nucleic acid container wall and does not fill a portion of the cavity.

Any suitable nanoparticle precursor may be used to form the nanoparticles as described herein. Nanoparticle precursors can be used to initiate, catalyze and/or grow nanoparticles. In some cases, all or a portion of the material of the nanoparticle precursor can be incorporated into the resulting nanoparticles. In some embodiments, nanoparticle precursors can be used to form nanoparticles using a seed-mediated growth process. In some such embodiments, the nanoparticle precursor may be in solid form. For example, the nanoparticle precursor may be in the form of nanoparticles, such as inorganic nanoparticles. In some cases, the nanoparticle precursor comprises crystals. In particular instances, the 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, the nanoparticle precursor comprises a semiconductor (e.g., Rh, Ge, silicon compounds and alloys, cadmium selenide, cadmium sulfide, indium arsenide, and indium phosphide). In some cases, the nanoparticle precursor can include group II-VI (e.g., IV-VI) elements. In particular 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 doping compound. Combinations of these and other materials are also possible.

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 precursors. Alternatively, Ag, Pt and Pd nanoparticles may be used as precursors. For semiconductor and metal oxide nanoparticles (e.g., including Cd, Cr, Co, Ti, Zn, Mn, Ni, Mg, Fe or different materials described herein)One or more of) the corresponding material, clusters of the corresponding material or small-sized nanoparticles may be used as seeds. In other embodiments, enzymes or peptides may serve as nucleation sites if they can trigger the growth of nanoparticles (e.g., organic nanoparticles). Some catalysts such as Pt (PPh)₃）₂Cl₂May also be used to trigger the growth of polymers or nanoparticles.

The nanoparticle precursors can also be in the form of a solution, as described herein. The material composition of the resulting nanoparticles can be controlled by varying the type of precursor solution used. Any suitable solution may be used to form the nanoparticles, and may be selected using the instructions provided herein in combination with the general knowledge in the art. For example, HAuCl₄The precursor solution can be used to form gold nanoparticles, and AgNO₃The precursor solution may be used to form silver nanoparticles (optionally in combination with other solutions such as ascorbic acid). The combination of nanoparticle precursors and precursor solutions can also be determined by one of ordinary skill in the art in combination with the description provided herein.

In other embodiments, nanoparticle precursors can be used to form nanoparticles using a non-seed mediated growth process. For example, in some embodiments, the nanoparticle precursor may be in the form of a monomer or polymer, and formation of the nanoparticles may include polymerizing and/or crosslinking monomer and/or polymer units.

In some cases, the nanoparticle precursor can comprise an amino acid, a peptide, a nucleotide, or a nucleic acid (e.g., to form a nanoparticle that is substantially formed of an amino acid, a peptide, or a nucleic acid).

In other cases, the nanoparticle precursor may include other functionalities, such as binding sites, or may impart specific surface functionalities. For example, nanoparticle precursors can comprise a self-assembled monolayer to impart a specific surface chemistry to the precursor. In particular embodiments, the nanoparticle precursor may include a binding site or other suitable component to allow it to attach 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 the surface of the container.

The nanoparticle precursor can have any suitable size. In some embodiments, the nanoparticle precursor has at least one cross-sectional dimension that is between 0.5nm and 1 micron (e.g., between 0.5nm and 500nm, or between 0.5nm and 250 nm). In some embodiments, the 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 500nm, less than or equal to 250nm, less than or equal to 100nm, less than or equal to 75nm, less than or equal to 50nm, less than or equal to 40nm, less than or equal to 30nm, less than or equal to 20nm, less than or equal to 10nm, less than or equal to 5nm, less than or equal to 1nm, or less than or equal to 0.5 nm. In some embodiments, the nanoparticle precursor has at least one cross-sectional dimension that is greater than or equal to 0.5nm, greater than or equal to 1nm, greater than or equal to 2nm, greater than or equal to 5nm, greater than or equal to 10nm, greater than or equal to 20nm, or greater than or equal to 50 nm. Other sizes are also possible. Combinations of the above ranges are also possible.

In some cases, the size (e.g., volume) of the 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 cavity of the nanoparticle or container formed from the precursor. Other sizes are also possible.

In particular embodiments, the nanoparticle precursor has an average molecular weight of, for example, 20Da to 10kDa (e.g., 20Da to 5kDa, or 20Da to 1 kDa). The nanoparticle precursor has an average molecular weight of, for example, less than or equal to 10kDa, less than or equal to 5kDa, less than or equal to 1kDa, less than or equal to 500Da, less than or equal to 200Da, less than or equal to 100Da, less than or equal to 50Da, or less than or equal to 20 Da. In some embodiments, the nanoparticle precursor has an average molecular weight greater than or equal to 10Da, greater than or equal to 20Da, greater than or equal to 50Da, greater than or equal to 100Da, greater than or equal to 200Da, greater than or equal to 500Da, greater than or equal to 1kDa, or greater than or equal to 10 kDa. Other molecular weights are also possible. Combinations of the above ranges are also possible.

As described herein, nanoparticles having unique and/or predetermined shapes can be formed using the methods described herein. For example, a nanoparticle having a particular shape (and/or size) can be formed by designing the cavity of the nucleic acid container to have the complement of the desired shape (and/or size) of the nanoparticle. The cavity may then serve as a template for forming all portions of the nanoparticle. Non-limiting examples of shapes of nanoparticles include tubes, boxes, barrels, rectangles, rods, "T" -shaped, "L" -shaped branched structures, diamonds, stars, squares, parallelograms, triangles, pentagons, hexagons, polyhedrons, and rings, including shapes substantially similar thereto. In some cases, the nanoparticles have a non-spherical shape. In other cases, the nanoparticles have an arbitrary or irregular shape. In some embodiments, the nanoparticles have a symmetrical shape. In some embodiments, the symmetric shape can include at least 1, at least 2, at least 3, or at least 4 axes of symmetry. In some embodiments, the nanoparticles have an asymmetric shape (e.g., no axis of symmetry).

In some embodiments, the nanoparticles formed by the methods described herein are solid or solid-like (e.g., have a solid core), and are not hollow structures. In other embodiments, portions of the nanoparticles may be hollow, e.g., as described herein with respect to fig. 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 open).

The cross-section of the nanoparticles may have any suitable shape. For example, the cross-section may be in the shape of a rectangle, a bar, a "T," an "L," a branched structure, a diamond, a star, a square, a parallelogram, a triangle, a pentagon, a hexagon, or a toroid, including shapes substantially similar thereto. Other shapes are also possible. In some cases, the cross-section of the nanoparticle has a non-spherical shape. In some embodiments, each cross-section of the nanoparticle has a non-spherical shape. In other cases, the cross-section of the nanoparticle has an arbitrary or irregular shape, a symmetric shape, or an asymmetric shape. In a particular embodiment, each cross-section of the nanoparticle has a symmetrical shape. In other embodiments, each cross-section of the nanoparticle has an asymmetric shape.

In some embodiments, the nanoparticles can have a 3-dimensional shape that includes a plurality of numbers of different sides. For example, the nanoparticles may be in the shape of prisms comprising four sides. In particular embodiments, the nanoparticles can include, for example, 3-10⁶Side (e.g., 3-100, 3-70, 3-50, or 3-30, 3-25, 3-20, 3-15, 6-15, 3-10, 6-10, 3-9, 3-5, 3-8, 20-50, 50-100, 100-10)³、10³-10⁴、10⁴-10⁵Or 10⁵-10⁶One side). In some cases, the nanoparticle comprises 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 10³At least 10⁴Or at least 10⁵And different sides.

As described herein, in some embodiments, a nanoparticle comprises at least one surface portion having a shape that is complementary to a shape of an interior surface portion of a nucleic acid container. A surface portion generally refers to a surface portion having a surface area greater than the surface area of a single atom. In some cases, the surface portion can have at least 1nm²At least 2nm²At least 5nm²At least 10nm²At least 15nm²At least 20nm²At least 25nm²At least 30nm²At least 50nm²At least 200nm²At least 200nm²At least 500nm²Or at least 1000nm²Wherein the largest surface portion is the surface area of the entire nanoparticle.

In some cases, the nanoparticle has at least one surface portion that is complementary in shape to an interior surface portion of the nucleic acid container at a sub-nanometer (e.g., 0.5 nm) level. For example, the growth of nanoparticles within the container may stop or significantly slow down upon reaching the boundary of the inner surface portion of the container. In some such embodiments, the surface chemical and/or physical interaction between the nanoparticle and the interior 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 nanoparticles or nanoparticle precursors can prevent further growth of the nanoparticles. The particular orientation of the atoms of the inner surface portion of the nucleic acid container, wherein the shapes of the inner surface portion and the surface portion of the resulting nanoparticle are complementary, e.g., at a sub-nanometer level, may determine the final shape of the resulting nanoparticle.

In some embodiments, a relatively high percentage of the surface area of the nanoparticle is complementary to the shape of the interior surface (e.g., cavity) of the nucleic acid container at a 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 nanoparticle surface area can be complementary to the shape of the interior surface of the nucleic acid container on a sub-nanometer level. In other embodiments, 100% of the nanoparticle surface area may be complementary to the shape of the inner surface of the nucleic acid container on a sub-nanometer level.

In other embodiments, the resulting nanoparticles have at least one surface portion that is complementary in shape to an interior surface portion of the nucleic acid container on a nanoscale (e.g., 1nm or greater) level. For example, the growth of nanoparticles within the container may stop or significantly slow before reaching the boundary of the interior surface portion of the container, such that the resulting nanoparticles have a volume less than the volume of the cavity. In some such embodiments, the nanoparticles have a shape substantially similar to the shape of the cavity and are complementary to the interior walls of the container on a nanoscale level, but are not complementary to the interior walls of the container on a sub-nanometer level.

In some embodiments, a relatively high percentage of the surface area of the nanoparticles is complementary to the shape of the interior surface portion of the nucleic acid container on a nanoscale level (e.g., 1nm or greater). 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 the shape of the interior surface portion of the nucleic acid container on a nanoscale level. In other embodiments, 100% of the nanoparticle surface area may be complementary to the shape of the interior surface portion of the nucleic acid container on a nanoscale level.

Nanoparticles having complementarity on both the molecular (e.g., angstrom) and nanoscale levels for different portions of the nanoparticle are also possible.

In still other embodiments, the resulting nanoparticles comprise one or more surface portions that are not complementary to an interior surface portion of the nucleic acid container. For example, constructing a nanoparticle in a nucleic acid container that includes two open ends can result in a nanoparticle having a middle portion that is partially complementary (e.g., at the molecular level or at the nanoscale level) to an interior surface of the nucleic acid container, but has ends that are not complementary to any portion of the nucleic acid container. In some such embodiments, the nanoparticles may grow out of the cavity of the nucleic acid container and may be shaped by other factors.

In a particular embodiment, the nanoparticle comprises at least two opposing surface portions each having a shape complementary to the shape of the interior surface portion of the nucleic acid container. Complementarity may be at the molecular level as described above or at the nanoscale level. For example, as shown in the embodiment illustrated in FIG. 1A, two opposing surface portions of the nucleic acid container cavity are shown as inner surface portions 26A and 26B. In some embodiments, the opposing surface portions may be parallel to each other. After the nanoparticles 38 are formed, the surface portions of the nanoparticles at these portions may be complementary to the interior surface portions 26A and 26B of the nucleic acid container.

In other embodiments, the nanoparticle comprises at least two adjacent surface portions each having a shape complementary to the shape of the interior 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 formation of the nanoparticles 38. Accordingly, the nanoparticles 38 may include adjacent surface portions that are complementary to the interior surface portions of the cavity at these locations.

The nanoparticles may be formed of any suitable material. In some embodiments, the nanoparticles formed by the methods described herein may be inorganic nanoparticles. In some cases, the nanoparticles comprise 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, the nanoparticles comprise a semiconductor (e.g., Rh, Ge, silicon compounds and alloys, cadmium selenide, cadmium sulfide, indium arsenide, and indium phosphide). In some cases, the nanoparticles may include group II-VI (e.g., IV-VI) elements. In particular embodiments, the nanoparticles comprise a metal oxide or a metal fluoride. In some cases, the nanoparticles comprise an alloy. In some cases, the nanoparticles comprise a doping compound. Combinations of these and other materials are also possible. The nanoparticles may be electrically and/or thermally conductive in some embodiments, or non-electrically and/or non-thermally conductive in other embodiments.

In other embodiments, the nanoparticles formed by the methods described herein may be organic nanoparticles. In some cases, the nanoparticles may comprise a polymer, which may be crosslinked 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 polymethacrylates and degradable polymers such as polylactic acid, polyglycolic acid and copolymers thereof. Examples of natural polymers include hyaluronic acid, chitosan, and collagen. In some embodiments, a conductive polymer may be used. In particular embodiments, the nanoparticle does not include a polymeric material (e.g., it is non-polymeric). In some cases, the nanoparticle may comprise a protein, an enzyme, or a peptide.

The surface of the nanoparticle may include materials for forming the interior portion of the nanoparticle, or may additionally impart specific surface functionalities such as binding sites or other components. For example, the nanoparticles may comprise a self-assembled monolayer to impart a specific surface chemistry to the nanoparticles. In some cases, the nanoparticle surface may be passivated by one or more chemicals to promote attachment of the components.

The nanoparticles may be of any suitable size. In some embodiments, the nanoparticles have at least one cross-sectional dimension (or at least two cross-sectional dimensions) that is between 2nm and 1 micron (e.g., between 2nm and 500nm, or between 2nm and 250 nm). In some embodiments, the nanoparticles have at least one cross-sectional dimension (or at least two cross-sectional dimensions) that is, for example, less than or equal to 1 micron, less than or equal to 500nm, less than or equal to 250nm, less than or equal to 100nm, less than or equal to 75nm, less than or equal to 50nm, less than or equal to 40nm, less than or equal to 30nm, less than or equal to 20nm, less than or equal to 10nm, or less than or equal to 5 nm. In particular embodiments, the nanoparticles have at least one cross-sectional dimension (or at least two cross-sectional dimensions) that is greater than or equal to 2nm, greater than or equal to 5nm, greater than or equal to 10nm, greater than or equal to 50nm, or greater than or equal to 100 nm. Combinations of the above ranges are also possible. In some cases, the cross-sectional dimension is a maximum cross-sectional dimension.

In some cases, the nanoparticles have, for example, 8nm³-1μm³（1*10⁹nm³) The volume of (a). In particular embodiments, the nanoparticles have, for example, at least 20nm³At least 50nm³At least 100nm³At least 500nm³At least 1X 10³nm³At least 5X 10³nm³At least 1X 10⁴nm³At least 5X 10⁴nm³At least 1X 10⁵nm⁴At least 5X 10⁵nm³At least 1X 10⁶nm³At least 5X 10⁶nm³At least 1X 10⁷nm³At least 5X 10⁷nm³At least 1X 10⁸nm³Or at least 5X 10⁸nm³The volume of (a). In some embodiments, the nanoparticles have, for example, less than 1 x 10⁹nm³Less than 5X 10⁸nm³Less than 1X 10⁸nm³Less than 5X 10⁷nm³Less than 1X 10⁷nm³Less than 5X 10⁶nm³Less than 1X 10⁶nm³Less than 5X 10⁵nm³Less than 1X 10⁵nm³Less than 5X 10⁴nm³Less than 1X 10⁴nm³Less than 5X 10³nm³Less than 1X 10³nm³Less than 500nm³Less than 100nm³Less than 50nm³Or less than 20nm³The volume of (a). Other ranges are also possible. Combinations of the above ranges are also possible. In particular embodiments, such volumes are based on the use of a single nucleic acid scaffold. Larger volumes may be possible using multiple nucleic acid scaffolds.

As described herein, the nanoparticles can have a size, dimension (e.g., length, width, height), cross-sectional dimension, and/or volume substantially similar to that of the vessel cavity in which the nanoparticles are grown. In other embodiments, the nanostructures may have a size, dimension (e.g., length, width, height), cross-sectional dimension, and/or volume that is less than that of the container cavity in which the nanoparticles are grown. For example, the growth of the nanoparticles may be stopped before the nanoparticles reach one or more sides of the container. In other embodiments, the nanostructures may have a size, dimension (e.g., length, width, height), cross-sectional dimension, and/or volume that is greater than that of the container cavity in which the nanoparticles are grown. For example, the walls of the container may be designed to be elastic, so that nanoparticles growing therein cause the walls of the container to expand during the growth process. Other configurations of nanoparticles and/or containers are also possible.

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 the nanoparticle precursor used to form the nanoparticle. The volume of the resulting nanoparticle may depend, at least in part, on the size of the nucleic acid container cavity, which may be varied as described herein. It is understood that nanoparticles having a volume less than or greater than the nucleic acid container cavity are also possible. For example, portions of the nanoparticles may be constructed within the container and other portions of the nanoparticles may be grown outside of the container.

In some cases, the nanoparticles 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. Other values of aspect ratio are also possible. As used herein, "aspect ratio" refers to the ratio of length to width, where length and width are measured perpendicular to each other, and length refers to the longest linear measurement.

As described herein, in some cases, all or a portion 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 nanoparticle surface area can be encapsulated or coated by the nucleic acid nanostructures. In some cases, the entire surface area of the nanoparticle is encapsulated or coated by the nucleic acid nanostructure. In some such embodiments, all or a portion of the nanoparticle may be attached to the nucleic acid nanostructure. For example, all or a portion of the nanoparticles can be physically adsorbed onto the nucleic acid nanostructures. In other embodiments, all or a portion of the nanoparticle is not attached to the nucleic acid nanostructure, but is only adjacent to the nucleic acid nanostructure. The nanoparticles may or may not be in contact with the nucleic acid nanostructures during the encapsulation process.

Also provided are compositions comprising nanoparticles having one or more of the features described herein. For example, in some cases, the 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, do not have an axis of symmetry, are partially complementary (at the molecular level or at the atomic level) to an interior surface of the nucleic acid container, or are encapsulated/coated by the nucleic acid container.

In another set of embodiments, one or more surfaces of the nanoparticles can be patterned (e.g., in two dimensions and/or three dimensions) from one or more components. This may be accomplished, for example, by structuring a pattern of one or more components on the interior surface of the nucleic acid container used to form the nanoparticles, and then triggering the growth of the nanoparticles within the container. The growth of the nanoparticles within the container may result in the mode of incorporation of one or more components onto the surface of the nanoparticles. Using this approach, nanoscale structures can be constructed with details on the nanometer or sub-nanometer level. Examples of such structures include metal coins and arbitrarily shaped nanoscale electronic devices. Other structures such as core-shell structures in the form of alloys or polymer/metal hybrid structures may 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 pentagonal core. The methods described herein are a more rational way to construct any pre-designed structure than certain prior methods.

In some embodiments, the structured nanoparticles can be used as templates to structure other materials that cannot be prepared directly by DNA-directed synthesis (e.g., those that require high temperature or pressure, and/or those that are formed in organic solutions). In some embodiments, the at least partially hollow nanoparticles formed by the methods described herein can be used as a template to form secondary nanostructures within the hollow portions (e.g., cavities) of the nanoparticles. In other embodiments, the outer surface of the nanoparticle may serve as a template to form larger secondary nanostructures. The nanoparticles used as templates may optionally be removed after the formation of the secondary nanostructures. Nanoparticles formed from or comprising, for example, gold, silver or platinum may be suitable for use as templates. Other materials for use as a template may also be possible. The secondary nanostructures formed using the nanoparticles as templates 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.

The hollow portions (e.g., cavities) of the nanoparticles and/or secondary nanostructures formed using the nanoparticles may be of any suitable size. In some embodiments, the hollow portion (e.g., cavity) of the nanoparticle and/or secondary nanostructure has at least one cross-sectional dimension (or at least two cross-sectional dimensions) that is between 1nm and 1 micron (e.g., between 1nm and 500nm, or between 1nm and 250 nm). In some embodiments, the hollow portion (e.g., cavity) of the nanoparticle and/or secondary nanostructure has at least one cross-sectional dimension (or at least two cross-sectional dimensions) that is, for example, less than or equal to 1 micron, less than or equal to 500nm, less than or equal to 250nm, less than or equal to 100nm, less than or equal to 75nm, less than or equal to 50nm, less than or equal to 40nm, less than or equal to 30nm, less than or equal to 20nm, less than or equal to 10nm, or less than or equal to 5 nm. In particular embodiments, the hollow portion (e.g., cavity) of the nanoparticle and/or secondary nanostructure has at least one cross-sectional dimension (or at least two cross-sectional dimensions) that is greater than or equal to 2nm, greater than or equal to 5nm, greater than or equal to 10nm, greater than or equal to 50nm, or greater than or equal to 100 nm. Combinations of the above ranges are also possible. In some cases, the cross-sectional dimension is a maximum cross-sectional dimension.

In some cases, the hollow portion of the nanoparticle and/or secondary nanostructure has, for example, 1nm³-1μm³（1*10⁹nm³) The volume of (a). In particular embodiments, the hollow portion of the nanoparticle and/or secondary nanostructure has, for example, at least 20nm³At least 50nm³At least 100nm³At least 500nm³At least 1X 10³nm³At least 5X 10³nm³At least 1X 10⁴nm³At least 5X 10⁴nm³At least 1X 10⁵nm⁴At least 5X 10⁵nm³At least 1X 10⁶nm³At least 5X 10⁶nm³At least 1X 10⁷nm³At least 5X 10⁷nm³At least 1X 10⁸nm³Or at least 5X 10⁸nm³The volume of (a). In some embodiments, the hollow portion of the nanoparticle and/or secondary nanostructure has, for example, less than 1 x 10⁹nm³Less than 5X 10⁸nm³Less than 1X 10⁸nm³Less than 5X 10⁷nm³Less than 1X 10⁷nm³Less than 5X 10⁶nm³Less than 1X 10⁶nm³Less than 5X 10⁵nm³Less than 1X 10⁵nm³Less than 5X 10⁴nm³Less than 1X 10⁴nm³Less than 5X 10³nm³Less than 1X 10³nm³Less than 500nm³Less than 100nm³Less than 50nm³Or less than 20nm³The volume of (a). Other ranges are also possible. Combinations of the above ranges are also possible.

As described herein, nanoparticles that may optionally be coated or encapsulated by a nucleic acid container or nanostructure may have a variety of different shapes. In some embodiments, the unique shape of the nanoparticle may be used to localize one or more components (e.g., binding sites, labels, ligands, etc.) on different portions of the nanoparticle surface and/or the nucleic acid container surface to allow the nanoparticle or nanostructure to be addressed differently. For example, a nanoparticle having eight different sides can be functionalized with one or more different components on each of the eight different sides. In some cases, the components are in the form of separate components that can be added at unique locations on the nanoparticles or nanostructures. For example, a single discrete component may be positioned on a single side of the nanoparticle, where different sides of the nanoparticle each include a different single discrete component. These and other embodiments can be used to detect a variety of different targets, as described in more detail below.

In some embodiments, the location of the components on the precise location of the nanoparticles and/or nanostructures may be controlled by the particular chemistry of the nucleic acid container. For example, a nanoparticle having eight different sides may be surrounded (partially or completely) by a nucleic acid container having a lumen with eight different sides, and walls of different chemistry on each of the different sides. The composition may be designed such that it has an affinity for a particular part of the nucleic acid container or nanostructure on one of the sides of the coated or encapsulated nanoparticle side. Thus, by designing the nucleic acid container to have a specific sequence or chemistry at a specific location, the addition of a specific component to the surface of the nucleic acid container and/or the surface of the nanoparticle at one of these locations can be performed. Similarly, additional components may be specifically added to different locations on the surface of the nucleic acid container and/or the surface of the nanoparticle using this method.

As described herein, the nucleic acid container is generally porous such that small pores or holes through the wall thickness 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 may be designed to include a specific nucleic acid sequence, hydrophilicity, hydrophobicity, charge, and/or size that facilitates localization of a specific component into the pores or onto the container surface. Likewise, the component to be added may be designed to include a complementary nucleic acid sequence, hydrophilic, hydrophobic, charge, and/or size such that it has an affinity for a particular portion of the nucleic acid container. In some cases, after the component is matched to a particular portion of the nucleic acid container, the component may be inserted all the way through the aperture such that it contacts a surface portion of the nanoparticle.

In some such embodiments, the ends of the components may have a specific chemistry that allows them to attach to the surface of the nanoparticle. For example, the component may be functionalized with a thiol that allows it to be physically adsorbed to gold nanoparticles. In some embodiments in which the component is directly attached to the nanoparticle, all or part of the nucleic acid container surrounding the nanoparticle (partially or completely) can optionally be removed while the component remains attached to the surface of the nanoparticle. However, in other embodiments, the nucleic acid container may remain surrounding the nanoparticle even after the components are attached. 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 the surface of the nanoparticle and a portion of the nucleic acid container is also possible. Any suitable attachment method, e.g., to the surface of the nanoparticle and/or a portion of the nucleic acid container, can be used, such as covalent bonding, physisorption, chemisorption, or attachment by ionic interactions, hydrophobic and/or hydrophilic interactions, electrostatic interactions, van der waals interactions, or combinations thereof.

The components may have any suitable orientation with respect to the nanoparticle or nucleic acid container. For example, the components may be oriented substantially perpendicular to a wall of the nanoparticle or nucleic acid container. In some embodiments, the components may be oriented at a particular angle or range of angles (e.g., 0 ° -90 °,0 ° -15 °, 15 ° -45 °, 45 ° -60 °, or 60 ° -90 °) with respect to the walls of the nanoparticle or nucleic acid container.

In particular embodiments, inorganic nanoparticles comprising an isolated nucleic acid strand attached to the surface of an inorganic nanoparticle are provided, wherein the inorganic nanoparticle has a non-spherical shape. In certain embodiments, an inorganic nanoparticle coated by a nucleic acid container is provided, wherein the nucleic acid container comprises pores. The binding sites, e.g., isolated nucleic acid strands, may be attached to the surface of the inorganic nanoparticles and may extend from the surface of the inorganic nanoparticles through the pores of the nucleic acid container. In some cases, the length of the binding site (e.g., an isolated nucleic acid strand) is longer than the thickness of the nucleic acid container, such that the binding site extends outward from the nucleic acid container.

Control of many different parameters may be provided by using the localization of unique chemical directing components of the nucleic acid container (directly to the surface of the nanoparticle or nucleic acid container) around all or part of the nanoparticle. FIGS. 4A-4D show examples of different parameters that can be controlled when components are added to nucleic acid nanostructures and/or nanoparticles.

As shown in the embodiment illustrated in fig. 4A, the plurality of components 60 may vary along one or more surface portions of the nucleic acid container and/or nanoparticle. For example, in some embodiments, a single isolated component 60 may be positioned on a single side of a nucleic acid container and/or nanoparticle. In other embodiments, the two separate components may be positioned on a single side of the nucleic acid container and/or nanoparticle. In still other embodiments, three or more components 60 may be positioned on a single side of the nucleic acid container and/or nanoparticle.

As shown in the embodiment illustrated in fig. 4B, the distance between two or more components may also be controlled. For example, first component 60 and second component 62 can be positioned relatively distant from each other on the sides of the nucleic acid container and/or nanoparticle. In other embodiments, the two components may be positioned relatively close to each other, or on opposite sides of the nucleic acid container and/or nanoparticle.

Conformational control may also be provided as shown in the embodiment illustrated in fig. 4C. For example, the positioning of different components at unique locations on one or more sides of a nucleic acid container and/or nanoparticle may allow the components to interact with each other and/or portions of the nucleic acid container in order to provide a particular structural configuration of the components and/or alter the structural configuration of the container.

Furthermore, as shown in the embodiment illustrated in fig. 4D, the length of the components and/or the distance of the components from the surface of the nanoparticles may also be controlled. For example, if it is desired to include a component 60 positioned a short distance from the surface of the nanoparticle 38, a nucleic acid container having relatively thin walls may be used. If it is desired to include the component 20 relatively further away from the surface of the nanoparticle 38, a relatively thick wall may be used. In some embodiments, a relatively thick wall may be obtained by coating all or a portion of the nucleic acid container 20 with the second layer 70. 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 the component 60 can be varied to control the distance between the component ends and the surface of the nucleic acid container and/or the surface of the nanoparticle.

It should be understood that the types of parameters that can be controlled as shown in fig. 4A-4D are merely examples, and that other parameters may be possible in terms of the positioning of the components relative to the nucleic acid container and/or nanoparticles. In addition, it is understood that homogeneous or heterogeneous multiple components (e.g., binding sites) can be positioned on the same surface in a controlled conformation or relative orientation.

Fig. 4E and 4F show examples of how the surface addressability of a structure may be used to form components (e.g., higher level structures) using a particular orientation of the structure. For example, a nucleic acid container 20 having different sides a-f can include nanostructures 37 attached to the interior surface of the container. The nanostructures may fill a portion of the nucleic acid container cavity. The nanoparticles 38 may be formed within the cavity as described herein using the nucleic acid container and the nanostructures 37 as a template. The components 60 on each side of the structure may be unique and designed to bind specific components on the sides of other structures to form an assembly having a particular configuration, as illustratively shown in fig. 4F. Additional examples of components that use surface-specific interactions are described in more detail below.

In particular embodiments involving the positioning of the separation components on the nucleic acid container and/or nanoparticle, the components may be "separated" in the following sense: it is located a specific distance away from another component attached to the same nucleic acid container and/or nanoparticle, such that the separated component can be uniquely identified and distinguished from the other component (e.g., on a nanoscale level). In some cases, separating a component may facilitate the binding or attachment of other entities to the component because it is separate and avoids or reduces the amount of steric interaction with other nearby components attached to the same nucleic acid container and/or nanoparticle.

In some cases, a first component (e.g., an isolated component) is positioned at a distance of at least 2nm, at least 5nm, at least 10nm, at least 15nm, at least 20nm, at least 30nm, at least 40nm, or at least 50nm away from a nearest second component that is attached to the same nucleic acid container and/or nanoparticle. In particular instances, a first component (e.g., a separated component) is positioned at a distance of less than or equal to 500nm, less than or equal to 200nm, less than or equal to 100nm, less than or equal to 50nm, less than or equal to 40nm, less than or equal to 30nm, less than or equal to 20nm, less than or equal to 15nm, less than or equal to 10nm, or less than or equal to 50nm away from a nearest second component. Other distances are also possible. Combinations of the above ranges are also possible. In other embodiments, the 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 separated and/or indistinguishable from one another (e.g., on a nanometer scale).

Any suitable number of components (whether separate or not) can be attached to the nucleic acid container and/or the nanoparticle. In some embodiments, the nucleic acid nanostructures and/or nanoparticles may include, for example, 2-500 components (e.g., 2-100, 2-50, or 2-20 components). In some embodiments, the nucleic acid nanostructures and/or nanoparticles may comprise 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 nanostructures and/or nanoparticles comprise 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, the components are each separated from each other as described herein. Other numbers of components are also possible. Combinations of the above ranges are also possible.

As described herein, two or more components positioned on the surface of a nucleic acid container and/or nanoparticle can be positioned in any suitable orientation with respect to each other. In some embodiments, at least two components are attached to opposing surface portions of the nucleic acid container and/or nanoparticle. In other embodiments, the at least two components are located on adjacent surface portions of the nucleic acid container and/or the nanoparticle. In some cases, the first component is positioned on a surface of the nanoparticle and the second component is attached to a surface of the nucleic acid container. Other configurations and orientations are also possible.

The methods described herein may be used to "print" components such as proteins, organic molecules, and inorganic nanoparticles onto the surface of nanoparticles formed by the methods described herein. The components may first be disposed in a predesigned pattern onto the inner surface of the nucleic acid container and later transferred to the outer surface of the nanoparticles grown within the nucleic acid container by the interactions described herein (e.g., physisorption, covalent bonding, van der waals interactions, etc.) while maintaining the predesigned pattern. For example, nanoparticles grown within the container may reach the inner walls of the container where they contact the component and allow the component to attach to the nanoparticle surface. In some embodiments, the components may remain on the surface of the nanoparticle even after the nucleic acid container is removed. In other embodiments, the components may be attached to staple chains (e.g., nucleic acids) that hybridize to the container holder and hold the holder in the target shape. Since different staple chains may be used to hold different parts of the container together, the staple chains may be individually activated for attaching different components.

A variety of different components may be attached to the surface of the nucleic acid container and/or nanoparticle as described herein. In some embodiments, the component comprises a binding site. Sometimes, a component may contain two binding sites-one for the nanoparticle and one for the target separate from the nanoparticle. The term "binding" refers to an interaction, typically a specific or non-specific binding or interaction, including biochemical, physiological and/or pharmaceutical interactions, between a corresponding pair of molecules that exhibit affinity or binding capability for each other. Biological binding defines the type of interaction that occurs between pairs of biomolecules, including proteins, peptides, nucleic acids, glycoproteins, carbohydrates, hormones, and the like. Specific examples include antibodies/antigens, antibodies/haptens, enzymes/substrates, enzymes/inhibitors, enzymes/cofactors, binding proteins/substrates, carrier proteins/substrates, lectins/carbohydrates, receptors/hormones, receptors/effectors, complementary strands of nucleic acids, proteins/nucleic acid repressors/inducers, ligands/cell surface receptors, viruses/ligands, aptamers/proteins, and the like. Such molecules are examples of components that may be used with the nanoparticles and nucleic acid containers described herein.

In some embodiments, the binding site comprises a nucleic acid. For example, the nucleic acid can 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-stranded and double-stranded portions are also possible. Examples of nucleic acids are provided in more detail below.

In particular embodiments, the component comprises a label. Examples of labels include readout (or detectable) labels, such as luminescent probes, fluorophores or molecules or compounds of fluorophore labels, chromophores or molecules or compounds of chromophore labels, and the like. In some cases, the label comprises a nanoparticle (e.g., a quantum dot). In particular embodiments, the label comprises a reporter, such as a Surface Enhanced Raman Scattering (SERS) reporter.

In some embodiments, the labels and corresponding binding sites are attached to the surface of the nanoparticles and/or nucleic acid containers. The label/binding site pair may be specific and unique to the target molecule, as described in more detail below. In some such embodiments, each label may be used to represent a particular binding site. In some cases, the nanoparticle and/or nucleic acid container comprises a plurality of label/binding site pairs, wherein each label/binding site pair is different from each other, thereby allowing multiplexing. Other combinations of labels and binding sites are also possible.

In some embodiments, positioning with specifically directed components with respect to the nucleic acid container and/or nanoparticle surface allows for control of the self-assembly of multiple nanostructures into higher-level components. For example, as shown in the embodiment illustrated in FIG. 5, the assembly 75 includes a plurality of nanostructures 40A-40D positioned in a particular arrangement relative to one another. The specific arrangement of the nanostructures with respect to each other may be obtained by using unique components placed at specific locations of the nucleic acid container and/or the nanoparticle. For example, nanostructure 40A may be assembled with nanostructure 40B using components 60A and 60B, which components 60A and 60B are attached to nanostructures 40A and 40B, respectively. In some embodiments, components 60A and 60B may be binding sites that are complementary to each other, such that they selectively bind to each other and not to other components, e.g., 61A, 61B, 62A, or 62B. As described herein, the components can be designed to include a suitable length, be positioned at a suitable distance from the surface of the coated nanoparticle, and/or be positioned on a particular side of the nucleic acid container and/or nanoparticle. Similarly, nanostructure 40B may be assembled with nanostructure 40C using a pair of components 61A and 61B, and nanostructure 40B may be assembled with nanostructure 40D using a pair of components 62A and 62B. As shown in this exemplary embodiment, each surface of the nanostructure can be tagged with a different binding site, allowing different nanostructures to be attached on each different surface in three-dimensional space.

As described herein, each nanostructure (including nucleic acid containers and/or nanoparticles) used in the assembly can have any suitable shape. For example, as illustratively shown in fig. 5, nanostructures 40A may be in the form of rhomboids, nanostructures 40B may be in the form of pentagons, nanostructures 40C may be in the form of rhomboids having different orientations with respect to nanostructures 40A, and nanostructures 40D may be in the form of hexagons. In other embodiments, other shapes may be used. In addition, it should be understood that any suitable material may be used in each of the nanostructures. For example, the nanoparticles 38A-38D may be formed of the same material or different materials, such as those described herein. In addition, the components 60-62 may vary and may be the same type or different types of components.

In some embodiments, an assembly, such as the one shown in fig. 5, may be formed by synthesizing a plurality of nucleic acid-coated nanoparticles, each formed by growing a nanoparticle from a nanoparticle precursor positioned within a nucleic acid container, and then assembling the nucleic acid-coated nanoparticles. In other embodiments, higher level structures may be formed by: nucleic acid containers each having a nanoparticle precursor positioned therein are assembled, and nanoparticles are subsequently synthesized from the nanoparticle precursors within the nucleic acid containers to form nucleic acid-coated nanoparticles. Combinations of such methods are also possible.

As described herein, any suitable component or binding site can be used for assembly, and the component or binding site can be bound to the nanoparticle and/or nucleic acid portion of the nanostructure. In some embodiments, the nucleic acid-coated nanoparticles are attached to each other through a component or binding site that is attached to the nucleic acid portion of the nucleic acid-coated nanoparticles. In other embodiments, the nucleic acid-coated nanoparticles are attached to each other through a component or binding site that is attached to the nanoparticle portion of the nucleic acid-coated nanoparticles. In some cases, nucleic acid-coated nanoparticles are attached to each other using a thermal process (e.g., using heat to cause attachment or bonding between two nanoparticles, or two or more components to bond with a nanoparticle). In other cases, the nucleic acid-coated nanoparticles are attached to each other using a photophysical process. In certain instances, the nucleic acid-coated nanoparticles are attached to each other using a binding process.

After assembly, all or a portion of the nucleic acid container can optionally be removed from the nucleic acid-coated nanoparticle. In some such embodiments, the surface of the nanoparticles may be passivated before, during, or after the removal step. The nanoparticles may remain attached to each other in the assembly after the removing step. In other embodiments, the nucleic acid container is not removed after assembly of the multiplex nanostructure.

Different types of components may be formed. In some cases, the components contain electronic circuitry. In some embodiments, the components are in the form of a two-dimensional array. In other embodiments, the components are in the form of a three-dimensional array. In some cases, the nanostructures may be assembled hierarchically based on surface-specific binding.

An example of a process for forming a line is shown in the embodiment illustrated in fig. 6A and 6B. Fig. 6A shows a process 100 that includes the assembly of nanostructures 40A, 40B, and 40C with nanostructures 40E to form electronic circuitry. Fig. 6B shows different orientations of the structure in the form of nanorods that include conductive nanoparticles 38 that can be used to form electronic circuitry. As illustratively shown in the figures, a variety of nanostructures can be used as building blocks to be placed at specified locations, in some embodiments, due to the specificity of binding between different surfaces, as described herein. For example, in fig. 6B, the junctions between structures can be specifically aligned at specified locations on the nanorods. As also described herein, different strategies may be used to form different structures. For example, in one embodiment, the assembly may be formed by assembling a nucleic acid container and then triggering the growth of a conductive material within the container. In another embodiment, conductive materials can be grown in each building block using nucleic acid containers, and the resulting nanostructures can then be assembled into larger structures. In some cases, the merging of the ends of the building blocks, for example via thermal/opto-physical methods, may create a continuous conductive network that may be used as electronic circuitry.

The components may be of any suitable size and may be on a nanoscale, microscale, intermediate-scale, or large-scale. In some cases, the component has a length and/or at least one cross-sectional dimension that is less than or equal to 1mm, 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 500nm, less than or equal to 100nm, less than or equal to 50nm, less than or equal to 10nm, or less than or equal to 1 nm. In other cases, the component has a length and/or at least one cross-sectional dimension that is greater than or equal to 1nm, greater than or equal to 10nm, greater than or equal to 100nm, 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 ranges are also possible. The length of the assembly can be determined by measuring the distance between the two outermost portions of the most distally spaced nanostructures forming the assembly. Similarly, the cross-sectional dimensions of the assembly can be determined by: a cross-section between two outermost portions of the nanostructures forming the assembly is obtained and the distance between the outermost portions is measured.

The assembly of nanostructures may have any suitable configuration. For example, in some embodiments, the modules may have a rectilinear shape (e.g., aaaabbbbbbbbbbb, where a and B are different nanostructure building blocks, or alternating chains such as ABABABAB). In other cases, the assembly may have a star shape (e.g., five B's surrounding one a). In other embodiments, the assembly may form a three-dimensional structure such as a tube, a box, a barrel, a rectangle, a bar, a "T" shape, an "L" shape, a branched structure, a diamond, a square, a parallelogram, a rhomboid, a triangle, a pentagon, a hexagon, or a polyhedron, including shapes substantially similar thereto. Other configurations are also possible.

In some embodiments, the nanostructures described herein (e.g., nanoparticles coated or uncoated from a nucleic acid container) can be used for multiplex detection. In some embodiments, the detection may involve introducing a composition suspected of containing a target molecule (e.g., a biomolecule) into the plurality of nanostructures and allowing the target molecule (if present) to bind to the surface of at least two different nanostructures. An example of such a method is shown schematically in fig. 7. Process 80 involves the use of nanostructures 40A and nanostructures 40D that include different components attached to different portions/sides of the nanostructures. For example, nanostructure 40A includes components 81-84 positioned on different sides of the nanostructure, and nanostructure 40D includes components 85-90 positioned on different sides of the nanostructure. In some cases, the components of the nanostructures are each different from one another, although in other cases, some components of the nanostructures may be the same as one another, while others may be different from one another. After the target molecule 94 is introduced, binding between the portion of the target molecule and one of the components of structure 40A can occur, and binding between the portion of the target molecule and the component of structure 40D can occur. Depending on the particular binding sites included in the target molecule, different combinations of binding between the components of nanostructures 40A and 40D may occur. As illustratively shown in fig. 7, the target molecule can include a binding site specific for component 82 of nanostructure 40A and a binding site specific for component 90 of nanostructure 40D. Using such methods, each two surfaces (e.g., one surface from one nanostructure and another surface from a different nanostructure) can be used to detect one specific target molecule.

In some embodiments, the introduction of the target molecule may trigger the recognition of two specific surfaces, and a unique signal due to the recognition event may be generated and/or enhanced. For example, in one set of embodiments, each of the components shown in fig. 7 can be a reporter for SERS-based detection. Binding between a target molecule and two specific surfaces (one on each different nanostructure) can enhance the raman signal from the two specific reporters associated with binding. Using such methods, a variety of different target molecules can be detected using a relatively small number of nanostructures, as the signal from each target molecule binding will be unique and distinguishable from the others.

In some embodiments, the number of different target molecules that can be recognized by the nanostructures described herein may depend, at least in part, on the number of different components (e.g., binding sites) located on the nanostructure. As described herein, in some cases, the components are in the form of isolated components. In general, the number of different target molecules that can be identified using the nanostructures described herein can be determined using the formula (n x-1) n x/2 (assuming that detection of each target molecule involves binding to two nanostructures), where n is the number of nanostructures and x is the number of different binding sites (and/or sides) to which the nanostructures are each bound. For example, 2 nanostructures with 5 binding sites on each nanostructure can result in the detection of (2 x 5-1) x 2 x 5/2=45 different target molecules. The number of different target molecules that can be identified using the nanostructures described herein can be determined using the formula (n x-2) × (n x-1) × n x/3, assuming that detection of each target molecule involves binding to three nanostructures. In some embodiments, two nanostructures (or 3, 4,5, 6, etc. nanostructures) can be used to detect, for example, 2-2,000 different target molecules (e.g., 2-1,000, 2-500, 2-200, 2-100, or 2-50 different target molecules). For example, 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 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, the composition comprises 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 a different target molecule.

It should be understood that other detection methods than SERS-based detection may be used using the nanostructures described herein. For example, other metal-surface enhanced luminescent probes can be used, wherein a luminescent group is used in place of the raman signaling reporter.

In the context of the present invention, nucleic acids include DNA and RNA as well as 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 (DNA backbone enantiomers known in the literature), Peptide Nucleic Acids (PNA), double PNA clips, pseudo-complementary PNA, Locked Nucleic Acids (LNA), or co-nucleic acids (co-nucleic acids) such as DNA-LNA co-nucleic acids as described above. It is understood that the nucleic acids used in the embodiments described herein may be homogeneous or heterogeneous in nature. For example, they may be entirely DNA in nature, or they may comprise 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 interaction of such polymers more or less stable under certain conditions.

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 naturally occurring nucleic acids or may represent non-naturally occurring nucleic acids (e.g., due to the presence of elements not found in naturally occurring nucleic acids). Methods for harvesting nucleic acids from 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.

The nucleic acid may have a homogeneous backbone (e.g., entirely phosphodiester or entirely phosphorothioate) or a heterogeneous (e.g., chimeric) backbone. Phosphorothioate backbone modifications render the nucleic acid less susceptible to nucleases and therefore more stable under specific conditions (compared to native phosphodiester backbone nucleic acids). Other linkages that may provide more stability to nucleic acids include, but are not limited to, dithiophosphate linkages, methylphosphonate linkages, methylthiophosphate linkages, borophosphonate linkages, peptide linkages, alkyl linkages, dephosphorylate linkages, and the like.

Nucleic acids having modified backbones, e.g., backbones comprising phosphorothioate linkages, and including nucleic acids comprising chimeric modified backbones, can be synthesized using automated techniques employing phosphoramidate or H-phosphonate chemistry. (F.E. Eckstein, "Oligonucleotides and nucleic acids-A Practical ApproachIRL Press，Oxford，UK1991, and m.d. matteucci and m.h. carothers,Tetrahedron Lett.21，719（1980）) Aryl and alkyl-phosphonate linkages can be prepared, for example, as described in U.S. Pat. No. 4,469,863; and alkylphosphotriester linkages in which the charged oxygen moiety is alkylated, such 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 Rev90: 544; goodchild J (1990) Bioconjugate Chem1: 165; crook ST et al (1996) Annu Rev Pharmacol Toxicol36: 107-129; and Hunziker J et al (1995) Mod Synth Methods7: 331-417.

The nucleic acids described herein may additionally or alternatively comprise modifications in their sugars. For example, the beta-ribose unit or the beta-D-2 ' -deoxyribose unit can be replaced by a modified sugar unit, wherein the modified sugar unit is selected from, for example, beta-D-ribose, alpha-D-2 ' -deoxyribose, L-2 ' -deoxyribose, 2 ' -F-2 ' -deoxyribose, arabinose, 2 ' -F-arabinose, 2 ' -O- (C)₁-C₆) Alkyl-ribose, preferably 2' -O- (C)₁-C₆) The alkyl-ribose being 2 '-O-methyl ribose, 2' -O- (C)₂-C₆) Alkenyl-ribose, 2' - [ O- (C)₁-C₆) alkyl-O- (C)₁-C₆) Alkyl radical]Ribose, 2' -NH₂-2' -deoxyribose, β -D-xylofuranose, α -arabinofuranose, 2, 4-dideoxy- β -D-erythro-hexapyranose, and carbocyclic (e.g. as described in Froehler J (1992) Am Chem Soc114: 8320), and/or open chain sugar analogues (e.g. as described in Vanderriessche et al (1993) Tetrahedron49: 7223), and/or bicyclic sugar analogues (e.g. as described in Tarkov M et al (1993) Helv Chim Acta76: 481).

The nucleic acid may comprise modifications in its bases. Modified bases include modified cytosines (e.g., 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' -propenocytosine or phenoxazine), And uracil and derivatives thereof (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-deaza-7-substituted guanines (e.g., 7-deaza-7- (C2-C6) alkynylguanine), 7-deaza-8-substituted guanines, hypoxanthine, N2-substituted guanines (e.g., N2-methyl-guanine), 5-amino-3-methyl-3H, 6H-thiazolo [4,5-d ] pyrimidine-2, 7-diketones, 2, 6-diaminopurine, 2-aminopurine, purines, indoles, adenine, substituted adenines (e.g., N6-methyl-adenine, 8-oxo-adenine), 8-substituted guanines (e.g., 8-hydroxyguanine and 8-bromoguanine), and 6-thioguanine. The nucleic acid 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).

As used herein, when the terms "bind" and "interact" refer to nucleic acids, they generally 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 cycler) such that a thermodynamically stable state of the hybridization elements (or a state relatively close thereto) is formed. According to particular embodiments described herein, the interaction between nucleic acids is specific and is generally controlled by the sequence of the interacting strand. 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.

It is understood that in some embodiments, other components, such as small molecules, proteins, and labels, can be attached to the nucleic acids described herein.

The following examples are intended to illustrate specific embodiments of the invention, but should not be construed as limiting and not to exemplify the full scope of the invention.

Example 1

This example shows a method for forming gold nanoparticles in a nucleic acid container as described herein.

Step 1: formation of a nucleic acid container. The nucleic acid container is formed by folding DNA using a DNA origami method. The nucleic acid container was designed using the cadano software. To fold the DNA strands into the designed shape, 16.7uL200nM M13 nucleic acid scaffold (P8064 mutation) (SEQ. ID. NO.1), 20uL500nM staple strands (obtained following the procedure described in Dietz et al, Science, 325: 725-₂) And 44uL of water. The resulting solution was rapidly heat denatured, then slowly cooled from 80 degrees celsius to 61 degrees celsius over 100 minutes, and then slowly cooled from 60 degrees celsius to 24 degrees celsius over 72 hours. Nucleic acid containers in 2% agarose gel (0.5 XTBE +10mM MgCl)₂) Purify in an ice water bath at 70V for 3 hours. The gel was stained with Sybr Gold and the nucleic acid container was extracted from the gel using a collision immersion method.

Step 2: and arranging seeds. A 5nm mono-DNA functionalized gold nanoparticle precursor was used as a seed for growing template nanoparticles. To introduce seeds into the cavity of the nucleic acid container, the purified container (at 0.5 × TBE +10mM MgCl₂2nM in buffer) was mixed with 50nM5nM mono-DNA functionalized gold nanoparticles. The solution was incubated at 37 degrees for 16 hours and then slowly annealed to 24 degrees (1 degree/step, 20 minutes/step). To include a single gold nanoparticle in the cavity of a nucleic acid containerA particle, the mono-DNA from the gold nanoparticle designed to hybridize to a single complementary DNA sequence attached to the inner surface of the nucleic acid container.

Excess unbound gold nanoparticles were removed via spin centrifugation. 20uL of the seed-deposited nucleic acid container solution was mixed with 180uL of water and loaded into an Amicon centrifugal filter (MWCO =100kDa from Millipore) and centrifuged at 14,000g for 3 minutes. This step was repeated twice under the same conditions. The filter was then inverted and spun at 1,000g for 2 minutes. The residual solution was collected.

And step 3: growth of gold nanoparticles within a nucleic acid container. In order to grow gold nanoparticles having a cross section in a hexagonal shape, the above steps 1 and 2 are first performed to synthesize a nucleic acid container 20 including a cavity having a cross section in a hexagonal shape, as shown in fig. 8A. Subsequently, 10uL of the purified seed-disposing solution was mixed with 1uL of 14mM HAuCl₄The precursor solutions are combined. 1uL of 20mM ascorbic acid was then added. After 2 minutes, 3.5uL of the final solution was impregnated onto a copper mesh. After 2 minutes, the solution was wiped off with filter paper, leaving the nanostructures on the grid. 3.5uL2% uranium form was then added to the grid to stain the resulting nanostructure. After 45 seconds, the solution was wiped off with filter paper. The grid is then left to air dry in order to dry the resulting nanostructure.

Fig. 8B and 8C show TEM images of the resulting nanostructures after purification. As shown in fig. 8C, the nanoparticles 38 formed of gold have cross-sectional dimensions of 38nm and 34 nm.

This example shows that gold nanoparticles can be formed in a nucleic acid container that serves as a template during the growth of the nanoparticles. The resulting nanoparticles have a shape that is complementary to the shape of the cavity of the nucleic acid container.

Example 2

This example shows a method for forming silver nanoparticles in a nucleic acid container as described herein.

Steps 1 and 2 as 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 silver nanoparticles in the cavity of the nucleic acid container, 10uL of purified seed placement solution was mixed with 1uL100mM Mg (Ac)₂And (4) combining the solutions. To this mixture was then added 1uL14mM AgNO₃And 1uL20mM ascorbic acid. 3.5uL of the resulting solution was impregnated onto a copper mesh. After 2 minutes, the solution was wiped off with filter paper, leaving the nanostructures on the grid. 3.5uL2% uranium form was then added to the grid to stain the resulting nanostructure. After 45 seconds, the solution was wiped off with filter paper. The grid is then left to air dry in order to dry the resulting nanostructure.

Fig. 9B-9D show TEM images of the resulting nanostructures after purification. As shown in fig. 9B, the nanoparticles 38 formed of silver have cross-sectional dimensions of 17nm and 18 nm. The wall thickness of the nucleic acid container 20 is about 4-6 nm. Fig. 9C and 9D show two nanostructures that dimerize. Dimerization structures were formed using steps 1 and 2 described in example 1. However, before growing silver nanoparticles in the cavities of the nucleic acid containers, the two nucleic acid containers were attached to each other using complementary DNA strands.

This example shows that silver nanoparticles can be formed in a nucleic acid container that serves as a template during the growth of the nanoparticles. The resulting nanoparticles have a shape that is complementary to the shape of the cavity of the nucleic acid container. This example also shows that the nucleic acid containers can be attached to each other to form larger nanostructures.

Example 3

This example shows a method for forming silver nanoparticles in a nucleic acid container having quantum dots bound to an interior surface of the container. The quantum dots are used to block the opening of the nucleic acid container.

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 20uL of the seed-set origami solution, 0.5uL of a 2uM quantum dot (from Invitrogen) solution was added and incubated at 35 ℃ for 16 hours, and then slowly cooled to room temperature for an additional 2 hours. Seed purification and silver growth were performed according to the method described in example 2.

Fig. 10 is a TEM image showing the resulting nanostructures after purification. Nanoparticles 38 made of silver are formed within the nucleic acid container 20. Quantum dots 39 are located at both ends of the nucleic acid container, the quantum dots having a size of about 20 nm.

This example shows that when both ends of the container are blocked by quantum dots, silver nanoparticles can be formed in the nucleic acid container, which serves as a template during the growth of the nanoparticles. The resulting nanoparticles have a shape complementary to the shape of the cavity formed by the nucleic acid container and the quantum dots. This example also shows that the nucleic acid container can be used to prepare heterostructures that can potentially be applied in solar cells and hydrogen production from water.

Example 4

This example shows a method for designing an open nucleic acid container and the use of the container to guide the shape of silver nanoparticles during their formation. In this example, an M13 nucleic acid scaffold (P8064 mutation) (seq. id. No. 1) was used.

Hollow DNA containers used as molds were designed using a 3D DNA origami strategy as described herein (fig. 11). To ensure structural rigidity of the nucleic acid (DNA) container 20, a multi-layered square grid is designed to form walls 25 (e.g., sidewalls). Two-layer designs made of 16-helix bundle, three-layer designs made of 18-helix bundle and four-layer designs made of 24-helix bundle were tested in different shaped DNA containers (fig. 11). The cross-sectional design of the chamber 30 of the vessel has different shapes of sub-25 nm, including rectangular, square, triangular and annular shapes (fig. 11A); this unique property gives programmability, a feature that existing hard templates lack. The thickness of the DNA container was also fine-tuned from 10nm to 30 nm. To ensure metal growth within the central cavity of a single defined DNA container, 5-nm gold nanoparticles used as seeds 34 for silver or gold growth were conjugated to the inner surface of the DNA container via DNA hybridization (fig. 11B). The 21-nt single stranded DNA was immobilized on the seed surface and the stoichiometric ratio between the gold seed and the surface DNA was 1:1 in the reaction buffer. Multiple 21-nt ssDNA ranging from 3-25 was immobilized into the inner surface of a DNA container that had sequence complementarity to those on the surface of the seed. Notably, in some embodiments, direct reduction of precious metals, such as silver and gold, can result in metallization at the outer surface of the DNA container without the use of seeds. The constrained growth of the metal nanostructures 38 within each DNA mold for the specifically specified shape and size is produced by seed-mediated subsequent reduction of the metal precursor (fig. 11C).

In this experiment, portions of the DNA container, such as the DNA bucket and lid, were folded by slowly annealing the staple/rack mixture from 80 ℃ to 24 ℃ over a 72 hour period. The crude material was then subjected to agarose gel electrophoresis (1.5% agarose gel) using 0.5XTBE/10mM MgCl₂As a running buffer. The purified structure was extracted from the gel and subsequently recovered via centrifugation. Seed placement was performed by incubating open DNA containers (e.g., buckets) with an excess of 5-nm gold particles (stoichiometric ratio between gold and DNA containers ranging from 2:1 to 5: 1) at 35C for 16 hours, and then annealed to 24C over 3 hours. Excess gold nanoparticles were removed by using a size exclusion spin column. To form the closed cavity, the DNA lid was mixed with the seed-placed DNA bucket at 35C for 16 hours, and then annealed to 24C over 3 hours. Metal precursors such as silver nitrate for silver nanoparticles and chloroauric acid for forming gold nanoparticles are then added to the purified gold-DNA bucket conjugate followed by the addition of a reducing agent such as Ascorbic Acid (AA). After several minutes to several hours of growth at 4C or room temperature in the dark, the solution was impregnated onto a copper mesh and stained with a uranium salt for TEM imaging.

Example 5

This example shows a method for designing a closed nucleic acid container (e.g., a cassette) and the use of the container to guide the shape of silver nanoparticles during their formation. In this example, an M13 nucleic acid scaffold (P8064 mutation) (seq. id. No. 1) was used.

The three-dimensional constrained growth of silver nanoparticles was examined using a box-shaped DNA container. Each DNA cassette container 20 is designed with three separate components: a tub 47 and two square lids 50 and 52 (fig. 12). The cavity 30 surrounded by the lid and the inside surface of the pail is designed to have a square or rectangular cross-section. Rectangular DNA buckets were assembled from 88 parallel double helices. The cross-sectional dimension of the central lumen is designed to be 8 spirals by 6 spirals. The side wall is constructed of 16 helical bundles and 18 helical bundles. The barrel lengths were set to 6 and 9 double helix angles, respectively. A square DNA bucket was assembled from 108 double helices. Each side wall is constructed from 18 helical bundles. The cross-sectional dimension of the central lumen is designed to be 6 spirals by 6 spirals with a length of 7 double spiral turns. The three-layer DNA lid design has a width of 18 helices, a thickness of 3 helices and a length of 15 helix turns.

To attach the lid to the barrel, 6 or 16 15-nt single stranded binding sites were introduced at both ends of the 18-helix bundle in a rectangular DNA barrel; whereas in the square DNA cassette, 13 15-nt single-stranded binding sites 49 were immobilized at each end of the DNA side wall (FIG. 12A). The binding sites at each bundle show the same sequence. On one side of the DNA lid, 20 15-nt single stranded DNAs having sequences complementary to those on the DNA barrel were introduced. The spacing between the two different sequences of DNA was set at 20nm, consistent with the spacing of the binding sites on the barrel.

For rectangular tubs and lids, the formation yield is about 10-20%, whereas for square tubs, the folding yield is much lower by 5% due to dimerization of the tubs by sticky ends. TEM imaging dictates the formation of the shape of the design. The cross-sectional dimensions of the central cavity were 20nm x 15nm for the two rectangular barrels, consistent with 2.5 nm/double helix, and 15nm x 15nm for the square cavity. However, due to partial dehydration and structural deformation during TEM sample preparation for imaging, small deviations of 2 or 3nm were observed, as well as angular deviations from 90 degrees and/or rough inner surfaces. TEM imaging also revealed a seed placement yield of about 74-91% (N > 100) for different shaped DNA buckets.

The unpurified reaction solution was then imaged with TEM after the lid was closed to determine formation yield (FIG. 12B-12D left, N > 100). Rectangular DNA buckets with dimensions of 20-15-30nm were used to optimize the formation yield of DNA cassette containers for seed placement. At a 3:1 lid to bucket stoichiometry ratio, including 6 binding sites on each end of a rectangular bucket, produced less than 10% box formation yield; while increasing binding sites to 16, box formation yield promoted to 31% (fig. 12B, left). Increasing the lid to pail stoichiometric ratio from 2:1 to 6:1 resulted in a slight increase in box formation yield from 28% to 33%. At a relatively high stoichiometric ratio, i.e., 6:1, in some cases, each end of the barrel may be connected to two lids, which may prevent a proper lid closing process. The yield of defect structures also increased from 20% in the 2:1 stoichiometric ratio to 50% in the 6:1 stoichiometric ratio. The formation of defect structures prevents further increases in the formation yield of DNA cassettes at high stoichiometric ratios. At a stoichiometric ratio of 3:1, the cassette closure yields were found to be 13% and 21% for DNA buckets with sizes of 20-15-20nm and 15-15-25, respectively (FIG. 12C-12D left). The DNA cassette container in the seed arrangement was further reduced by a factor of about 20% using the barrel of the seed arrangement compared to that of the DNA cassette container, which is consistent with the formation yield of the DNA barrel in the seed arrangement. Agarose gel electrophoresis was tested to purify the reaction solution of the seed-arranged DNA cassette containers. However, after strip extraction of the DNA cassette containers corresponding to the seed arrangement, TEM imaging revealed the presence of open and closed seed arrangement DNA cassette containers, which resulted from small mobility differences or structural deformations during gel extraction.

The growth of silver nanoparticles was triggered by the addition of silver nitrate (1.4 mM) and ascorbic acid (2 mM). After 4-10 minutes of growth at room temperature, the silver nanoparticles 38 grown in the DNA cassettes were imaged by TEM (fig. 12B right). TEM images indicated the presence of a 4-8 nm-thick sidewall after silver growth, suggesting that the DNA container remained intact after silver growth. In a rectangular DNA cassette container with a 20-15-30nm sized cavity, silver nanoparticles were grown to 20-16-30nm size (FIG. 12B, right). Rectangular cross sections as well as rounded corners were observed in the TEM images. Silver nanoparticles with similar rectangular cross-section and 20-nm thickness were observed when the cavity size was reduced to 20-15-20nm (fig. 12C, right). The different maximum values allow the thickness of the silver nanoparticles in the DNA cassette container to confirm the constraint of the DNA cassette container in the thickness direction. The cross-sectional dimensions of the DNA cassette container were changed from 20nm x 15nm to 15nm x 15nm, resulting in silver nanoparticles with a square cross-section (fig. 12D right). Each edge was measured at about 16nm in the TEM image. The larger size of the silver nanoparticles compared to those of the cavity results from DNA double helix compression by silver nanoparticle growth.

In some cases, defective DNA structures were also observed during the silver growth process. In the growth direction constrained by two-layer DNA sidewalls made of 16-helix bundles in a rectangular DNA cassette container, defect structures with sidewall bending and cavity enlargement were observed; whereas in the growth direction constrained by the two three-layer DNA sidewalls made of 18-helix bundles, defect structures with enlarged size sizes, e.g. from 20nm to about 25nm, are mainly observed. TEM images indicated that the defect yield for the two-layer sidewall was 5 times higher than that of the three-layer sidewall (N > 50). In a square DNA cassette container with three layers of DNA sidewalls, the defect structure is mainly due to the enlargement of the cavity size, e.g., from 15nm to about 20 nm. The defect ratio also depends on the reaction time and the reactant concentration. When the reaction time was 4 minutes, 0.3mM AgNO3 and 0.5mM AA were used as reactants, the defect ratio with respect to the square boxes was reduced 2/3.

Several other designs have also been tested with respect to the lid and pail to construct cavities having different shapes. A16-helix bundle of DNA having a length of 10 or 15nm is introduced on top of the 30-helix bundle. After purification, TEM images indicated the formation of 30 helical bundles. However, in this particular experiment, the two 16-helix bundles are not tightly connected to the 30-helix bundle and cannot be used for cassette formation. A DNA bucket with a triangular top was also constructed. After seed placement and lid closing, a well-defined spacing was observed at one vertex, consisting of several 10-nm DNA helices. Although triangular tips were observed in the confined silver nanoparticles, the spacing between the 10-nm DNA helices expanded. This is due to the unstable bonding of 1 or 2 staple exchanges in the 10-nm DNA helix compared to 4-5 staple exchanges in the 30-nm DNA helix. The twisted DNA barrel further demonstrates that the rigid and stable DNA sidewalls constrain metal growth.

12A-12D show constrained growth of silver nanoparticles within DNA cassette containers. Fig. 12B shows the design (top) and TEM images (bottom) for silver nanoparticles grown in rectangular DNA cassettes with dimensions of 20-15-30 nm. From left to right: a DNA cassette, a seed-deployed DNA cassette, and silver grown within the cassette. Fig. 12C shows the design (top) and TEM images (bottom) for silver nanoparticles grown in rectangular DNA cassettes with dimensions of 20-15-20 nm. From left to right: a DNA cassette, a seed-deployed DNA cassette, and silver grown within the cassette. FIG. 12D shows the design (top) and TEM images (bottom) for silver nanoparticles grown in square DNA cassettes with dimensions of 15-15-25 nm. From left to right: a DNA cassette, a seed-deployed DNA cassette, and silver grown within the cassette. FIG. 12E shows magnified TEM images for silver nanoparticles grown in rectangular DNA cassettes with dimensions of 20-15-30 nm.

Example 6

This example shows a method for designing an open nucleic acid container and the use of the container to direct the shape of silver and gold nanoparticles during their formation. In this example, an M13 nucleic acid scaffold (P8064 mutation) (seq. id. No. 1) was used.

The generality of constrained growth of metal nanostructures in DNA molds was tested in an open nucleic acid container 20 (e.g., a bucket) to confirm cross-sectional controllability (fig. 13). Four layers of DNA are spirally connected to form a wall 25 surrounding a specifically shaped cavity within the DNA container. The cavity 30 is designed to have three different cross-sectional shapes, such as an equilateral triangle (FIG. 13A), a right-angled triangle (FIG. 13B), and a disk shape (FIG. 13C). Three 21-nt single stranded binding sites were introduced at the inner surface of the container to immobilize the seeds 34.

Gel purification indicates 5-10% folding yield of the DNA container. TEM images show that DNA channels in the shape of equilateral triangles exhibit edge lengths of 25nm with a thickness of 15nm (FIG. 13A left). In the case of right triangles, two different sets of edge dimensions have been observed, for example 20-24-31nm (FIG. 13B left) and 15-29-33nm (not shown). Shape diversity is attributed to the presence of a 16 base single stranded region at the edge of the DNA container (not shown). The single-stranded regions may exhibit different lengths under different stretching conditions, which in turn may result in variable container sizes. For the DNA circle, the inner diameter of the disc-shaped container was determined to be 25nm and the thickness to be 10nm (FIG. 13C left). After gold seed placement, TEM images revealed placement yields of 60-75% for each vessel (N > 100). Although multiple binding sites were present on the inner surface of the container, most DNA containers were conjugated to one seed (in fig. 13A-C), which was attributed to steric repulsion in the nanoparticles within the container.

Silver nanoparticles grown in DNA containers (e.g. 4-8 minutes at room temperature) were imaged by TEM. It was found that about 5-10% of the formed silver nanoparticles had the shape of the cross section of the vessel in which the designed nanoparticles were grown (the remaining nanoparticles had the shape of spheres). In an equilateral triangle shaped container, the fully constrained silver nanoparticles exhibit an equilateral triangle shaped cross section with each edge having a length of 25nm and three rounded vertices (fig. 13A right). The DNA container remained intact after silver growth and was found to completely surround the side surface of the growing silver nanoparticles. No significant bending or curvature of the DNA sidewalls was observed. In the center of the silver nanoparticles, a circular shadow with a diameter of 5-nm is attributed to the gold seed arranged in the DNA container. In the right triangle shaped channel with the size of 20-24-31nm, right triangle silver nanoparticles with the size of 19-24-29nm were grown (fig. 13B right). Similar to that in the equilateral triangular channels, rounded vertices were also observed in the right-angled triangular silver nanoparticles. In the disk-shaped channel, silver spheres with a cross-sectional diameter of 25nm were observed (fig. 13C right).

In an open vessel, only laterally, not vertically, growing particles are confined to the shape of the vessel, which results in a 5-10% constrained yield. Most unconstrained particles exhibit a spherical shape.

Four layers of DNA helices were observed to increase sidewall rigidity. In the case of DNA containers of equilateral triangular shape, the number of defective structures with curved side walls is lower than those in two-and three-layer rectangular DNA containers. It was also observed that right triangular vessels having dimensions of 15-29-33nm also bound the silver grown therein. However, nanoparticles grown in a container having a sharp apex including an angle of 30 degrees are not well formed, compared to that formed in a container having an angle of 60 degrees in an equilateral triangle, or a container having an angle of 50 degrees in a right triangle.

Several other open containers were also tested. An equilateral triangle-shaped DNA container with 15-nm edges exhibits less rigid sidewalls than one with 25-nm edges due to less staple exchange interconnecting the DNA double helix. The silver nanoparticles grown therein produced triangular shaped silver nanoparticles (1%, N > 100), but most nanostructures were spheroid-like. The honeycomb grid is used for constructing a hexagonal DNA container; however, after silver growth, a directional translation of the double helix in the DNA sidewalls was observed, indicating a less rigid structure of the honeycomb grid compared to that of the square grid.

Gold nanoparticles were also grown in open rectangular DNA containers. Compared to that of silver nanoparticles, gold was present at 0.5XTBE/10mM Mg (NO 3)₂The kinetics of growth in buffer are much slower. After thirty minutes of reaction, it was not observed for the nanoparticles withinA significant size increase was observed due to the chelating effect of EDTA on the gold precursor. Removal of EDTA from the reaction buffer significantly promoted growth kinetics. The thirty minute reaction produced 15nm x 20nm rectangular cross-shaped gold nanoparticles in a rectangular bucket (fig. 13D right).

Example 7

This example describes a method of performing directed self-assembly of nucleic acid (e.g., DNA) containers as described herein. In this example, an M13 nucleic acid scaffold (P8064 mutation) (seq. id. No. 1) was used.

DNA containers, including those containing inorganic nanoparticles, provide not only structural constraints, but also surface addressability information. For example, for DNA containers containing nanoparticles therein, due to the sequence specificity of the 3D DNA origami, each staple strand (e.g., DNA strand) located near the surface of the silver nanoparticles can be independently addressed and adjusted, which allows surface addressability resolution down to 2.5nm x 3.4nm on the surface of the nanoparticles. Each staple strand can be further modified with different binding characteristics, including biotin or a variety of different sequence single-stranded regions, controlled orientation, and stoichiometric ratios. Unlike previous post-assembly strategies known in the art, the surface addressability of DNA nanostructures allows metal growth within the pre-assembled container network. Based on this feature, branched metal trimers and Quantum Dot (QD) -silver heterostructures were constructed as shown in fig. 14.

To assemble the Y-shaped DNA container 51 from three individual DNA bucket containers 20, six single-stranded connectors were arranged in two parallel rows at one end of each rectangular bucket by extending the specific staple strand at positions 3 and 5. Each row consists of three 15-nt single stranded DNAs of different sequences and is hybridized with its complementary strand in the other partner bucket. Incubation of separately prepared and purified DNA buckets (3 nM) in the presence of 10nM gold seeds 34 produced seed-arranged Y-buckets. TEM imaging indicated a 5% yield of formation of Y-buckets of seed placement (fig. 14A, left and center). Polymers such as pentamers and hexamers are also observed in the unpurified solution. Silver grown in Y-barrels produces individual nanoparticles within each barrel, and a Y-orientation with respect to the trimer, as constrained by the orientation of the DNA barrel container (fig. 14A, right). The widths of the silver nanoparticles 38 within each barrel were determined to be 20, 22 and 23 nm. The slightly increased width over the width of the rectangular barrel cavity (20 nm) is attributed to the pitch enlargement in the DNA double helix by metal growth. At the center of the Y-shaped bucket where the growth front of the silver nanoparticles is encountered, three well-defined particle interfaces are observed, confirming that the silver nanoparticles so formed are initiated and assembled by three separate silver segments. The presence of particle interfaces also indicates low growth kinetics of differently oriented facets at the center due to the absence of seeds at the center of the Y-shaped bucket.

To construct a heterogeneous Quantum Dot (QD) -silver nanoparticle-quantum dot sandwich structure 53 as shown in fig. 14B, 5 or 6 biotin groups were introduced at each end of an open rectangular barrel container 20 having a size of 20-15-30nm and having a cavity. The quantum dots serve as a lid for the container to constrain the growth of silver nanoparticles within the container. Biotinylation of the DNA container barrel was achieved by extending the selected staple strand at position 5 via the TT spacer. The biotinylated rectangular bucket container (5 nM) was first incubated with gold seeds 34 (10 nM) for 17 hours and then in the presence of excess quantum dots 53 (streptavidin-coated QD (50 nM)) for another 17 hours. Excess quantum dots 57 and gold seeds were removed via spin column purification. TEM imaging revealed the formation of a designed sandwich between the QDs and the seed-arranged barrel ((fig. 14B, left and center). after staining, a white sphere with a diameter of 15-20nm was attributed to the PEG and streptavidin shell surrounding the QD core, 70% of the seed-arranged barrel was found to be conjugated with two QDs at both ends (N > 100). notably, no QDs were found to be attached to the side surfaces of the DNA barrel, silver nanoparticles were generated between the two QDs by arranged gold seed-mediated growth, with a designed QD-Ag-QD heterostructure (fig. 14B, right). the size of the Ag nanoparticles inside the DNA barrel was determined to be 21nm x 30nm, which is consistent with the size of the container cavity.

Nucleic acid sequences

M13 nucleic acid scaffold (P8064 mutation) (SEQ ID NO: 1)

GAATTCGAGCTCGGTACCCGGGGATCCTCAACTGTGAGG

AGGCTCACGGACGCGAAGAACAGGCACGCGTGCTGGCAGAA

ACCCCCGGTATGACCGTGAAAACGGCCCGCCGCATTCTGGC

CGCAGCACCACAGAGTGCACAGGCGCGCAGTGACACTGCGC

TGGATCGTCTGATGCAGGGGGCACCGGCACCGCTGGCTGCA

GGTAACCCGGCATCTGATGCCGTTAACGATTTGCTGAACACA

CCAGTGTAAGGGATGTTTATGACGAGCAAAGAAACCTTTACC

CATTACCAGCCGCAGGGCAACAGTGACCCGGCTCATACCGC

AACCGCGCCCGGCGGATTGAGTGCGAAAGCGCCTGCAATGA

CCCCGCTGATGCTGGACACCTCCAGCCGTAAGCTGGTTGCGT

GGGATGGCACCACCGACGGTGCTGCCGTTGGCATTCTTGCG

GTTGCTGCTGACCAGACCAGCACCACGCTGACGTTCTACAAG

TCCGGCACGTTCCGTTATGAGGATGTGCTCTGGCCGGAGGC

TGCCAGCGACGAGACGAAAAAACGGACCGCGTTTGCCGGAA

CGGCAATCAGCATCGTTTAACTTTACCCTTCATCACTAAAGG

CCGCCTGTGCGGCTTTTTTTACGGGATTTTTTTATGTCGATG

TACACAACCGCCCAACTGCTGGCGGCAAATGAGCAGAAATTT

AAGTTTGATCCGCTGTTTCTGCGTCTCTTTTTCCGTGAGAGC

TATCCCTTCACCACGGAGAAAGTCTATCTCTCACAAATTCCG

GGACTGGTAAACATGGCGCTGTACGTTTCGCCGATTGTTTCC

GGTGAGGTTATCCGTTCCCGTGGCGGCTCCACCTCTGAAAGC

TTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAAC

CCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCT

TTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCG

CCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTT

TGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGC

TGGAGTGCGATCTTCCTGAGGCCGATACTGTCGTCGTCCCCT

CAAACTGGCAGATGCACGGTTACGATGCGCCCATCTACACCA

ACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCA

CGGAGAATCCGACGGGTTGTTACTCGCTCACATTTAATGTTG

ATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTG

ATGGCGTTCCTATTGGTTAAAAAATGAGCTGATTTAACAAAA

ATTTAATGCGAATTTTAACAAAATATTAACGTTTACAATTTAA

ATATTTGCTTATACAATCTTCCTGTTTTTGGGGCTTTTCTGAT

TATCAACCGGGGTACATATGATTGACATGCTAGTTTTACGAT

TACCGTTCATCGATTCTCTTGTTTGCTCCAGACTCTCAGGCA

ATGACCTGATAGCCTTTGTAGATCTCTCAAAAATAGCTACCC

TCTCCGGCATTAATTTATCAGCTAGAACGGTTGAATATCATA

TTGATGGTGATTTGACTGTCTCCGGCCTTTCTCACCCTTTTG

AATCTTTACCTACACATTACTCAGGCATTGCATTTAAAATATA

TGAGGGTTCTAAAAATTTTTATCCTTGCGTTGAAATAAAGGC

TTCTCCCGCAAAAGTATTACAGGGTCATAATGTTTTTGGTAC

AACCGATTTAGCTTTATGCTCTGAGGCTTTATTGCTTAATTTT

GCTAATTCTTTGCCTTGCCTGTATGATTTATTGGATGTTAATG

CTACTACTATTAGTAGAATTGATGCCACCTTTTCAGCTCGCG

CCCCAAATGAAAATATAGCTAAACAGGTTATTGACCATTTGC

GAAATGTATCTAATGGTCAAACTAAATCTACTCGTTCGCAGA

ATTGGGAATCAACTGTTATATGGAATGAAACTTCCAGACACC

GTACTTTAGTTGCATATTTAAAACATGTTGAGCTACAGCATT

ATATTCAGCAATTAAGCTCTAAGCCATCCGCAAAAATGACCT

CTTATCAAAAGGAGCAATTAAAGGTACTCTCTAATCCTGACC

TGTTGGAGTTTGCTTCCGGTCTGGTTCGCTTTGAAGCTCGAA

TTAAAACGCGATATTTGAAGTCTTTCGGGCTTCCTCTTAATCT

TTTTGATGCAATCCGCTTTGCTTCTGACTATAATAGTCAGGG

TAAAGACCTGATTTTTGATTTATGGTCATTCTCGTTTTCTGAA

CTGTTTAAAGCATTTGAGGGGGATTCAATGAATATTTATGAC

GATTCCGCAGTATTGGACGCTATCCAGTCTAAACATTTTACT

ATTACCCCCTCTGGCAAAACTTCTTTTGCAAAAGCCTCTCGC

TATTTTGGTTTTTATCGTCGTCTGGTAAACGAGGGTTATGAT

AGTGTTGCTCTTACTATGCCTCGTAATTCCTTTTGGCGTTATG

TATCTGCATTAGTTGAATGTGGTATTCCTAAATCTCAACTGAT

GAATCTTTCTACCTGTAATAATGTTGTTCCGTTAGTTCGTTTT

ATTAACGTAGATTTTTCTTCCCAACGTCCTGACTGGTATAAT

GAGCCAGTTCTTAAAATCGCATAAGGTAATTCACAATGATTA

AAGTTGAAATTAAACCATCTCAAGCCCAATTTACTACTCGTT

CTGGTGTTTCTCGTCAGGGCAAGCCTTATTCACTGAATGAGC

AGCTTTGTTACGTTGATTTGGGTAATGAATATCCGGTTCTTG

TCAAGATTACTCTTGATGAAGGTCAGCCAGCCTATGCGCCTG

GTCTGTACACCGTTCATCTGTCCTCTTTCAAAGTTGGTCAGT

TCGGTTCCCTTATGATTGACCGTCTGCGCCTCGTTCCGGCTA

AGTAACATGGAGCAGGTCGCGGATTTCGACACAATTTATCAG

GCGATGATACAAATCTCCGTTGTACTTTGTTTCGCGCTTGGT

ATAATCGCTGGGGGTCAAAGATGAGTGTTTTAGTGTATTCTT

TTGCCTCTTTCGTTTTAGGTTGGTGCCTTCGTAGTGGCATTA

CGTATTTTACCCGTTTAATGGAAACTTCCTCATGAAAAAGTC

TTTAGTCCTCAAAGCCTCTGTAGCCGTTGCTACCCTCGTTCC

GATGCTGTCTTTCGCTGCTGAGGGTGACGATCCCGCAAAAGC

GGCCTTTAACTCCCTGCAAGCCTCAGCGACCGAATATATCGG

TTATGCGTGGGCGATGGTTGTTGTCATTGTCGGCGCAACTAT

CGGTATCAAGCTGTTTAAGAAATTCACCTCGAAAGCAAGCTG

ATAAACCGATACAATTAAAGGCTCCTTTTGGAGCCTTTTTTTT

GGAGATTTTCAACGTGAAAAAATTATTATTCGCAATTCCTTTA

GTTGTTCCTTTCTATTCTCACTCCGCTGAAACTGTTGAAAGTT

GTTTAGCAAAATCCCATACAGAAAATTCATTTACTAACGTCT

GGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGG

GCTGTCTGTGGAATGCTACAGGCGTTGTAGTTTGTACTGGTG

ACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTG

CTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGT

TCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCC

TGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAA

CCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCC

CGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAA

TACTTTCATGTTTCAGAATAATAGGTTCCGAAATAGGCAGGG

GGCATTAACTGTTTATACGGGCACTGTTACTCAAGGCACTGA

CCCCGTTAAAACTTATTACCAGTACACTCCTGTATCATCAAA

AGCCATGTATGACGCTTACTGGAACGGTAAATTCAGAGACTG

CGCTTTCCATTCTGGCTTTAATGAGGATTTATTTGTTTGTGAA

TATCAAGGCCAATCGTCTGACCTGCCTCAACCTCCTGTCAAT

GCTGGCGGCGGCTCTGGTGGTGGTTCTGGTGGCGGCTCTGA

GGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGCT

CTGAGGGAGGCGGTTCCGGTGGTGGCTCTGGTTCCGGTGAT

TTTGATTATGAAAAGATGGCAAACGCTAATAAGGGGGCTATG

ACCGAAAATGCCGATGAAAACGCGCTACAGTCTGACGCTAAA

GGCAAACTTGATTCTGTCGCTACTGATTACGGTGCTGCTATC

GATGGTTTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAAT

GGTGCTACTGGTGATTTTGCTGGCTCTAATTCCCAAATGGCT

CAAGTCGGTGACGGTGATAATTCACCTTTAATGAATAATTTC

CGTCAATATTTACCTTCCCTCCCTCAATCGGTTGAATGTCGC

CCTTTTGTCTTTGGCGCTGGTAAACCATATGAATTTTCTATTG

ATTGTGACAAAATAAACTTATTCCGTGGTGTCTTTGCGTTTCT

TTTATATGTTGCCACCTTTATGTATGTATTTTCTACGTTTGCT

AACATACTGCGTAATAAGGAGTCTTAATCATGCCAGTTCTTT

TGGGTATTCCGTTATTATTGCGTTTCCTCGGTTTCCTTCTGGT

AACTTTGTTCGGCTATCTGCTTACTTTTCTTAAAAAGGGCTTC

GGTAAGATAGCTATTGCTATTTCATTGTTTCTTGCTCTTATTA

TTGGGCTTAACTCAATTCTTGTGGGTTATCTCTCTGATATTAG

CGCTCAATTACCCTCTGACTTTGTTCAGGGTGTTCAGTTAAT

TCTCCCGTCTAATGCGCTTCCCTGTTTTTATGTTATTCTCTCT

GTAAAGGCTGCTATTTTCATTTTTGACGTTAAACAAAAAATC

GTTTCTTATTTGGATTGGGATAAATAATATGGCTGTTTATTTT

GTAACTGGCAAATTAGGCTCTGGAAAGACGCTCGTTAGCGTT

GGTAAGATTCAGGATAAAATTGTAGCTGGGTGCAAAATAGCA

ACTAATCTTGATTTAAGGCTTCAAAACCTCCCGCAAGTCGGG

AGGTTCGCTAAAACGCCTCGCGTTCTTAGAATACCGGATAAG

CCTTCTATATCTGATTTGCTTGCTATTGGGCGCGGTAATGAT

TCCTACGATGAAAATAAAAACGGCTTGCTTGTTCTCGATGAG

TGCGGTACTTGGTTTAATACCCGTTCTTGGAATGATAAGGAA

AGACAGCCGATTATTGATTGGTTTCTACATGCTCGTAAATTA

GGATGGGATATTATTTTTCTTGTTCAGGACTTATCTATTGTTG

ATAAACAGGCGCGTTCTGCATTAGCTGAACATGTTGTTTATT

GTCGTCGTCTGGACAGAATTACTTTACCTTTTGTCGGTACTT

TATATTCTCTTATTACTGGCTCGAAAATGCCTCTGCCTAAATT

ACATGTTGGCGTTGTTAAATATGGCGATTCTCAATTAAGCCC

TACTGTTGAGCGTTGGCTTTATACTGGTAAGAATTTGTATAA

CGCATATGATACTAAACAGGCTTTTTCTAGTAATTATGATTCC

GGTGTTTATTCTTATTTAACGCCTTATTTATCACACGGTCGGT

ATTTCAAACCATTAAATTTAGGTCAGAAGATGAAATTAACTA

AAATATATTTGAAAAAGTTTTCTCGCGTTCTTTGTCTTGCGAT

TGGATTTGCATCAGCATTTACATATAGTTATATAACCCAACCT

AAGCCGGAGGTTAAAAAGGTAGTCTCTCAGACCTATGATTTT

GATAAATTCACTATTGACTCTTCTCAGCGTCTTAATCTAAGCT

ATCGCTATGTTTTCAAGGATTCTAAGGGAAAATTAATTAATA

GCGACGATTTACAGAAGCAAGGTTATTCACTCACATATATTG

ATTTATGTACTGTTTCCATTAAAAAAGGTAATTCAAATGAAAT

TGTTAAATGTAATTAATTTTGTTTTCTTGATGTTTGTTTCATC

ATCTTCTTTTGCTCAGGTAATTGAAATGAATAATTCGCCTCTG

CGCGATTTTGTAACTTGGTATTCAAAGCAATCAGGCGAATCC

GTTATTGTTTCTCCCGATGTAAAAGGTACTGTTACTGTATATT

CATCTGACGTTAAACCTGAAAATCTACGCAATTTCTTTATTTC

TGTTTTACGTGCAAATAATTTTGATATGGTAGGTTCTAACCCT

TCCATTATTCAGAAGTATAATCCAAACAATCAGGATTATATT

GATGAATTGCCATCATCTGATAATCAGGAATATGATGATAAT

TCCGCTCCTTCTGGTGGTTTCTTTGTTCCGCAAAATGATAAT

GTTACTCAAACTTTTAAAATTAATAACGTTCGGGCAAAGGAT

TTAATACGAGTTGTCGAATTGTTTGTAAAGTCTAATACTTCTA

AATCCTCAAATGTATTATCTATTGACGGCTCTAATCTATTAGT

TGTTAGTGCTCCTAAAGATATTTTAGATAACCTTCCTCAATTC

CTTTCAACTGTTGATTTGCCAACTGACCAGATATTGATTGAG

GGTTTGATATTTGAGGTTCAGCAAGGTGATGCTTTAGATTTT

TCATTTGCTGCTGGCTCTCAGCGTGGCACTGTTGCAGGCGGT

GTTAATACTGACCGCCTCACCTCTGTTTTATCTTCTGCTGGT

GGTTCGTTCGGTATTTTTAATGGCGATGTTTTAGGGCTATCA

GTTCGCGCATTAAAGACTAATAGCCATTCAAAAATATTGTCT

GTGCCACGTATTCTTACGCTTTCAGGTCAGAAGGGTTCTATC

TCTGTTGGCCAGAATGTCCCTTTTATTACTGGTCGTGTGACT

GGTGAATCTGCCAATGTAAATAATCCATTTCAGACGATTGAG

CGTCAAAATGTAGGTATTTCCATGAGCGTTTTTCCTGTTGCA

ATGGCTGGCGGTAATATTGTTCTGGATATTACCAGCAAGGCC

GATAGTTTGAGTTCTTCTACTCAGGCAAGTGATGTTATTACT

AATCAAAGAAGTATTGCTACAACGGTTAATTTGCGTGATGGA

CAGACTCTTTTACTCGGTGGCCTCACTGATTATAAAAACACT

TCTCAGGATTCTGGCGTACCGTTCCTGTCTAAAATCCCTTTA

ATCGGCCTCCTGTTTAGCTCCCGCTCTGATTCTAACGAGGAA

AGCACGTTATACGTGCTCGTCAAAGCAACCATAGTACGCGCC

CTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGC

GCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCT

CCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCT

TTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCC

GATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATT

TGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACG

GTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGT

GGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCG

GGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGAA

CCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCG

TGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAG

GGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACC

ACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTG

GCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTG

GAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGC

TCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGG

CTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACA

CAGGAAACAGCTATGACCATGATTAC

Staple strand sequence for the DNA container in fig. 8.

Oligonucleotide 1 (SEQ ID NO: 2)

TAACCACCCACTACGTGAACCACGTCAAAGGGCGAACCGCCT

Oligonucleotide 2 (SEQ ID NO: 3)

CGGGCGCAGGTGCCGTAAAGCCAGTTTGGAACAAGGTTTGCC

Oligonucleotide 3 (SEQ ID NO: 4)

CGGCGAACGATTTAGAGCTTGATAAATCAAAAGAAAAATCGG

Oligonucleotide 4 (SEQ ID NO: 5)

GTTTTTTTATTAAAGAACGTGTGCAGCAAGCGGTCTGGGCGC

Oligonucleotide 5 (SEQ ID NO: 6)

CTAAAGGAGATAGGGTTGAGTTCCTGTTTGATGGTTTAATGA

Oligonucleotide 6 (SEQ ID NO: 7)

AAAGTTGTTCACTAAATGAAAGCGTTAGAATCAGAGCGAATC

GT

Oligonucleotide 7 (SEQ ID NO: 8)

TTCAGAGAGTGACTCCAATCACCCGGTCACGACTATGG

Oligonucleotide 8 (SEQ ID NO: 9)

CAGGGTGAAAGTGTAAAGCCTTTTTCACGGTCATCGTGTGTT

Oligonucleotide 9 (SEQ ID NO: 10)

ATCGGCCATTAATTGCGTTGCCCTGTGCACTCTGTCTGCAGC

Oligonucleotide 10 (SEQ ID NO: 11)

CGAGCCGTTTCCTGTGTGAAATCATAAACATCCCTGCCCTGC

Oligonucleotide 11 (SEQ ID NO: 12)

TGCTGCGTATCACGCTGAGTCCACGGGGTCGTAGGGCGACGTATA

Oligonucleotide 12 (SEQ ID NO: 13)

GTGAGCTCGGCCAGAATGCGGCGGCATCAGATGCCCAATCCG

Oligonucleotide 13 (SEQ ID NO: 14)

ACTAGCTGCAGGTTCCGTAGCCCGGAGCCCCCGTGGCGAACAGGA

Oligonucleotide 14 (SEQ ID NO: 15)

CAGCAAAGGTATGAGCCGGGTGGTCTGGTCAGCAGGTCTCGT

Oligonucleotide 15 (SEQ ID NO: 16)

CAGCGGTCATTGCAGGCGCTTGTCGGTGGTGCCATACGATGC

Oligonucleotide 16 (SEQ ID NO: 17)

GGCTGGTGTAGAACGTCAGCGGGCCAGAGCACATCGCGGATCTCAC

Oligonucleotide 17 (SEQ ID NO: 18)

CCGGGCGAAGAATGCCAACGGGCAAACGCGGTCCGGGGCGGTATTT

Oligonucleotide 18 (SEQ ID NO: 19)

ACCTCGCACTGGGTTACGGTGCTGAACTCACAACGCGCGCGA

Oligonucleotide 19 (SEQ ID NO: 20)

TCCTGGTGCTCACTGTTTACACTGATAGCTGGAAGCATGTTT

Oligonucleotide 20 (SEQ ID NO: 21)

CGCTGGCGCTCATTTCCGTGGGTTTTCCCTTCGCTTGCC

Oligonucleotide 21 (SEQ ID NO: 22)

TGATTGCTAAAAAAGCCATGTGCTTTCATCAGGCTTCGC

Oligonucleotide 22 (SEQ ID NO: 23)

AGCAGTTTTTTTTCCAACCGCCGGTTGCTCGTTAACGGGCCGGGGG

Oligonucleotide 23 (SEQ ID NO: 24)

AAAAAAAAAGTTAACCCACGCGCGGGGTGCCGGTGCTGCGCGGCTC

Oligonucleotide 24 (SEQ ID NO: 25)

GGAATTAAGTCGAAAGGGGCGCATGGGATAGATTGTAAGAAGATTCAAT

Oligonucleotide 25 (SEQ ID NO: 26)

GTGTAAAACGAAGGGCGACAGTATCGTCGGATGTTAAACATATGTAGAG

Oligonucleotide 26 (SEQ ID NO: 27)

TCGACGGGAAAGGCAAAGGCACCGTAGCCAGAAATAATAAGAGAACATT

Oligonucleotide 27 (SEQ ID NO: 28)

CGCCAGGTGAAGGGATAGCTCAAACTTAAATTTCTAGCC

Oligonucleotide 28 (SEQ ID NO: 29)

GTGCCAATTACCAGTCCCGGATGTGTACATCGACACGTTCCGCAGC

Oligonucleotide 29 (SEQ ID NO: 30)

AGTAAACGGCTTAAAATTCAGAAATAGCTGAAAAGATTTAAA

Oligonucleotide 30 (SEQ ID NO: 31)

ACTAAATGTGAACCAATTCGTAAAGATCTACCCCTCATATTA

Oligonucleotide 31 (SEQ ID NO: 32)

GCTTTCCGCGCCATTCGCCATGAGGTGG

Oligonucleotide 32 (SEQ ID NO: 33)

CCGTAATCGTAACCGTGCATCATTACGCCAGCTGGTGGGTAA

Oligonucleotide 33 (SEQ ID NO: 34)

TGGGAACTTGAGGGGACGACGATCGGTGCGGGCCTCAGTCAC

Oligonucleotide 34 (SEQ ID NO: 35)

AACAACCCGGCCTCAGGAAGAGCGCAACTGTTGGGACGGCCA

Oligonucleotide 35 (SEQ ID NO: 36)

ACGGTAAAGGAACGCCATCAACTTTCATCAACATTCCAGCCA

Oligonucleotide 36 (SEQ ID NO: 37)

ATGCCGGACCCCGGTTGATAATCGCATTAAATTTTTTCTCCG

Oligonucleotide 37 (SEQ ID NO: 38)

TAAGCAACAAAAGGGTGAGAAATATTCAACCGTTCAGCCCCA

Oligonucleotide 38 (SEQ ID NO: 39)

GCCAAAGGTGGCATCAAACATGTTTTAAATACCTTTAA

Oligonucleotide 39 (SEQ ID NO: 40)

CAAAAACATATTTTAAATGCAAGCTATTTTTGAGAACTAGCA

Oligonucleotide 40 (SEQ ID NO: 41)

TGAAATAACCTGTTTAGTCATTCCATATAACCCAGACC

Oligonucleotide 41 (SEQ ID NO: 42)

ATACTTTAAAAATTTTTAGAAAAAGGCTATCAGGTTCGATGA

Oligonucleotide 42 (SEQ ID NO: 43)

GTAGCATTGAATATAATGCTGAAGAGGTCATTTTTAGAAAAC

Oligonucleotide 43 (SEQ ID NO: 44)

TGATCAGAGCATAAAGCAGTAATGTGTAGGTTAAATTA

Oligonucleotide 44 (SEQ ID NO: 45)

GGGGCGCAAAGTACGGTGTCTACAGGTCAGGATTACTGACTA

Oligonucleotide 45 (SEQ ID NO: 46)

TTTCGCATCCCAATTCTGCGATAATTCGAGCTTCAATTAAGA

Oligonucleotide 46 (SEQ ID NO: 47)

TTGCTCCATCAAAAATCAGGTAATACTGCGGAATCATGCAGA

Oligonucleotide 47 (SEQ ID NO: 48)

GGAAGCAAAAGCGGATTGCATCCAGAGGGGGTAATGAGCAAC

Oligonucleotide 48 (SEQ ID NO: 49)

TTGCAAAAAGAAGCGAAAGTTGATAATGGTCCCCTGTA

Oligonucleotide 49 (SEQ ID NO: 50)

ACCACATAACATTATTACAGGCAAATCAACGTAACTCAAGAG

Oligonucleotide 50 (SEQ ID NO: 51)

CTAGTCATAAACCATAATTTTGATTAGCTCATTCTACTGCAAAAT

Oligonucleotide 51 (SEQ ID NO: 52)

TACATAAAATAAAACGAACTACTCATTCAGTGAATGCATAGG

Oligonucleotide 52 (SEQ ID NO: 53)

ACGAGGCTTGGGAAGAAAAATGCCCTGACGAGAAATGAACGG

Oligonucleotide 53 (SEQ ID NO: 54)

TAAAGTAAAACAGAAGCAACTCCAGGAAGTTCTATATTGTTGTAC

Oligonucleotide 54 (SEQ ID NO: 55)

ACTATCACTCATTATACCAGTACGAGTAGTAAATTCCAACTT

Oligonucleotide 55 (SEQ ID NO: 56)

CAGACGATTATGCGATTTTAAAGATGGTTTAATTTATCATAA

Oligonucleotide 56 (SEQ ID NO: 57)

AAAGCGAGAGCGAAAGACGCGTTTACGAGTAGACCATTGAAGCCT

Oligonucleotide 57 (SEQ ID NO: 58)

GTTCGCCAAAAGCGTCCCTTTACCGAGAGTATGCAACTGAGC

Oligonucleotide 58 (SEQ ID NO: 59)

TAATCTTCCCAGCGATTATACAGAGGCAAAAGAATATAACCGCAAT

Oligonucleotide 59 (SEQ ID NO: 60)

CTGGCTGGAAACAAAGTACAAAAGGCACCAACCTATGAG

Oligonucleotide 60 (SEQ ID NO: 61)

TGTACAGTTGTATCATCGCCTAAAATACGTAATGCGGCCGCTAGGT

Oligonucleotide 61 (SEQ ID NO: 62)

TGAAAGATGTGTCGAAATCCGGGAAGTTTCCATTAACCC

Oligonucleotide 62 (SEQ ID NO: 63)

GGGAACCCTCCATGTTACTTAGGACTAAAGACTTTCATCGGAAAAA

Oligonucleotide 63 (SEQ ID NO: 64)

TGACGACCTGGAACTGAGGGCTTGGAACTGGTAACCCTAGTT

Oligonucleotide 64 (SEQ ID NO: 65)

CGGTCGCAAACGAACAAGCGCACCTTCAAAAGCTGACGGAACTCAA

Oligonucleotide 65 (SEQ ID NO: 66)

GCTTGATACCTGCTAAATAGCGTAGTTTAGTGGATAAGTACT

Oligonucleotide 66 (SEQ ID NO: 67)

AGTTAAACACTACGCGGAGATACCAGGCAAGGCTTCTAC

Oligonucleotide 67 (SEQ ID NO: 68)

GATCGTCAACGGGTGATAAATGGACAGACACCAGACAGGACGATAG

Oligonucleotide 68 (SEQ ID NO: 69)

TCACGGTTTAAGGAACAAAACTACCACCCTCAGAGAAGGTGC

Oligonucleotide 69 (SEQ ID NO: 70)

TAGCAACGCTTTGAGCCGGAAACGGTCACAACTTTAATTACCCGAT

Oligonucleotide 70 (SEQ ID NO: 71)

GACTGAATTTTGTCGTCGTGTATCTTGATATGCTTTTGACCGTTCACCA

Oligonucleotide 71 (SEQ ID NO: 72)

GAATTCAGCGTCCACAGAACCGCCGGGTTTTGGGTCAGATCCTCACTCA

Oligonucleotide 72 (SEQ ID NO: 73)

GGAGAATAATACTGAGTCATTTTCTTAAGAGGCCCCCTAGGCAGGACCA

Oligonucleotide 73 (SEQ ID NO: 74)

GAATTGCGCCTTTAATTGTATGCAGCGAAAGACAGTTCA

Oligonucleotide 74 (SEQ ID NO: 75)

TCATAGTCAACTTTCAACAGTTTTCTTAAACAGCTTGCAGGG

Oligonucleotide 75 (SEQ ID NO: 76)

GAGAGGGACCGTACTCAGGAGACGATCTAAAGTTTTCTGTAT

Oligonucleotide 76 (SEQ ID NO: 77)

ATTAGCGACCCTCAGAACCGCAACGCCTGTAGCATGAGTGAG

Oligonucleotide 77 (SEQ ID NO: 78)

CTCCTCAAGAGCCACCACCCTTTCGTCACCAGTACACTAAAG

Oligonucleotide 78 (SEQ ID NO: 79)

GAAAGTAAGGGATAGCAAGCCCCATGTACCGTAACAATTTTT

Oligonucleotide 79 (SEQ ID NO: 80)

GGTGAAAGCGGCCTCCCCCCCCTTCCATTTGGGGAGGG

Oligonucleotide 80 (SEQ ID NO: 81)

TTTAACGGCTCAGTACCAGGCACCGCCACCCTCAGACAGCCC

Oligonucleotide 81 (SEQ ID NO: 82)

CCGTTGATATGCCACCACGTCAGATACCATTTTACCAG

Oligonucleotide 82 (SEQ ID NO: 83)

AGCCGCCTCAGACGATTGGCCTATAAACAGTTAATGCTGAGA

Oligonucleotide 83 (SEQ ID NO: 84)

GTCATAGTCAGAGCCGCCACCTTAAAGCCAGAATGAATAAGT

Oligonucleotide 84 (SEQ ID NO: 85)

ACTTGAGATTAGCGTTTGCCACCACCACCGGAACCCAGTCTC

Oligonucleotide 85 (SEQ ID NO: 86)

GCACCATCTGTAGCGCGTTTTCCGCCACCCTCAGATCACAAA

Oligonucleotide 86 (SEQ ID NO: 87)

CCATCGAGTAATCAGTAGCGACCACCACCAGAGCCTTGACAG

Oligonucleotide 87 (SEQ ID NO: 88)

TATTCATTAATAACGGAATACCCGAACAAAGTTACTCAAAAA

Oligonucleotide 88 (SEQ ID NO: 89)

AAGGTAAAACTGGCATGATTATTAAGAAAAGTAAGTTTACAG

Oligonucleotide 89 (SEQ ID NO: 90)

ATTCAACTTATTACGCAGTATGCTATCTTACCGAAAAACAGG

Oligonucleotide 90 (SEQ ID NO: 91)

CGCCAAAAACGTAGAAAATACAGAAACAATGAAATGGGAGAA

Oligonucleotide 91 (SEQ ID NO: 92)

GGTAGCAAGGCCGGAAAAAGTTTGCCTTTAGCCCTCAG

Oligonucleotide 92 (SEQ ID NO: 93)

AAAATTCAAGGTGGCAACATATTAAGCCCAATAATTGAACAA

Oligonucleotide 93 (SEQ ID NO: 94)

TTTAGACTCCCGATTGAGGAATTAGAGCCAGATTTTCG

Oligonucleotide 94 (SEQ ID NO: 95)

AGAGAATAAAATAAACAGCCACAAATCAGATATAGAACCAAG

Oligonucleotide 95 (SEQ ID NO: 96)

TTAACTGAACGCTAACGAGCGAGGCGTTTTAGCGAATAATCG

Oligonucleotide 96 (SEQ ID NO: 97)

CGCCCAAAGAACAAGCAAGCCAGAGAATATAAAGTCATGTAA

Oligonucleotide 97 (SEQ ID NO: 98)

AAGTATTATTAGCAGCCCAGATAGCCAAAAGATATTGAGTCACCG

Oligonucleotide 98 (SEQ ID NO: 99)

ATTCTAATCATTCCAAGAACGAGACGACGACAATACAGTAGG

Oligonucleotide 99 (SEQ ID NO: 100)

GCGTCTTTCCATTAGACAGCAATAGTTAGCAGACAAAAACCAGTA

Oligonucleotide 100 (SEQ ID NO: 101)

GGGAGGTCGAGCATGTAGAAAGCCTGTTTATCAACATGCGTT

Oligonucleotide 101 (SEQ ID NO: 102)

AAGCAGCTACGGGTAATAATTGAGTAAAAGAGTCACAAAATGAAA

Oligonucleotide 102 (SEQ ID NO: 103)

TACCGCAAAAGGTAAAGTAATCGCCATATTTAACATAGTTAA

Oligonucleotide 103 (SEQ ID NO: 104)

GCTGTCTTGTTCAGCTAATGCCAGTATAAAGCCAAACCGACC

Oligonucleotide 104 (SEQ ID NO: 105)

TCAACCTCCCTCTTACCAACACCCAAGAGCAATACATAATAT

Oligonucleotide 105 (SEQ ID NO: 106)

TTTAGGCAAAACTTTTTCAAATGCTGATGCAAATCATTA

Oligonucleotide 106 (SEQ ID NO: 107)

AATTCTGTCCGGTATTAAAGGCTTCAGTTACAACATAAGCCC

Oligonucleotide 107 (SEQ ID NO: 108)

GCTTAATCTAAATTTAATGGTTTTAACCTCCGGCTGAGTGAAAGCA

Oligonucleotide 108 (SEQ ID NO: 109)

ATACAAAGCGTTAAATAAGAAAATAGTGAATTTATTTTTCCCTACA

Oligonucleotide 109 (SEQ ID NO: 110)

TTTCATCGGTTATATAACTATAGTACATAAACATCTTGC

Oligonucleotide 110 (SEQ ID NO: 111)

GTGTGATATAGGTCTGAGAGATAAATCGATTATTCGTTT

Oligonucleotide 111 (SEQ ID NO: 112)

CCTAATTACAAACCTACTACTTCTTAATAGAAAATATCCGAA

Oligonucleotide 112 (SEQ ID NO: 113)

TGGAAACATGTAAATATATTTACGCCAAACCGACACTCATCGTAGC

Oligonucleotide 113 (SEQ ID NO: 114)

GCTTCTGCTACCTTTTGAAATCGCTCAAAACAACATTCCTTAGAAC

Oligonucleotide 114 (SEQ ID NO: 115)

TAATTAACAAAATCAAATAAGTTCTTACAGAACGCCCAA

Oligonucleotide 115 (SEQ ID NO: 116)

CCTTGAAAAGAGTCTAAACACGTATCATAATAGATTAATTTATTTG

Oligonucleotide 116 (SEQ ID NO: 117)

ATGAAACAAATCAATATATGTTAGGTTGTTCTGACTGAG

Oligonucleotide 117 (SEQ ID NO: 118)

AAAAGAAATTGATGATGAGAAGTATTGGCAAGAACCACCTGA

Oligonucleotide 118 (SEQ ID NO: 119)

AAACAGTAACCCACCAGATCCTTTGCTGAACTTAACACAGTA

Oligonucleotide 119 (SEQ ID NO: 120)

ACGTAATCCTAGATAATGGAATTGTCGCCATACGTGGCTGGT

Oligonucleotide 120 (SEQ ID NO: 121)

AACTCATCATCAATTCGCCTCAATACAGAGGGCCAACAGAAA

Oligonucleotide 121 (SEQ ID NO: 122)

AGATCATTTTAATTTTAAAAAATCCCACGCTAGATTCATCTG

Oligonucleotide 122 (SEQ ID NO: 123)

GGAATTAGTCAGATGAATATATCGCGCAGAGGCGATCGCTAT

Oligonucleotide 123 (SEQ ID NO: 124)

AGGATTTGCAATTCATCAATATAAAACAGAAATAAGAAGATG

Oligonucleotide 124 (SEQ ID NO: 125)

TTATCTATTAGAGCCGTCAATGATTGTTTGGATTACATATCA

Oligonucleotide 125 (SEQ ID NO: 126)

TGGTCAGTTAGACTTTACAAAATTCCTGATTATCAGCGTAGA

Oligonucleotide 126 (SEQ ID NO: 127)

TCACCTTGCCCGAACGTTATTGCGGAACAAAGAAAAGTACCT

Oligonucleotide 127 (SEQ ID NO: 128)

CTGTGAATGGAACTCAAATAACATGCGCTTAATGCGCC

Oligonucleotide 128 (SEQ ID NO: 129)

GTCAGTACTCAAATATCAAACACAACTCGTATTAAAAGGAGC

Oligonucleotide 129 (SEQ ID NO: 130)

TAAGAATTAAAAATACCGAACATCAACAGTTGAAAACATTTG

Oligonucleotide 130 (SEQ ID NO: 131)

CCTTACCGCCTCACGCAGACGAGCCTGGCAAGTGTAGCAAATCAA

Oligonucleotide 131 (SEQ ID NO: 132)

ATACTACATTTTTTTATGGAGCTAAGAAAGGAAGGGAACGGAACC

Oligonucleotide 132 (SEQ ID NO: 133)

GAGGCCAGCTCATGGAAATACAAAGGGACATTCTGTGAGGCG

Oligonucleotide 133 (SEQ ID NO: 134)

GCTACAGTTCTTTGATTAGTAACTATCGGCCTTGCACAGACA

Oligonucleotide 134 (SEQ ID NO: 135)

TTGCTTTAATTAACCGTTGTAATCCAGAACAATATGAAAGCG

Oligonucleotide 135 (SEQ ID NO: 136)

ACGTGCTAAAGAGTCTGTCCAAGCCATTGCAACAGGAGATAG

Oligonucleotide 136 (SEQ ID NO: 137)

GGCCGATAATCCTGAGAAGTGTTGACGCTCAATCGCCAGTCA

Oligonucleotide 137 (SEQ ID NO: 138)

CCGAGCTCGAATTCGTAATCA

Oligonucleotide 138 (SEQ ID NO: 139)

GGCCCTGTTTTCACCAGTGAGCAACATA

Oligonucleotide 139 (SEQ ID NO: 140)

AAAACAGACGTTAATATTTTGGGATTGA

Oligonucleotide 140 (SEQ ID NO: 141)

ATGAGGCCGGAGAATTAAATAGTA

Oligonucleotide 141 (SEQ ID NO: 142)

GAGAATGATATTCATTGAATCTAGGAAT

Oligonucleotide 142 (SEQ ID NO: 143)

GGGATTTGATAGTTGCGCCGAATATATT

Oligonucleotide 143 (SEQ ID NO: 144)

TGAATTTATGATACAGGAGTGTGCCGTC

Oligonucleotide 144 (SEQ ID NO: 145)

GAGTCTTTTCTATCACCCGGAAAT

Oligonucleotide 145 (SEQ ID NO: 146)

TGAAAATTATCCCAATCCAAAATTACCG

Oligonucleotide 146 (SEQ ID NO: 147)

AAATTATAAGAAAACAAAATTTTTTTAA

Oligonucleotide 147 (SEQ ID NO: 148)

ATATTTTATAGCCCTAAAACAAGGAAGG

Oligonucleotide 148 (SEQ ID NO: 149)

AATGCAATACGGCGCGTCTGCGCG

Oligonucleotide 149 (SEQ ID NO: 150)

GGCCCTGTTTTCACCAGTGAGCAACATATTCCTCTACCACCTACATCAC

Oligonucleotide 150 (SEQ ID NO: 151)

AAAACAGACGTTAATATTTTGGGATTGATTCCTCTACCACCTACATCAC

Oligonucleotide 151 (SEQ ID NO: 152)

ATGAGGCCGGAGAATTAAATAGTATTCCTCTACCACCTACATCAC

Oligonucleotide 152 (SEQ ID NO: 153)

GAGAATGATATTCATTGAATCTAGGAATTTCCTCTACCACCTACATCAC

Oligonucleotide 153 (SEQ ID NO: 154)

GGGATTTGATAGTTGCGCCGAATATATTTTCCTCTACCACCTACATCAC

Oligonucleotide 154 (SEQ ID NO: 155)

TGAATTTATGATACAGGAGTGTGCCGTCTTCCTCTACCACCTACATCAC

Oligonucleotide 155 (SEQ ID NO: 156)

GAGTCTTTTCTATCACCCGGAAATTTCCTCTACCACCTACATCAC

Oligonucleotide 156 (SEQ ID NO: 157)

TGAAAATTATCCCAATCCAAAATTACCGTTCCTCTACCACCTACATCAC

Oligonucleotide 157 (SEQ ID NO: 158)

AAATTATAAGAAAACAAAATTTTTTTAATTCCTCTACCACCTACATCAC

Oligonucleotide 158 (SEQ ID NO: 159)

ATATTTTATAGCCCTAAAACAAGGAAGGTTCCTCTACCACCTACATCAC

Oligonucleotide 159 (SEQ ID NO: 160)

AATGCAATACGGCGCGTCTGCGCGTTCCTCTACCACCTACATCAC

Oligonucleotide 160 (SEQ ID NO: 161)

CAAAATCAAACCTGTCGTGCCGCCCGCT

Oligonucleotide 161 (SEQ ID NO: 162)

AGCCGCCGCGAAACGTACAGCATCCCGT

Oligonucleotide 162 (SEQ ID NO: 163)

GCCCAAGGATTGCGGGAAGATACA

Oligonucleotide 163 (SEQ ID NO: 164)

GGAAGCCGCTTTTGCAAAAGACGTTTAC

Oligonucleotide 164 (SEQ ID NO: 165)

TCACGTTAAAAAAAAGGCTCCACGAGGG

Oligonucleotide 165 (SEQ ID NO: 166)

GAGGTTGGCCTATTTCGGAACGAAACAT

Oligonucleotide 166 (SEQ ID NO: 167)

GAACAGAATCCGTCACCTCAATAG

Oligonucleotide 167 (SEQ ID NO: 168)

AGTCAGAAATTTTATCCTGAAGACTTGC

Oligonucleotide 168 (SEQ ID NO: 169)

TTTACATTTTGAATACCAAGTTTAGAAT

Oligonucleotide 169 (SEQ ID NO: 170)

CACGACCCGCCTGCAACAGTGTAAAGCA

Oligonucleotide 170 (SEQ ID NO: 171)

AAAACGCCAGTAAAGGGGGAAAGC

Oligonucleotide 171 (SEQ ID NO: 172)

CAAAATCAAACCTGTCGTGCCGCCCGCTTATCTTCCTCACACTCCCAAA

Oligonucleotide 172 (SEQ ID NO: 173)

AGCCGCCGCGAAACGTACAGCATCCCGTTATCTTCCTCACACTCCCAAA

Oligonucleotide 173 (SEQ ID NO: 174)

GCCCAAGGATTGCGGGAAGATACATATCTTCCTCACACTCCCAAA

Oligonucleotide 174 (SEQ ID NO: 175)

GGAAGCCGCTTTTGCAAAAGACGTTTACTATCTTCCTCACACTCCCAAA

Oligonucleotide 175 (SEQ ID NO: 176)

TCACGTTAAAAAAAAGGCTCCACGAGGGTATCTTCCTCACACTCCCAAA

Oligonucleotide 176 (SEQ ID NO: 177)

GAGGTTGGCCTATTTCGGAACGAAACATTATCTTCCTCACACTCCCAAA

Oligonucleotide 177 (SEQ ID NO: 178)

GAACAGAATCCGTCACCTCAATAGTATCTTCCTCACACTCCCAAA

Oligonucleotide 178 (SEQ ID NO: 179)

AGTCAGAAATTTTATCCTGAAGACTTGCTATCTTCCTCACACTCCCAAA

Oligonucleotide 179 (SEQ ID NO: 180)

TTTACATTTTGAATACCAAGTTTAGAATTATCTTCCTCACACTCCCAAA

Oligonucleotide 180 (SEQ ID NO: 181)

CACGACCCGCCTGCAACAGTGTAAAGCATATCTTCCTCACACTCCCAAA

Oligonucleotide 181 (SEQ ID NO: 182)

AAAACGCCAGTAAAGGGGGAAAGCTATCTTCCTCACACTCCCAAA

Oligonucleotide 182 (SEQ ID NO: 183)

CCAGCAGGGGGAGAGGCGGTTCTAATGA

Oligonucleotide 183 (SEQ ID NO: 184)

GACGTTGAGAGATAGACTTTCTGCCGCC

Oligonucleotide 184 (SEQ ID NO: 185)

TGTCAATTCAGCTCATTTTTTAGCGAGT

Oligonucleotide 185 (SEQ ID NO: 186)

GGTATGCCTGTAAATCGTTCATTT

Oligonucleotide 186 (SEQ ID NO: 187)

TTATAGTTGTTTAGACTGGATAGGAATT

Oligonucleotide 187 (SEQ ID NO: 188)

AATAGAATCAGCTTGCTTTCGTTTGCGG

Oligonucleotide 188 (SEQ ID NO: 189)

CAAATAATGCCTTGAGTAACAGATTAGG

Oligonucleotide 189 (SEQ ID NO: 190)

GAACATCGGCCAAAATCGGGCGAC

Oligonucleotide 190 (SEQ ID NO: 191)

GAAGCGCAGAGCCTAATTTGCATCCGGT

Oligonucleotide 191 (SEQ ID NO: 192)

TTTTCAGATTTCAATTACCTGTAACCTT

Oligonucleotide 192 (SEQ ID NO: 193)

AACCCTTCAGCAGAAGATAAACAATATC

Oligonucleotide 193 (SEQ ID NO: 194)

AACCCGAGTATTCCTCGAAAGGAG

Oligonucleotide 194 (SEQ ID NO: 195)

CCAGCAGGGGGAGAGGCGGTTCTAATGATAACATTCCTAACTTCTCATA

Oligonucleotide 195 (SEQ ID NO: 196)

GACGTTGAGAGATAGACTTTCTGCCGCCTAACATTCCTAACTTCTCATA

Oligonucleotide 196 (SEQ ID NO: 197)

TGTCAATTCAGCTCATTTTTTAGCGAGTTAACATTCCTAACTTCTCATA

Oligonucleotide 197 (SEQ ID NO: 198)

GGTATGCCTGTAAATCGTTCATTTTAACATTCCTAACTTCTCATA

Oligonucleotide 198 (SEQ ID NO: 199)

TTATAGTTGTTTAGACTGGATAGGAATTTAACATTCCTAACTTCTCATA

Oligonucleotide 199 (SEQ ID NO: 200)

AATAGAATCAGCTTGCTTTCGTTTGCGGTAACATTCCTAACTTCTCATA

Oligonucleotide 200 (SEQ ID NO: 201)

CAAATAATGCCTTGAGTAACAGATTAGGTAACATTCCTAACTTCTCATA

Oligonucleotide 201 (SEQ ID NO: 202)

GAACATCGGCCAAAATCGGGCGACTAACATTCCTAACTTCTCATA

Oligonucleotide 202 (SEQ ID NO: 203)

GAAGCGCAGAGCCTAATTTGCATCCGGTTAACATTCCTAACTTCTCATA

Oligonucleotide 203 (SEQ ID NO: 204)

TTTTCAGATTTCAATTACCTGTAACCTTTAACATTCCTAACTTCTCATA

Oligonucleotide 204 (SEQ ID NO: 205)

AACCCTTCAGCAGAAGATAAACAATATCTAACATTCCTAACTTCTCATA

Oligonucleotide 205 (SEQ ID NO: 206)

AACCCGAGTATTCCTCGAAAGGAGTAACATTCCTAACTTCTCATA

Staple strand sequence for the DNA container in fig. 9.

Oligonucleotide 1 (SEQ ID NO: 207)

ACATCGTGAATACATTAGCGACCAGAG

Oligonucleotide 2 (SEQ ID NO: 208)

TTAGAAGGTCAATACCGAACACTTTTTA

Oligonucleotide 3 (SEQ ID NO: 209)

CCGTACTAGTATAGCCTAAATTATGTAA

Oligonucleotide 4 (SEQ ID NO: 210)

CGACGTTTTTTGCAATGTTTAGAAGAGAA

Oligonucleotide 5 (SEQ ID NO: 211)

CGAGCATCCCGTCGGGAGTTAGGCGCATA

Oligonucleotide 6 (SEQ ID NO: 212)

CCATATGCACTCCAACTAAAAAATTGGGCTTGAG

Oligonucleotide 7 (SEQ ID NO: 213)

GCGTGCCATTAAAGGCCGTTCATATTACGGTAATC

Oligonucleotide 8 (SEQ ID NO: 214)

AGGTGAGTTAACACTAACGTCATAGCAGCCTTTAC

Oligonucleotide 9 (SEQ ID NO: 215)

AGCCAGCAAATCTAAACAGGGGACGGGAGAATTAA

Oligonucleotide 10 (SEQ ID NO: 216)

GTTATCTTAGGAGCAATAAGAATGAAATAGCAATA

Oligonucleotide 11 (SEQ ID NO: 217)

AAAAGCCTGAGCAATACCTTTCCACCCTCAGAGCC

Oligonucleotide 12 (SEQ ID NO: 218)

CGCCATGTTTACCAAACATAGATCAAAAGCGTCAT

Oligonucleotide 13 (SEQ ID NO: 219)

AAACGTATGCAAATATTTCATGTTAAATAACACTG

Oligonucleotide 14 (SEQ ID NO: 220)

ACGCCGAATAAACAAATTCTTGTAACGAATTTTGC

Oligonucleotide 15 (SEQ ID NO: 221)

TTTGAGGGGACGACAACAAGATGCCCTGAACCGAT oligonucleotide 16 (SEQ ID NO: 222)

TCGGCTAATTCTGTATCAACAGCTTGCTCAACAAC

Oligonucleotide 17 (SEQ ID NO: 223)

GCGAGGTTTTTGTTAAATCAGATTGTATCGCCTGT

Oligonucleotide 18 (SEQ ID NO: 224)

GACACCACGGAATAACATACAACAAAGATGAGGAT

Oligonucleotide 19 (SEQ ID NO: 225)

TCCAACAGGTCTGAAGCCAGTTTTGATCAGAATGA

Oligonucleotide 20 (SEQ ID NO: 226)

AAAACCAGGATTAGCGGGGTTAAGTATTATCGGCG

Oligonucleotide 21 (SEQ ID NO: 227)

AAAGAGCTCCTGTACGTGGGACACATCCTAATTTA

Oligonucleotide 22 (SEQ ID NO: 228)

GCAGTGTTCAATCAAAGGCTAAATTGAGCGATGCCG

Oligonucleotide 23 (SEQ ID NO: 229)

AACAATTCTCGTCAAAACCGATCAAAAGGGCTTACC

Oligonucleotide 24 (SEQ ID NO: 230)

ATCATAGCATCAGCAGTTTGAACCCTGTGACTCCTT

Oligonucleotide 25 (SEQ ID NO: 231)

CAGTAGTGCCGGACAAACAGATCTACTAGGAAGGTA

Oligonucleotide 26 (SEQ ID NO: 232)

CCCTTAGACGCAGATGCCGCCGAAGCCCCTTCAAAG

Oligonucleotide 27 (SEQ ID NO: 233)

GAGAGATCGGAAAACTGACTAAAGATTAAGCCGTTC

Oligonucleotide 28 (SEQ ID NO: 234)

GGTGCGGGCCTCTTAACGCTCAATCTACCAGTTTCA

Oligonucleotide 29 (SEQ ID NO: 235)

ATAATCGATCGAGAGGGATCGAGGCTTTGAGTGTAC

Oligonucleotide 30 (SEQ ID NO: 236)

GATAGGTACAAACGCCGGATATCATCAAGAGTAATCT

Oligonucleotide 31 (SEQ ID NO: 237)

AGTCATAAGTTGCCACATTATTCATCAGTTGAGTTATACC

Oligonucleotide 32 (SEQ ID NO: 238)

TCTTCGCCTCCTCTCAAAAACTGGCCTAGACGGTGGAACCG

Oligonucleotide 33 (SEQ ID NO: 239)

CCCTCACTTTACCAGAGAATCCTTGAAGTCCCGGCCTCACC

Oligonucleotide 34 (SEQ ID NO: 240)

CGCCTGTGCACTCTTGAACCTGAGAGTCCCCTGAACAAAGTC

Oligonucleotide 35 (SEQ ID NO: 241)

AATCAACAGTTGAACATCCCTAAGAATTAGAAAGGCCGGAGA

Oligonucleotide 36 (SEQ ID NO: 242)

CGGTAGCGCACTCAGCCATCCACCCAACGAATGCACTGGTCT

Oligonucleotide 37 (SEQ ID NO: 243)

CTTCTGAGAGGTGTTATGGTTAAAACATTAAAGAAACGCAAA

Oligonucleotide 38 (SEQ ID NO: 244)

CGGCCTTTAGTGATTCCGGCAATAAGAGCTGAATATACCCTC

Oligonucleotide 39 (SEQ ID NO: 245)

GCTCATTAACAGCGGCTCTCAAGACTTTAGCCGCCGCCAGTG

Oligonucleotide 40 (SEQ ID NO: 246)

TGAGAAGGAATAACCTTGCTTTTTTAATCTCATTAAGGCAGG

Oligonucleotide 41 (SEQ ID NO: 247)

CCAATCGCAAGACAGGAAACAAAGAGGCTAAACAGTTCAGAA

Oligonucleotide 42 (SEQ ID NO: 248)

ATGCTGACCTTTTTATTCTGAGCCCGTATAAACAGAGTGCCT

Oligonucleotide 43 (SEQ ID NO: 249)

TGGGAAGTTCGCCAAGTCAGGATTTTAAGAACTGGTGTGAAT

Oligonucleotide 44 (SEQ ID NO: 250)

GCAAAGCCACCGCTTACCTTAAATTTCAACTTTAACAAAGCT

Oligonucleotide 45 (SEQ ID NO: 251)

CGTGCATTTGGTGTGCTCATTTTACCCAAATCAACACAAGAA

Oligonucleotide 46 (SEQ ID NO: 252)

TCATTCCATTAAACGAAAGACCGAGGGTAGCAACGCATGAGG

Oligonucleotide 47 (SEQ ID NO: 253)

TTCATCAACCAACCGAAAGAGGACAGATGAACGGGGCCACTA

Oligonucleotide 48 (SEQ ID NO: 254)

CTATTTTGCACCATTTGCGGGTGTATCACCCCCAGCGATTAT

Oligonucleotide 49 (SEQ ID NO: 255)

AACCCACTACACTGTTCTTTGCGACAACTTTTAAAGGGGTCA

Oligonucleotide 50 (SEQ ID NO: 256)

ATCACCATCAATATAATGCCTTAGAACCTTTTACCTTTATTT

Oligonucleotide 51 (SEQ ID NO: 257)

GAGTAATGTGTAGGCAGTCAAGAGAGATAGAGGGTTCAGGTC

Oligonucleotide 52 (SEQ ID NO: 258)

TAAAGATGGAAACGTGATTAAAATACTTTGTACCATACCAGC

Oligonucleotide 53 (SEQ ID NO: 259)

CAAAAGAACTGGCACAATAATTAAAGGTGTGTGTTGTTGGCA

Oligonucleotide 54 (SEQ ID NO: 260)

AGAGCATGGGCAAAAATTACGAATAAATATTTTCAGCTGGTC

Oligonucleotide 55 (SEQ ID NO: 261)

CAATAGAGACGGAACGACTTGAGCCAATAATAAAGGATTATA

Oligonucleotide 56 (SEQ ID NO: 262)

ACTGTAGCGCGTTTTAGCACCCAATAACCGTCAGATGAATAT

Oligonucleotide 57 (SEQ ID NO: 263)

CCACCACACCACCCGTAGGATTAGAGAGAAGAAGACAAAATC

Oligonucleotide 58 (SEQ ID NO: 264)

AGAACCGAATTGCTAGACCGGTCTCTGAATTTAAGAGCAGTT

Oligonucleotide 59 (SEQ ID NO: 265)

GATACAGGAGTGTAATAAATCGGAAACATTTCATTTGAATTA

Oligonucleotide 60 (SEQ ID NO: 266)

AACGAGTAACATGATTGCTCATACAGACGACGATATTAGTTA

Oligonucleotide 61 (SEQ ID NO: 267)

TTACAGGGAAGAAAAACAGTAGGGCTCAGGCGATCAGGCGAT

Oligonucleotide 62 (SEQ ID NO: 268)

GTAGCATTCCACAGTTTTGTCATATGCGGAGGCATTTTCGAG

Oligonucleotide 63 (SEQ ID NO: 269)

AAACGGCACCAGTACGCCAACATGTAATAAGGTAATAATTTT

Oligonucleotide 64 (SEQ ID NO: 270)

TCGGTTTATAGAACGAGTAGTGGAATTGCTTTCAAGTTAATA

Oligonucleotide 65 (SEQ ID NO: 271)

CACTAAAACACTCACGAAGGCACATTAAATGTGAACAAATCA

Oligonucleotide 66 (SEQ ID NO: 272)

TCTTTGATCGCCTGATAAATTGCGAACCGATATAGCCGAGCT

Oligonucleotide 67 (SEQ ID NO: 273)

ATCAAAATGGCTTAGATAACTATTAATGGCGACCGTTACAAAC

Oligonucleotide 68 (SEQ ID NO: 274)

AAGAACGCGAGAAAAACGACGACGGGAAGGATAGCTTGAATCC

Oligonucleotide 69 (SEQ ID NO: 275)

TCCCGACTTTGTTAAAATTCGAATTGTACGAACTGAACGAACC

Oligonucleotide 70 (SEQ ID NO: 276)

ATTCGCCTGAACAAAATTAACAAGTACATATGTGAGTAGTCAAT

Oligonucleotide 71 (SEQ ID NO: 277)

ACCAGCTGCTGCGAATAAGAGCAAACAAGAGAATATTGCCTCAAATAT

Oligonucleotide 72 (SEQ ID NO: 278)

GAGAGGTTGAGAGCTAGCATTGTACCCCGGTTGCTTCACGGATCCAGC

Oligonucleotide 73 (SEQ ID NO: 279)

GAAGCCAAGTTACCAGTATGGGCAACATATAATGGTAACATCTTTACA

Oligonucleotide 74 (SEQ ID NO: 280)

ATTACGCAGAAGGTATAGATTAGAGCCTATTAGATATCATTAATTATC

Oligonucleotide 75 (SEQ ID NO: 281)

ATCGGTTTGCGGGTTATTAATCGTATTAAATCCTTAATGGGAACGGAA

Oligonucleotide 76 (SEQ ID NO: 282)

AATATTAAATTCACCATTCCTGATTATTTGTTTGAAATTGCACAGTAA

Oligonucleotide 77 (SEQ ID NO: 283)

CGAACCCCTTTTGAAATTTCAATTACCGCACAGGGGGCGGTTAATTTT

Oligonucleotide 78 (SEQ ID NO: 284)

CAGTAAATCAGGTAATGCTTTGAGACTCCTCACTCGGATAAAATTTGT

Oligonucleotide 79 (SEQ ID NO: 285)

GGATTAAAATAGCGCAACACCCACCACCCTCATTTTCAGACGAGGCAT

Oligonucleotide 80 (SEQ ID NO: 286)

CGGATAACCTATTAACCTCCCATAGGTCTGAGAGAAGACGCTGAGTAA

Oligonucleotide 81 (SEQ ID NO: 287)

GCGGAGTGAGACGACGTTGGTAGAAAGCAGGATAGCAAGCCTGCTGCA

Oligonucleotide 82 (SEQ ID NO: 288)

AGACCAAAGGCCGCACGCATACGAGAAACACCCAATAGATACCAATCA

Oligonucleotide 83 (SEQ ID NO: 289)

CGAATTCTAATGCGAACGTTAGAGCCTAATTTGCCCAATCCAGCCAGAA

Oligonucleotide 84 (SEQ ID NO: 290)

CGTGGCATTTTGAATATCCTGACGCTAACGAGCGTTTTTGTTCGCCTGC

Oligonucleotide 85 (SEQ ID NO: 291)

AACAGGGAGAAGATTAGTCTTAAAGCGTTAGCAAGGCAAGCCACGTAAT

Oligonucleotide 86 (SEQ ID NO: 292)

CAAACCCCACTGCGTGCGGCGAATACCGATAGCCCCCGGGTAAAGGCTT

Oligonucleotide 87 (SEQ ID NO: 293)

TGCCCTGCGGCATCTTACCTGCAGCCATCTGGTCACAGCAAAAATATCA

Oligonucleotide 88 (SEQ ID NO: 294)

GGAGCGGTTGCGGATAAAGGTTTAGCAAACGTAGACAGATAGGATAATA

Oligonucleotide 89 (SEQ ID NO: 295)

ACGGCTGATAATGGGCACGTATTGTAGAATCCTCAGCGCAGAGGAAGTT

Oligonucleotide 90 (SEQ ID NO: 296)

GTAGATTCCGTCACATTATTCATTAAAGTATTTTGTGGCAATACCAGAA

Oligonucleotide 91 (SEQ ID NO: 297)

GGCAGCCCGGTCCGTGCAACTGCTGTAGCTCAACATTAATTGGTCATTT

Oligonucleotide 92 (SEQ ID NO: 298)

TAACGGAAACGTCAGTGGCATCATTTGGGAATTAGTTAGCAAGCGTCAG

Oligonucleotide 93 (SEQ ID NO: 299)

ACAAACAACAGGAGTCAGAGCCGCCACCCACCGGATTTGCCATTCGGTC

Oligonucleotide 94 (SEQ ID NO: 300)

TAGGCATTATACACCGGAATATAAGGCCTTCTGACCCGGAAGTACCAGG

Oligonucleotide 95 (SEQ ID NO: 301)

GAGAATACTCCAAACAAAAGGAGCCTTTTGAATTTGAACGCGTTCCTTA

Oligonucleotide 96 (SEQ ID NO: 302)

ATTCTCAACAGTTGAGGATCCTAAAACATAAGCAAAAATAAACAGATAA

Oligonucleotide 97 (SEQ ID NO: 303)

TCAGAAAACAGGAAGCTCATTTAGGAACTCCATGTGAACGAGGCGGCAA

Oligonucleotide 98 (SEQ ID NO: 304)

GTAAAAATCTACAATAGCGGTGCCGGTTCAGACGTCATACCGCCAGCAC

Oligonucleotide 99 (SEQ ID NO: 305)

CGATGAATTTATCCAGTTACAATATTTACATTAAACGTTTTAGTGTCGA

Oligonucleotide 100 (SEQ ID NO: 306)

AGAGAGAAACGATTCTTTCCAATCAGCTACAATTTTGGCTATAAAACAG

Oligonucleotide 101 (SEQ ID NO: 307)

GCCCAATACTAACAACTAAAAAGGAATTACCTTGCGTTGCCACGCTGAG

Oligonucleotide 102 (SEQ ID NO: 308)

GCTATTGGAGTTAACTGAACATGGAATAACATAAAAAGCATCGAGGAAG

Oligonucleotide 103 (SEQ ID NO: 309)

TTTAAATGCGATATTCGCTGATAAATTACTTCGTTAACGGCTGGTTTGA

Oligonucleotide 104 (SEQ ID NO: 310)

ACCGATTGCCAAAGCCAGCTTTTGCAGGCGCTTTCCCGAACGAGAAGCC

Oligonucleotide 105 (SEQ ID NO: 311)

GAGGGAGATAGTAGTGAAAAGCCAATGAACAGAATCAATTCTGCGAACG

Oligonucleotide 106 (SEQ ID NO: 312)

AGCATTATTATTTAAGGGTTAGAACCTCACGCAAACAAAAGAAAGCTAA

Oligonucleotide 107 (SEQ ID NO: 313)

AAAATCAATTATCATTCAGGTCAATATAATCCTGACAGATGATCACAAT

Oligonucleotide 108 (SEQ ID NO: 314)

AACCATCAGAGCACACGTCAGCGTGGTGTATCAAAAACATCCACATTCA

Oligonucleotide 109 (SEQ ID NO: 315)

GCAAAACTTAGTTTGACCATTTTAAATATTTTTTCTTGCCGTGAAGGGT

Oligonucleotide 110 (SEQ ID NO: 316)

TAGCCCCCACAGTTGATTCCCAAGTTTGCCTTTAGGCCGGAACCCTTTT

Oligonucleotide 111 (SEQ ID NO: 317)

GGTGTCTGGCGAATTATTCCGTCCGGCCGATAGCATTTGGGGCGCGAGC

Oligonucleotide 112 (SEQ ID NO: 318)

GCCTCCCTCAGAGCCGCCAATAAAGTACAGTAGATCGTAATCAGTAGCG

Oligonucleotide 113 (SEQ ID NO: 319)

CGGAACCTCATTCCATATATTCAAGTTATGATGAACCAAATCCCGTAAA

Oligonucleotide 114 (SEQ ID NO: 320)

TCAAAATAGAACCACGCCGCCATTGGCCCAAACAACTGGTAACGGGGTC

Oligonucleotide 115 (SEQ ID NO: 321)

ATTCGAGGAAAGACCATCAAATTATAGTATATTCAGTCCAATTAGTAAA

Oligonucleotide 116 (SEQ ID NO: 322)

TCAGACGAGCATTGTCAAGAAATTGCTTGGAGAAAATTACCAAGCCAGC

Oligonucleotide 117 (SEQ ID NO: 323)

ACATGGCAATGGAAATCGACATAAAATTCTGTAAATTAGATTACTACAG

Oligonucleotide 118 (SEQ ID NO: 324)

CCATAACGATAGCTCGTCGCTATTAATTGTGTACAGCGCAGAAGCAAAC

Oligonucleotide 119 (SEQ ID NO: 325)

TTAATGCTTTCGGAAGTGCCGTGATATACAGGAGGCCACCCTCAGAACC

Oligonucleotide 120 (SEQ ID NO: 326)

AAAGAAGGAATTACTAATGCAGATACATAGGAATAGGTAACGCAACTGT

Oligonucleotide 121 (SEQ ID NO: 327)

AGTAAGAGAGAGGCGTAAACTTTTTCAAGGGGATGCAATAGGAACATTA

Oligonucleotide 122 (SEQ ID NO: 328)

TTTACGGTCAGAACCGCCACCGTACCGTAAAGCTGGCGAAAGATATATT

Oligonucleotide 123 (SEQ ID NO: 329)

TCACCAGGTGATAACATAATTACTAGAATAGTATCGTCTTTCTAAATGA

Oligonucleotide 124 (SEQ ID NO: 330)

TCATAGTTAAACTAACGGAACAACCCATCTCAGAGTATCATAACCCTCG

Oligonucleotide 125 (SEQ ID NO: 331)

TAAACAACGAATAAAGCTCAGGAAGATCTTAACAATAAAGCCCGCTATT

Oligonucleotide 126 (SEQ ID NO: 332)

ATTTTCTTGAAAATTAAAGTAACGACAATAAACAATAATGCACTTAAAC

Oligonucleotide 127 (SEQ ID NO: 333)

TTCACGTGTATGGGTCTAAAGACAGCCCAGTTTCGGCCACCCTGTATCA

Oligonucleotide 128 (SEQ ID NO: 334)

AAGGCTAAAATAATATCCCGCTGCCAGTTTCCGGGCTAATTGAGAATCG

Oligonucleotide 129 (SEQ ID NO: 335)

ATATTCGGTCGCCACAGTGAAATGGTTTTGATAGAAAGGAACAGCCAGC

Oligonucleotide 130 (SEQ ID NO: 336)

CATCGCCCTTTTGCACAGCGAGTAACAAGTAGAAAAGTCCTGGACAGTA

Oligonucleotide 131 (SEQ ID NO: 337)

TGCGCCGCAGCAGCCAAGTACTTTTCATATTACCGGAAGCCTTTAGTTG

Oligonucleotide 132 (SEQ ID NO: 338)

GGCTGGCAACTTTTAAAACGAAGAATAAATCCGCGACCTGCGCAAGAAC

Oligonucleotide 133 (SEQ ID NO: 339)

AACTGACCTGACCTTTTGAGGCTTGCAGGATTCTCGCCAGCTATCCGGT

Oligonucleotide 134 (SEQ ID NO: 340)

ACTAAAGACTTTTTGCTACAGTCACCCTACAATGATTCGAGGAATTGTA

Oligonucleotide 135 (SEQ ID NO: 341)

GTAAAATGTTTTTACGCACTCGCTGTCTCCTGTTTCCAGACGCCGACAA

Oligonucleotide 136 (SEQ ID NO: 342)

TAAACGGACCAAGCGTACAACGGAGATTAGGTTTTCGCCCAAAAGAATA

Equivalent scheme

While several embodiments of the 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 appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and 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.

As defined and used herein, all definitions should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

As used herein in the specification and in the claims, the indefinite articles "a" and "an" should be understood to mean "at least one" unless explicitly indicated to the contrary.

It will also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as "comprising," including, "" carrying, "" having, "" containing, "" involving, "" containing, "" consisting of … … and the like are to be understood to be open-ended, i.e., to mean including but not limited to. The transition phrases "consisting of … …" and "consisting essentially of … …" shall be closed or semi-closed transition phrases, respectively, as shown in the U.S. Patent Office Patent examination program Manual of Patent application products, section 2111.03. The following are claimed:

Claims

1. A method, comprising:

nanoparticles are formed from nanoparticle precursors positioned within a nucleic acid container having a predetermined three-dimensional structure.

2. An article, comprising:

a nanoparticle positioned within a nucleic acid container having a predetermined three-dimensional structure, wherein the nanoparticle comprises at least one surface portion having a shape complementary to a shape of an interior surface portion of the nucleic acid container.

3. A method, comprising:

forming a nanoparticle comprising at least one surface portion having a shape complementary to a shape of an inner surface portion of a nucleic acid container having a predetermined three-dimensional structure on a sub-nanometer level.

4. A method, comprising:

providing a nucleic acid container as a template for forming nanoparticles, 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 nanoparticles within the nucleic acid container; and

attaching the plurality of components to the nanoparticle.

5. An article, comprising:

a nanoparticle comprising at least two opposing surface portions each having a shape complementary to a shape of a surface portion of a nucleic acid nanostructure.

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. A method, comprising:

the isolated nucleic acid strands are attached to the surface of inorganic nanoparticles having a non-spherical shape.

8. An article, comprising:

an inorganic nanoparticle coated by a nucleic acid container, wherein the nucleic acid container comprises a pore; 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. A method, comprising:

providing inorganic nanoparticles coated from the nucleic acid container, wherein the nucleic acid container comprises pores;

introducing a nucleic acid strand through a well of the nucleic acid container; and

attaching a portion of the nucleic acid strand to a surface of the inorganic nanoparticle.

10. An article, comprising:

an assembly of nucleic acid coated nanoparticles, wherein the nucleic acid coated nanoparticles are attached to each other by complementary binding sites.

11. A method, comprising:

forming an assembly of nucleic acid-coated nanoparticles, wherein the nucleic acid-coated nanoparticles are attached to each other by complementary binding sites.

12. A composition, comprising:

a plurality of nanoparticles, wherein two of the plurality of nanoparticles can be used to detect at least 12 different target molecules.

13. A method, comprising:

two non-spherical nanoparticles were used to detect at least 12 different target molecules.

14. A composition, comprising:

a plurality of nanoparticles, wherein at least 90% of the nanoparticles vary in maximum cross-sectional dimension by less than 0.5 standard deviation from the median maximum cross-sectional dimension of all nanoparticles in the composition, and wherein each of the plurality of nanoparticles comprises at least 6 distinct sides.

15. A nanoparticle formed by the method of any one of the preceding claims.

16. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle precursor comprises an inorganic nanoparticle.

17. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle precursor comprises a metal.

18. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle precursor comprises a semiconductor.

19. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle precursor comprises Au, Ag, Cd, Zn, Cu, Pb, Mn, Ni, Mg, Fe, Pd, and/or Pt.

20. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle precursor comprises a monomer of an organic polymer.

21. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle precursor has a cross-sectional dimension of less than or equal to 50nm, less than or equal to 25nm, less than or equal to 10nm, less than or equal to 5nm, less than or equal to 3nm, less than or equal to 2nm, less than or equal to 1nm, or less than or equal to 0.1 nm.

22. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle comprises at least one surface portion having a shape that is complementary to a shape of an interior surface portion of the nucleic acid container.

23. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle has a shape that is complementary to a shape of an interior surface of the nucleic acid container.

24. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle is formed from template-assisted synthesis within a nucleic acid container.

25. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle has 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.

26. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle comprises a cross-section in the shape of a rectangle, a rod, a T, an L, a branched structure, a diamond, a star, a square, a parallelogram, a triangle, a pentagon, or a hexagon.

27. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticles are in the shape of a polyhedron.

28. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle has a non-spherical shape.

29. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle has an asymmetric shape.

30. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle is an inorganic nanoparticle.

31. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle comprises a metal.

32. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle is an alloy.

33. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle comprises a semiconductor.

34. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle comprises a polymer.

35. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle has at least one cross-sectional dimension that is less than or equal to 1 micron, less than or equal to 500nm, less than or equal to 250nm, less than or equal to 100nm, less than or equal to 75nm, less than or equal to 50nm, less than or equal to 40nm, less than or equal to 30nm, less than or equal to 20nm, less than or equal to 10nm, or less than or equal to 1 nm.

36. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle has at least one cross-sectional dimension that is greater than or equal to 1nm, greater than or equal to 5nm, greater than or equal to 10nm, greater than or equal to 50nm, or greater than or equal to 100 nm.

37. An article, a composition, or a method as in any one of the preceding claims, wherein the 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.

38. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle is encapsulated by a nucleic acid nanostructure.

39. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle comprises an isolated binding site attached to the surface of the nanoparticle.

40. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle comprises at least 2, at least 4, at least 6, at least 8, or at least 10 different isolated binding sites attached to the surface of the nanoparticle.

41. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle comprises an isolated nucleic acid strand attached to the surface of the nanoparticle.

42. An article, a composition, or a method as in any one of the preceding claims, wherein the isolated binding sites are located at least 2nm, at least 5nm, at least 10nm, at least 15nm, at least 20nm, or at least 30nm away from each other.

43. An article, a composition, or a method as in any one of the preceding claims, wherein the nucleic acid strand is a DNA strand or a DNA analog, or an RNA strand or an RNA analog.

44. An article, a composition, or a method as in any one of the preceding claims, wherein 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.

45. An article, a composition, or a method as in any one of the preceding claims, wherein the nucleic acid container comprises a cavity having a volume, and substantially all of the volume is filled with the nanoparticle.

46. An article, a composition, or a method as in any one of the preceding claims, wherein the nucleic acid container comprises a cavity in the shape of a polyhedron.

47. An article, a composition, or a method as in any one of the preceding claims, wherein the nucleic acid container comprises a cavity having a non-spherical shape.

48. An article, a composition, or a method as in any one of the preceding claims, wherein the nucleic acid container comprises a cavity having an asymmetric shape.

49. An article, a composition, or a method as in any one of the preceding claims, wherein the nucleic acid container comprises at least one open side, or at least two open sides.

50. An article, a composition, or a method as in any one of the preceding claims, wherein the container is substantially closed.

51. An article, a composition, or a method as in any one of the preceding claims, wherein the nucleic acid container comprises at least one lid that can be opened or closed.

52. An article, a composition, or a method as in any one of the preceding claims, wherein the nucleic acid container comprises a cavity, and the cavity can have a cross-sectional dimension of less than or equal to 1 micron, less than or equal to 500nm, less than or equal to 250nm, less than or equal to 100nm, less than or equal to 75nm, less than or equal to 50nm, less than or equal to 40nm, less than or equal to 30nm, less than or equal to 20nm, less than or equal to 10nm, or less than or equal to 1 nm.

53. An article, a composition, or a method as in any one of the preceding claims, wherein the nucleic acid container comprises a cavity, and the cavity has a cross-sectional dimension of greater than or equal to 1nm, greater than or equal to 5nm, greater than or equal to 10nm, greater than or equal to 20nm, greater than or equal to 30nm, greater than or equal to 40nm, greater than or equal to 50nm, greater than or equal to 100nm, greater than or equal to 500nm, or greater than or equal to 1 micron.

54. An article, a composition, or a method as in any one of the preceding claims, wherein the nucleic acid container comprises walls surrounding a cavity, and wherein the average thickness of the walls is less than or equal to 1 micron, less than or equal to 500nm, less than or equal to 250nm, less than or equal to 100nm, less than or equal to 75nm, less than or equal to 50nm, less than or equal to 40nm, less than or equal to 30nm, less than or equal to 20nm, less than or equal to 10nm, or less than or equal to 1 nm.

55. An article, a composition, or a method as in any one of the preceding claims, wherein the nucleic acid container comprises walls surrounding a cavity, and wherein the average thickness of the walls is greater than or equal to 1nm, greater than or equal to 10nm, greater than or equal to 25nm, greater than or equal to 50nm, greater than or equal to 100nm, greater than or equal to 500nm, or greater than or equal to 1 micron.

56. An article, a composition, or a method as in any one of the preceding claims, wherein the nucleic acid container comprises more than one layer.

57. An article, a composition, or a method as in any one of the preceding claims, wherein the nucleic acid container is formed from nucleic acids having a molecular weight of at least 640 kDa.

58. An article, a composition, or a method as in any one of the preceding claims, wherein the nucleic acid container is formed from a nucleic acid having a length of at least 1,000 bases.

59. An article, a composition, or a method as in any one of the preceding claims, wherein the component is formed by: assembling nucleic acid containers each having a nanoparticle precursor positioned therein, and then synthesizing nanoparticles from the nanoparticle precursors within the nucleic acid containers to form the nucleic acid-coated nanoparticles.

60. An article, a composition, or a method as in any one of the preceding claims, wherein the component is formed by: synthesizing a plurality of nucleic acid-coated nanoparticles, each by growing a nanoparticle from a nanoparticle precursor positioned within a nucleic acid container, and subsequently assembling the nucleic acid-coated nanoparticles.

61. An article, a composition, or a method as in any one of the preceding claims, wherein the nucleic acid-coated nanoparticles are attached to each other through binding sites that are attached to the nucleic acid portion of the nucleic acid-coated nanoparticles.

62. An article, a composition, or a method as in any one of the preceding claims, wherein the nucleic acid-coated nanoparticles are attached to each other through binding sites that are attached to the nanoparticle portion of the nucleic acid-coated nanoparticles.

63. An article, a composition, or a method as in any one of the preceding claims, wherein the nucleic acid-coated nanoparticles are attached to each other using a thermal process, a photophysical process, and/or a binding process.

64. An article, a composition, or a method as in any one of the preceding claims, further comprising removing a portion of the nucleic acid from the nucleic acid-coated nanoparticle.

65. An article, a composition, or a method as in any one of the preceding claims, further comprising substantially removing the nucleic acid coating from the nucleic acid-coated nanoparticle.

66. An article, a composition, or a method as in any one of the preceding claims, comprising passivating a surface of the nanoparticle before, during, or after the removing step.

67. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticles remain attached to each other in the assembly after the removing step.

68. An article, a composition, or a method as in any one of the preceding claims, wherein the component is an electronic circuit.

69. An article, a composition, or a method as in any one of the preceding claims, wherein the component is in the form of a two-dimensional array or a three-dimensional array.

70. An article, a composition, or a method as in any one of the preceding claims, wherein the component has at least one length and/or at least one cross-sectional dimension that is less than or equal to 1mm, 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 500nm, less than or equal to 100nm, less than or equal to 50nm, less than or equal to 10nm, or less than or equal to 1 nm.

71. An article, a composition, or a method as in any one of the preceding claims, wherein the component has at least one length and/or at least one cross-sectional dimension that is greater than or equal to 1nm, greater than or equal to 10nm, greater than or equal to 100nm, 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.

72. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle comprises a label attached to a surface of the nanoparticle.

73. An article, a composition, or a method as in any one of the preceding claims, wherein the label is isolated on the nanoparticle surface.

74. An article, a composition, or a method as in any one of the preceding claims, wherein the label comprises a nucleic acid strand, a fluorophore, a nanoparticle, an antibody, a peptide, or a reporter.

75. An article, a composition, or a method as in any one of the preceding claims, wherein the label is a surface-enhanced raman scattering reporter.

76. An article, a composition, or a method as in any one of the preceding claims, wherein the label is a luminescent probe.

77. An article, a composition, or a method as in any one of the preceding claims, wherein each label is adjacent to a binding site attached to the surface of the nanoparticle.

78. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle comprises a plurality of pairs of labels and binding sites, wherein the labels are each different from one another and the binding sites are each different from one another.

79. An article, a composition, or a method as in any one of the preceding claims, comprising 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 labels positioned on the surface of the nanoparticle.

80. An article, a composition, or a method as in any one of the preceding claims, wherein the components or markers are each separated from each other and positioned at a predetermined distance from each other.

81. A method as in any preceding claim, comprising attaching a predetermined number of components or labels to the nanoparticles.

82. The method of any one of the preceding claims, comprising using the article to detect a biomolecule.

83. The method of any one of the preceding claims, comprising using the article for multiplex detection of biomolecules.

84. An article, a composition, or a method as in any one of the preceding claims, wherein the detecting comprises introducing a target molecule to a plurality of nanoparticles and allowing the target molecule to bind to the surface of at least two different nanoparticles.

85. An article, a composition, or a method as in any one of the preceding claims, wherein the binding enhances raman signals from two reporter molecules bound to the surface of the nanoparticle.

86. The method of any one of the preceding claims, comprising 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.

87. An article, a composition, or a method as in any one of the preceding claims, wherein the nucleic acid-coated nanoparticles are each in the form of a nanoparticle positioned within a nucleic acid container.

88. The method of any one of the preceding claims, comprising synthesizing the nanoparticles by a seed-mediated growth process.

89. The method of any one of the preceding claims, comprising forming at least 10, at least 15, at least 20, at least 30, at least 50, or at least 100 inorganic nanoparticles each having a different shape in parallel.

90. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle is synthesized in the absence of a surfactant, or in the absence of an oxide template.

91. An article, a composition, or a method as in any one of the preceding claims, wherein the nucleic acid container is designed to include a cavity having a predetermined three-dimensional structure, and the shape of the nanoparticle is formed at least in part by contouring the cavity.

92. The method of any one of the preceding claims, comprising controlling ion diffusion kinetics to control growth kinetics and/or composition of the nanoparticles.

93. The method of any one of the preceding claims, comprising controlling the distribution of components in the nanoparticle alloy.

94. An article, a composition, or a method as in any one of the preceding claims, wherein the nucleic acid container comprises an inorganic nanostructure.

95. An article, a composition, or a method as in any one of the preceding claims, wherein the inorganic nanoparticle is hollow and comprises a cavity.

96. An article, a composition, or a method as in any one of the preceding claims, comprising using the inorganic nanoparticle as a template to construct secondary nanostructures in cavities of the nanoparticle.

97. An article, a composition, or a method as in any one of the preceding claims, wherein the nanoparticle has a complex arbitrary shape.