Systems and methods for biomolecule retention - Patents.com

Abstract

Compositions, systems, and methods are described for the presentation of analytes, such as biomolecules. Presentation of the analyte is accomplished by attachment of the analyte to a presentation molecule configured to associate with a surface or interface. Arrays of analytes can be formed from the described systems for use in assays and other methods.

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 159,500, filed March 11, 2021, and U.S. Provisional Application No. 63 / 256,761, filed October 18, 2021, each of which is incorporated by reference herein in its entirety.

[0002] Analytes and other molecules can be formed into structured or ordered arrays for a variety of purposes, including analytical techniques and other chemical purposes. For example, biomolecules can be patterned into single molecule arrays for purposes such as sequencing or molecular identification. High efficiency of analyte deposition on single molecule arrays can benefit from methods for preparing the analytes and the surfaces or interfaces onto which the analytes are deposited. Summary of the Invention

[0003] In aspects, provided herein are compositions comprising a structured nucleic acid particle (SNAP) comprising: (i) a presentation moiety configured to connect to an analyte; (ii) a capture moiety configured to connect to a surface; and (iii) a multifunctional moiety comprising a first functional group and a second functional group, wherein the multifunctional moiety is connected to the structured nucleic acid particle, the first functional group is connected to the presentation moiety, and the second functional group is connected to the capture moiety.

[0004] In another aspect, provided herein is a composition comprising a structured nucleic acid particle and a multifunctional site, wherein the multifunctional site is connected to a SNAP, and the multifunctional site is configured to form a continuous linker from the surface to an analyte.

[0005] In another aspect, provided herein is a structured nucleic acid particle (SNAP) complex comprising two or more SNAPs, wherein each SNAP of the two or more SNAPs is independently selected from the group consisting of a presentation SNAP, a utilization SNAP, or a combination thereof, wherein the presentation SNAP comprises a presentation moiety configured to connect to an analyte, and the utilization SNAP comprises a capture moiety configured to connect to a surface, and wherein the two or more SNAPs are connected to form a SNAP complex.

[0006] In another aspect, provided herein is a structured nucleic acid particle (SNAP) composition comprising a material including a surface, and two or more SNAPs, each SNAP of the two or more SNAPs independently selected from the group consisting of a presentation SNAP, a utilization SNAP, or a combination thereof, wherein the presentation SNAP comprises a presentation moiety configured to connect to an analyte, and wherein the two or more SNAPs are connected to the surface, and a first SNAP of the two or more SNAPs is connected to a second SNAP of the two or more SNAPs, thereby forming a SNAP complex.

[0007] In another aspect, provided herein is a composition comprising: a) an analyte; b) a presentation SNAP; and c) one or more SNAPs selected from the group consisting of a presentation SNAP, a utilization SNAP, and combinations thereof, wherein the presentation SNAP comprises a presentation moiety configured to connect to the analyte, wherein the presentation SNAP is connected to the analyte, and the presentation SNAP is connected to one or more SNAPs, thereby forming a SNAP complex.

[0008] In another aspect, provided herein is a structured nucleic acid particle composition comprising: a) a material comprising a surface; b) an analyte; c) a presentation SNAP, and one or more SNAPs selected from the group consisting of a presentation SNAP, a utilization SNAP, and combinations thereof, wherein the presentation SNAP comprises a presentation moiety configured to connect to the analyte, the presentation SNAP is connected to the analyte, and the presentation SNAP is connected to one or more SNAPs, thereby forming a SNAP complex, and the SNAP complex is connected to the surface.

[0009] In another aspect, provided herein is an array comprising a material comprising: a) a plurality of SNAP complexes; and b) a surface, wherein each of the SNAP complexes is attached to the surface, and wherein each SNAP complex of the plurality of SNAP complexes is attached to one or more other SNAP complexes of the plurality of SNAP complexes, and wherein each SNAP complex of the plurality of SNAP complexes comprises two or more SNAPs independently selected from the group consisting of a presentation SNAP, a utilization SNAP, and combinations thereof.

[0010] In another aspect, provided herein is a method of forming an array, comprising: a) providing a plurality of SNAP complexes; b) connecting each SNAP complex of the plurality of SNAP complexes to one or more additional SNAP complexes from the plurality of SNAP complexes; and c) connecting each SNAP complex of the plurality of SNAP complexes to a surface, each SNAP complex comprising a display SNAP and one or more utilization SNAPs, and each SNAP complex comprising a connection site that connects to the surface, thereby forming an array.

[0011] In another aspect, provided herein are compositions comprising: a) a structured nucleic acid particle comprising: i) a retention component; ii) a presentation component comprising a connecting group configured to attach an analyte, the presentation component being connected to the retention component; and iii) a capture component configured to connect to a surface, the capture component comprising a plurality of first surface-interactive oligonucleotides, each first surface-interactive oligonucleotide of the plurality of first surface-interactive oligonucleotides comprising a first nucleic acid strand connected to the retention component and a first surface-interactive component, the first surface-interactive component being configured to form a connecting interaction with the surface linking component, the capture component being constrained from contacting the presentation component by the retention component; and b) an analyte comprising a complementary connecting group configured to connect to the presentation component of the structured nucleic acid particle.

[0012] In another aspect, provided herein is a composition comprising: a) a structured nucleic acid particle comprising: i) a retention component; ii) a presentation component connected to the retention component; and iii) a capture component connected to the retention component, wherein the capture component comprises a plurality of oligonucleotides, each oligonucleotide of the plurality of oligonucleotides comprising a surface interaction component; and b) a solid support comprising a connecting surface, wherein the surface comprises a surface linkage component, wherein the surface interaction component of the plurality of surface interaction components is connected to a surface linkage, and wherein the presentation component is constrained from contacting the surface by the retention component.

[0013] In another aspect, provided herein is a method of identifying a polypeptide, the method comprising: a) providing a SNAP composition as described herein, wherein the polypeptide is linked to a presentation site; b) contacting a solid support with a plurality of detectable affinity reagents; c) detecting the presence or absence of binding of the plurality of detectable affinity reagents to the polypeptide; d) optionally repeating steps b)-c) with a second plurality of detectable affinity reagents; and e) identifying the polypeptide based on the presence or absence of binding of one or more of the affinity reagents.

[0014] In another aspect, provided herein is a method of sequencing a polypeptide, the method comprising: a) providing a SNAP composition as provided herein, wherein the polypeptide is attached to a presentation site; b) removing a terminal amino acid residue of the polypeptide by Edman-type degradation; c) identifying the terminal amino acid residue; and d) repeating steps b-c) until a sequence of amino acid residues for the polypeptide is identified.

[0015] In another aspect, provided herein is a single-analyte array comprising: a) a solid support comprising a plurality of addresses, each address of the plurality of addresses resolvable with single-analyte resolution, each address comprising a connecting surface, each connecting surface comprising one or more surface attachment sites; b) a plurality of structured nucleic acid particles, each structured nucleic acid particle comprising a connection site, the connection site comprising a plurality of oligonucleotides, each oligonucleotide of the plurality of oligonucleotides comprising a surface interaction site, each structured nucleic acid particle of the plurality of structured nucleic acid particles being connected to an address of the plurality of addresses by binding of the surface interaction sites of the plurality of oligonucleotides to the surface attachment sites of one or more complementary oligonucleotides, and each structured nucleic acid particle of the plurality of structured nucleic acid particles comprising a presentation site comprising a connection site connected to an analyte.

[0016] In another aspect, provided herein is a single-analyte array comprising: a) a solid support comprising a plurality of addresses, each address of the plurality of addresses resolvable from each other address with single-analyte resolution, and each address separated from each adjacent address by one or more interstitial regions; and b) a plurality of analytes, wherein a single analyte of the plurality of analytes is connected to an address of the plurality of addresses, each address of the plurality of addresses comprising no more than one single analyte, each single analyte being connected to a connecting surface of the address by a nucleic acid structure, wherein the nucleic acid structure blocks the single analyte from contacting the connecting surface.

[0017] In another aspect, provided herein is a nucleic acid nanostructure comprising at least 10 connected nucleic acids, the nucleic acid nanostructure comprising: a) a compressed region comprising high internal complementarity, wherein the high internal complementarity comprises at least 50% double-stranded nucleic acids and at least 1% single-stranded nucleic acids, the compressed region comprising a presentation site, the presentation site being connected or configured to connect to an analyte of interest; and b) a transmission region comprising low internal complementarity, wherein the low internal complementarity comprises at least about 50% single-stranded nucleic acids, the transmission region comprising a connection site, the connection site forming or configured to form a connecting interaction with a solid support.

[0018] In another aspect, provided herein are nucleic acid nanostructures comprising: a) a compressed structure, the compressed structure comprising a backbone strand and a first plurality of staple oligonucleotides, at least 80% of the nucleotides of the backbone strand hybridized to the nucleotides of the first plurality of staple oligonucleotides, the first plurality of staple oligonucleotides hybridizing to the backbone strand to form a plurality of tertiary structures, the plurality of tertiary structures including adjacent tertiary structures connected by single-stranded nucleic acid regions of the backbone, the relative positions of adjacent tertiary structures being positionally constrained; and b) a transmission structure, the transmission structure comprising a second plurality of staple oligonucleotides, the staple oligonucleotides connected to the backbone strand of the compressed structure, the transmission structure comprising at least 50% single-stranded nucleic acids, the transmission structure having an anisotropic three-dimensional distribution around at least a portion of the compressed structure.

[0019] In another aspect, provided herein are nucleic acid nanostructures comprising: a) a compressed structure, the compressed structure comprising a backbone strand and a first plurality of staple oligonucleotides, at least 80% of the nucleotides of the backbone strand are hybridized to the nucleotides of the first plurality of staple oligonucleotides, the first plurality of staple oligonucleotides hybridizing to the backbone strand to form a plurality of tertiary structures, the plurality of tertiary structures including adjacent tertiary structures connected by single-stranded regions of the backbone strand, the relative positions of the adjacent tertiary structures being positionally constrained, the compressed structure comprising an effective surface area; and b) a permeable structure, the permeable structure comprising a second plurality of staple oligonucleotides, the staple oligonucleotides being connected to the backbone strand of the compressed structure, the permeable structure comprising at least 50% single-stranded nucleic acids; and (i) the effective surface area of ​​the nucleic acid nanostructure is greater than the effective surface area of ​​the compressed structure, or ii) the ratio of the effective surface area to the volume of the nucleic acid nanostructure is greater than the ratio of the effective surface area to the volume of the compressed structure.

[0020] In another aspect, provided herein is a nucleic acid nanostructure comprising a plurality of nucleic acid strands, wherein each nucleic acid strand of the plurality of nucleic acid strands is hybridized to another nucleic acid strand of the plurality of nucleic acid strands to form a plurality of tertiary structures, wherein a nucleic acid strand of the plurality of nucleic acid strands comprises a first nucleotide sequence hybridized to a second nucleic acid strand of the plurality of nucleic acid strands, wherein a nucleic acid strand of the plurality of nucleic acid strands further comprises a second nucleotide sequence of at least 100 contiguous nucleotides, and wherein at least 50 nucleotides of the second nucleotide sequence are single-stranded.

[0021] In another aspect, provided herein is a composition comprising: a) a solid support comprising a plurality of sites; and b) a plurality of structured nucleic acid particles (SNAPs), each SNAP connected or configured to connect to an analyte, wherein each SNAP of the plurality of SNAPs is connected to a site of the plurality of sites, the plurality of sites comprising a first subset comprising a first amount of sites and a second subset comprising a second amount of sites, wherein each site of the first subset comprises two or more connected SNAPs, and each site of the second subset comprises only one connected SNAP, wherein the ratio of the amount of sites of the first subset to the amount of sites of the second subset is less than the ratio predicted by a Poisson distribution.

[0022] In another aspect, provided herein is an analyte array comprising: a) a solid support comprising a plurality of sites; and b) a plurality of nucleic acid nanostructures, each nucleic acid nanostructure being connected to an analyte of interest, each nucleic acid nanostructure of the plurality being connected to a site of the plurality of sites, and at least 40% of the sites of the plurality of sites comprising a single analyte of interest.

[0023] In another aspect, provided herein is a composition comprising: a) a solid support comprising sites configured to attach a nucleic acid nanostructure; b) a nucleic acid nanostructure, wherein the nucleic acid nanostructure is attached to the sites, the nucleic acid nanostructure is attached to an analyte of interest; and the nucleic acid nanostructure is configured to prevent contact between the analyte of interest and the solid support.

[0024] In another aspect, provided herein is a composition comprising: a) a solid support comprising a site configured to connect a nucleic acid nanostructure, the site comprising a surface area; and b) a nucleic acid nanostructure, the nucleic acid nanostructure connected to the site, the nucleic acid nanostructure connected or configured to connect to an analyte of interest; the nucleic acid nanostructure comprising a total effective surface area in its unbound configuration, the nucleic acid nanostructure comprising a compressed structure having an effective surface area, the effective surface area of ​​the compressed structure in its unbound configuration being less than 50% of the surface area of ​​the site, and the unbound configuration comprising a nucleic acid nanostructure that is disconnected from the site.

[0025] In another aspect, provided herein is a method for connecting nucleic acid nanostructures to array sites, the method comprising: a) contacting an array comprising the sites with a nucleic acid nanostructure, wherein the sites comprise a plurality of surface attachment sites and the nucleic acid nanostructure comprises a plurality of capture sites; b) connecting the nucleic acid nanostructure to the sites in an initial configuration, wherein the initial configuration does not comprise a stable configuration and the nucleic acid nanostructure is connected by connection of capture sites of a plurality of capture sites to surface attachment sites of the plurality of surface attachment sites; c) disconnecting connection of capture sites of a plurality of capture sites to surface attachment sites of the plurality of surface attachment sites; and d) modifying the nucleic acid nanostructure from the initial configuration to a stable configuration, wherein each capture site of the plurality of capture sites is connected to a surface attachment site of a plurality of surface attachment sites.

[0026] In another aspect, provided herein is a method of forming a multiplexed array of analytes, the method comprising: a) contacting an array comprising a plurality of sites with a first plurality of nucleic acid nanostructures, wherein each nucleic acid nanostructure of the first plurality of nucleic acid nanostructures is connected to an analyte of interest of a first plurality of analytes of interest; b) contacting the array comprising a plurality of sites with a second plurality of nucleic acid nanostructures, wherein each nucleic acid nanostructure of the second plurality of nucleic acid nanostructures is connected to an analyte of interest of a second plurality of analytes of interest; c) depositing the first plurality of nucleic acid nanostructures at a first subset of sites of the plurality of sites; and d) depositing the second plurality of nucleic acid nanostructures at a second subset of sites of the plurality of sites, wherein the first subset of sites and the second subset of sites comprise a random spatial distribution.

[0027] In another aspect, provided herein is a nanostructure comprising: (a) a compacted nucleic acid structure comprising a backbone strand hybridized to a first plurality of staple oligonucleotides, wherein the first plurality of staple oligonucleotides hybridize to the backbone strand to form a plurality of tertiary structures, the plurality of tertiary structures comprising adjacent tertiary structures connected by single-stranded regions of the backbone strand, wherein the relative positions of the adjacent tertiary structures are positionally constrained; (b) a transparent structure, wherein the transparent structure comprises a second plurality of staple oligonucleotides hybridized to the backbone strand; and (c) a solid support comprising surface-linked oligonucleotides, wherein the surface-linked oligonucleotides are bound to the surface of the solid support, and the surface-linked oligonucleotides are hybridized to the staple oligonucleotides of the transparent structure.

[0028] In another aspect, provided herein is a method for connecting nucleic acid nanostructures to an array, the method comprising: a) contacting a solid support with a nucleic acid nanostructure, the solid support comprising surface-linked oligonucleotides bound to the solid support, the nucleic acid nanostructure comprising: i) a compacted nucleic acid structure comprising a backbone strand hybridized to a first plurality of staple oligonucleotides, the first plurality of staple oligonucleotides hybridizing to the backbone strand to form a plurality of tertiary structures, the plurality of tertiary structures comprising adjacent tertiary structures connected by single-stranded regions of the backbone strand, the relative positions of the adjacent tertiary structures being positionally constrained; ii) a transparent structure, the transparent structure comprising a second plurality of staple oligonucleotides hybridized to the backbone strand; and b) hybridizing the surface-linked oligonucleotides to the staple oligonucleotides of the second plurality of staple oligonucleotides.

[0029] In another aspect, provided herein is a method for preparing an array of analytes, the method comprising: a) providing an array comprising a plurality of sites, each site comprising a surface-attached oligonucleotide; b) contacting the array with a plurality of analytes, each analyte being attached to a nucleic acid nanostructure, each nucleic acid nanostructure comprising a plurality of surface-attached oligonucleotides; and c) attaching a unique nucleic acid nanostructure to a site of the plurality of sites, wherein attaching the nucleic acid nanostructure comprises hybridizing the surface-attached oligonucleotide of the site to the surface-attached oligonucleotide of the nucleic acid nanostructure.

[0030] In another aspect, provided herein is an array of analytes of interest, comprising: a) a solid support comprising a plurality of sites, each site comprising a surface-attached oligonucleotide; b) a plurality of nucleic acid nanostructures, each nucleic acid nanostructure configured to attach an analyte, each nucleic acid nanostructure comprising a plurality of surface-attached oligonucleotides, each surface-attached oligonucleotide being non-self-complementary, each nucleic acid nanostructure of the plurality of nucleic acid nanostructures being connected to a site of the plurality of sites by hybridization of the surface-attached oligonucleotide to the surface-attached oligonucleotide; and c) a plurality of analytes of interest, each analyte of interest being attached to a nucleic acid nanostructure of the plurality of nucleic acid nanostructures.

[0031] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0032] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0033] [Figure 1] 1A and 1B show the angular offsets for two faces of a structured nucleic acid particle (SNAP), according to some embodiments. [Figure 2]A shows two sets of tertiary structures in a SNAP according to some embodiments. B shows a cross-section of a SNAP with multiple faces according to some embodiments. C shows two sets of tertiary structures in a SNAP according to some embodiments. D shows a cross-section of a SNAP with multiple faces according to some embodiments. [Figure 3A] 1 shows a SNAP comprising a multifunctional moiety, according to some embodiments. [Figure 3B] 1 shows the linkage site of a multifunctional moiety, according to some embodiments. [Figure 3C] 1 shows a SNAP comprising a multifunctional moiety attached to a solid support, according to some embodiments. [Figure 3D] 1 shows an analyte connected to a solid support by a multifunctional moiety, according to some embodiments. [Figure 4A] 1 shows a SNAP connected to a surface, according to some embodiments. [Figure 4B] 1 shows a SNAP connected to a surface, according to some embodiments. [Figure 4C] 1 shows a SNAP connected to a surface, according to some embodiments. [Figure 4D] 1 shows a SNAP connected to a surface, according to some embodiments. [Figure 4E] 1 shows a SNAP connected to a surface, according to some embodiments. [Figure 4F] 1 shows a SNAP connected to a surface, according to some embodiments. [Figure 4G] 1 shows a SNAP connected to a surface, according to some embodiments. [Figure 4H] 1 shows a SNAP connected to a surface, according to some embodiments. [Figure 5] A, B, C, and D show SNAPs with different capture surface conformations, according to some embodiments. [Figure 6]1 illustrates a square-shaped SNAP, according to some embodiments. [Figure 7A] 1 shows a multifunctional moiety comprising an alkyl group according to some embodiments. [Figure 7B] 1 shows a multifunctional site comprising a modified oligonucleotide, according to some embodiments. [Figure 8] A, B, C, and D show SNAPs containing multifunctional moieties, according to some embodiments. [Figure 9A] 1 illustrates a method for attaching an analyte to a surface according to some embodiments. [Figure 9B] 1 illustrates a method for attaching an analyte to a surface according to some embodiments. [Figure 9C] 1 illustrates a method for attaching an analyte to a surface according to some embodiments. [Figure 9D] 1 illustrates a method for attaching an analyte to a surface according to some embodiments. [Figure 9E] 1 illustrates a method for attaching an analyte to a surface according to some embodiments. [Figure 9F] 1 illustrates a method for attaching an analyte to a surface according to some embodiments. [Figure 10A] 1 shows a SNAP containing two multifunctional sites, according to some embodiments. [Figure 10B] 1 shows a SNAP containing two multifunctional sites, according to some embodiments. [Figure 10C] 1 shows a SNAP containing two multifunctional sites, according to some embodiments. [Figure 10D] 1 shows a SNAP containing two multifunctional sites, according to some embodiments. [Figure 11] A, B, C, and D show SNAPs containing multifunctional moieties, according to some embodiments. [Figure 12] A, B, and C show SNAP complexes containing tile-shaped SNAP, according to some embodiments. [Figure 13] A, B, C, and D show different SNAP symmetries according to some embodiments. [Figure 14] A and B show three-dimensional SNAP conformations according to some embodiments. [Figure 15] A and B show different orientations of connected SNAPs, according to some embodiments. [Figure 16] A and B show a three-dimensional SNAP complex, according to some embodiments. [Figure 17] A, B, and C show arrays formed from SNAP complexes, according to some embodiments. [Figure 18] A, B, and C show arrays formed from SNAP complexes, according to some embodiments. [Figure 19] A and B show SNAP complexes formed at the interface according to some embodiments. [Figure 20] 1 illustrates a method for isolating analyte fractions onto different SNAP species according to some embodiments. [Figure 21] A and B show SNAP-protein conjugate deposition on patterned arrays. [Figure 22] 1 shows an array comprising multiple species of SNAP, according to some embodiments. [Figure 23] A and B show an array containing multiple species of SNAP, according to some embodiments. [Figure 24] 1 shows an array comprising multiple species of SNAP, according to some embodiments. [Figure 25A] 1 shows a SNAP complex on a surface including surface roughness, according to some embodiments. [Figure 25B] 1 shows a SNAP complex on a surface including surface roughness, according to some embodiments. [Figure 25C] 1 shows a SNAP complex on a surface including surface roughness, according to some embodiments. [Figure 26A]1 shows multiple SNAP complexes on a single binding site, according to some embodiments. [Figure 26B] 1 shows multiple SNAP complexes on a single binding site, according to some embodiments. [Figure 26C] 1 shows multiple SNAP complexes on a single binding site, according to some embodiments. [Figure 27] A and B show arrays containing patterned binding sites, according to some embodiments. [Figure 28] A and B show SNAP complexes attaching to a patterned surface, according to some embodiments. [Figure 29] 1 shows a three-dimensional SNAP complex, according to some embodiments. [Figure 30A] HPLC data for SNAP-protein conjugate purification is shown. [Figure 30B] HPLC data for SNAP-protein conjugate purification is shown. [Figure 30C] HPLC data for SNAP-protein conjugate purification is shown. [Figure 30D] HPLC data for SNAP-protein conjugate purification is shown. [Figure 31] 1 provides a schematic diagram of a 5-tile DNA origami SNAP, according to some embodiments. [Figure 32] A, B, C, D, E, and F show fluorescence confocal scanning microscopy image data for SNAP deposition. [Figure 33] SNAP deposition under different solvent conditions is plotted. [Figure 34] A, B, and C show fluorescence confocal scanning microscopy image data for SNAP deposition. [Figure 35] SNAP deposition under different solvent conditions is plotted. [Figure 36A]1 illustrates a scheme for generating a SNAP, according to some embodiments. [Figure 36B] 1 illustrates a scheme for generating a SNAP, according to some embodiments. [Figure 37] A and B show SNAPs containing fully structured and partially structured regions, according to some embodiments. [Figure 38] A and B show a SNAP comprising multivalent sites in an internal volume region, according to some embodiments. [Figure 39] A and B show a SNAP comprising a chemically modified interior volume region, according to some embodiments. [Figure 40A] 1A-1C show various configurations of SNAP containing multiple surface interaction sites in contact with a connecting surface comprising multiple surface attachment sites, according to some embodiments. [Figure 40B] 1A-1C show various configurations of SNAP containing multiple surface interaction sites in contact with a connecting surface comprising multiple surface attachment sites, according to some embodiments. [Figure 40C] 1A-1C show various configurations of SNAP containing multiple surface interaction sites in contact with a connecting surface comprising multiple surface attachment sites, according to some embodiments. [Figure 41] A and B show different distributions of surface interaction sites on the capture site of SNAP, according to some embodiments. [Figure 42] 1 shows a scheme for providing multiple surface attachment sites on a solid support for the purpose of facilitating binding interactions with SNAP, according to some embodiments. [Figure 43] 1 shows detection of His-12 peptide SNAP arrays with B1 aptamer probes for dual His-12 SNAP on oligonucleotide-coated surfaces. [Figure 44]1 shows the detection of a His-12 peptide SNAP array with a B1 aptamer probe for a single His-12 SNAP on an oligonucleotide-coated surface. [Figure 45] 1 shows a comparison of His-12 detection with the B1 aptamer probe for SNAP on APTMS-coated and oligonucleotide-containing surfaces. [Figure 46] 1 shows fluorescence imaging data for unpatterned SNAP arrays formed on glass surfaces containing different surface concentrations of oligonucleotides and different SNAP concentrations. [Figure 47] Fluorescence imaging data for unpatterned SNAP arrays formed by direct conjugation of SNAP to azide-containing surfaces is shown. [Figure 48] 1 illustrates the difference between the effective surface area and footprint of a nucleic acid according to some embodiments. [Figure 49] A, B, C, D, and E show aspects of nucleic acid structure and conformation, according to some embodiments. [Figure 50A] 1 illustrates a method for forming a multiplexed single-analyte array, according to some embodiments. [Figure 50B] 1 illustrates a method for forming a multiplexed single-analyte array, according to some embodiments. [Figure 50C] 1 illustrates a method for forming a multiplexed single-analyte array, according to some embodiments. [Figure 50D] 1 illustrates a method for forming a multiplexed single-analyte array, according to some embodiments. [Figure 50E] 1 illustrates a method for forming a multiplexed single-analyte array, according to some embodiments. [Figure 50F] 1 illustrates a method for forming a multiplexed single-analyte array, according to some embodiments. [Figure 51] 1 shows a nucleic acid nanostructure comprising a backbone strand and a plurality of staple oligonucleotides, according to some embodiments. [Figure 52A] 1 illustrates various configurations of nucleic acid nanostructures, including compressed and transparent structures, according to some embodiments. [Figure 52B] 1 illustrates various configurations of nucleic acid nanostructures, including compressed and transparent structures, according to some embodiments. [Figure 52C] 1 illustrates various configurations of nucleic acid nanostructures, including compressed and transparent structures, according to some embodiments. [Figure 52D] 1 illustrates various configurations of nucleic acid nanostructures, including compressed and transparent structures, according to some embodiments. [Figure 52E] 1 illustrates various configurations of nucleic acid nanostructures, including compressed and transparent structures, according to some embodiments. [Figure 52F] 1 illustrates various configurations of nucleic acid nanostructures, including compressed and transparent structures, according to some embodiments. [Figure 52G] 1 illustrates various configurations of nucleic acid nanostructures, including compressed and transparent structures, according to some embodiments. [Figure 52H] 1 illustrates various configurations of nucleic acid nanostructures, including compressed and transparent structures, according to some embodiments. [Figure 53] A, B, C, D, and E show various configurations of nucleic acid nanostructures including permeation structures configured to form multivalent binding interactions, according to some embodiments. [Figure 54A] 1 illustrates a method for forming nucleic acid nanostructures having permeation structures, according to some embodiments. [Figure 54B] 1 illustrates a method for forming nucleic acid nanostructures having permeation structures, according to some embodiments. [Figure 54C] 1 illustrates a method for forming nucleic acid nanostructures having permeation structures, according to some embodiments. [Figure 55A]1 illustrates a method for forming multivalent binding interactions between a nucleic acid nanostructure and a solid support, according to some embodiments. [Figure 55B] 1 illustrates a method for forming multivalent binding interactions between a nucleic acid nanostructure and a solid support, according to some embodiments. [Figure 55C] 1 illustrates a method for forming multivalent binding interactions between a nucleic acid nanostructure and a solid support, according to some embodiments. [Figure 55D] 1 illustrates a method for forming multivalent binding interactions between a nucleic acid nanostructure and a solid support, according to some embodiments. [Figure 56A] 1A-1C show various configurations of nucleic acid nanostructures comprising permeation structures, according to some embodiments, where the permeation structures form multivalent binding interactions with a solid support. [Figure 56B] 1A-1C show various configurations of nucleic acid nanostructures comprising permeation structures, according to some embodiments, where the permeation structures form multivalent binding interactions with a solid support. [Figure 56C] 1A-1C show various configurations of nucleic acid nanostructures comprising permeation structures, according to some embodiments, where the permeation structures form multivalent binding interactions with a solid support. [Figure 57] 1 illustrates conformational changes for nucleic acid nanostructures upon surface binding interactions, according to some embodiments. [Figure 58A] 1 illustrates a method for reconfiguring the binding configuration of nucleic acid nanostructures connected to array sites, according to some embodiments. [Figure 58B] 1 illustrates a method for reconfiguring the binding configuration of nucleic acid nanostructures connected to array sites, according to some embodiments. [Figure 58C] 1 illustrates a method for reconfiguring the binding configuration of nucleic acid nanostructures connected to array sites, according to some embodiments. [Figure 59A] 1 shows atomic force microscopy images of nucleic acid nanostructures. [Figure 59B] 1 shows atomic force microscopy images of nucleic acid nanostructures. [Figure 59C] Various measurements of nucleic acid nanostructure yield and size are plotted. [Figure 59D] Various measurements of nucleic acid nanostructure yield and size are plotted. [Figure 60A] 1A-1C illustrate various configurations of array sites containing more than one type of bound surface moiety, according to some embodiments. [Figure 60B] 1A-1C illustrate various configurations of array sites containing more than one type of bound surface moiety, according to some embodiments. [Figure 60C] 1A-1C illustrate various configurations of array sites containing more than one type of bound surface moiety, according to some embodiments. [Figure 60D] 1A-1C illustrate various configurations of array sites containing more than one type of bound surface moiety, according to some embodiments. [Figure 61A] 1 illustrates steps in a method for attaching nucleic acid nanostructures to a solid support using unreacted functional groups, according to some embodiments. [Figure 61B] 1 illustrates steps in a method for attaching nucleic acid nanostructures to a solid support using unreacted functional groups, according to some embodiments. [Figure 61C] 1 illustrates steps in a method for attaching nucleic acid nanostructures to a solid support using unreacted functional groups, according to some embodiments. [Figure 61D] 1 illustrates steps in a method for attaching nucleic acid nanostructures to a solid support using unreacted functional groups, according to some embodiments. [Figure 61E] 1 illustrates steps in a method for attaching nucleic acid nanostructures to a solid support using unreacted functional groups, according to some embodiments. [Figure 62A] 1 illustrates a method of forming an array configured to generate a multiplexed array of analytes, according to some embodiments. [Figure 62B]1 illustrates a method of forming an array configured to generate a multiplexed array of analytes, according to some embodiments. [Figure 62C] 1 illustrates a method of forming an array configured to generate a multiplexed array of analytes, according to some embodiments. [Figure 62D] 1 illustrates a method for depositing more than one type of analyte to form a multiplexed array, according to some embodiments. [Figure 62E] 1 illustrates a method for depositing more than one type of analyte to form a multiplexed array, according to some embodiments. [Figure 63] 1 illustrates multiple sites of an array containing various defects or disruptions, according to some embodiments. [Figure 64] 1 illustrates an array of analytes formed by non-lithographic methods, according to some embodiments. [Figure 65] 1 illustrates a method for forming an array of analytes via charge-mediated interactions, according to some embodiments. [Figure 66] A, B, C, and D show various shapes and morphologies of array features formed according to some embodiments. [Figure 67A] 1 shows a schematic diagram of a functionalized array site, according to some embodiments. [Figure 67B] Fluorescence microscopy characterization of arrays formed by lithographic patterning is shown. [Figure 67C] 1 shows atomic force microscopy data of the surface roughness of array sites formed by lithographic patterning. [Figure 67D] Data are plotted for average array site diameter and site pitch for arrays formed by lithographic patterning. [Figure 67E] Data are plotted for average array site diameter and site pitch for arrays formed by lithographic patterning. [Figure 68]Fluorescence microscopy images of cycles of binding and stripping of fluorescently labeled oligonucleotides from functional nucleic acids are shown. [Figure 69] A, B, C, and D show fluorescence microscopy images of multiplexed arrays during binding and stripping of fluorescently labeled oligonucleotides with functional acids of structured nucleic acid particles. [Figure 70] Fluorescence microscopy images of arrays containing functional nucleic acids of different nucleotide sequence lengths during binding and stripping of fluorescently labeled oligonucleotides are shown. DETAILED DESCRIPTION OF THE INVENTION

[0034] Molecular ordering at the nanoscale is a key issue for many technologies, including analytical and biological analytical methods, catalysis and biocatalysis, micro- and nanofluidics, and micro- and nanoelectronics. Methods for arranging molecules at surfaces or interfaces are of particular interest, as the length scale of surface features or surface irregularities often approaches the length scale of the molecules arranged at the surface or interface. For example, single-molecule analytical techniques are of interest for numerous biological applications, including genomics, transcriptomics, and proteomics. The formation of single-analyte biomolecular arrays can be limited by nanoscale and / or single-molecule effects, which can alternately cause restricted or excessive biomolecule deposition at binding sites on single-analyte arrays. For example, defects in the nanoscale fabrication of solid surfaces can generate sites with aberrant binding properties, thereby creating localized defects in array patterning. Similarly, thermodynamic (e.g., entropy) and / or kinetic (e.g., slow dissociation) effects can cause unintended phenomena (e.g., molecular colocalization) at array sites given a sufficiently large sample of molecules. Consequently, methods for preparing consistent surfaces or interfaces and carefully controlling the deposition of molecules on the surfaces or interfaces are important when forming single-analyte arrays.

[0035] Many single-analyte array-based technologies favor the formation of substantially uniform arrays, both in terms of having a single analyte present at substantially all array sites of the single-analyte array (i.e., array site occupancy value > 0 analytes) and in terms of having no more than one single analyte at each array site of the single-analyte array (i.e., array site occupancy value = 1 analyte). The uniformity of single-analyte arrays can increase as the Poisson-like probability distribution narrows around the array site occupancy value of an analyte. Therefore, array formation methods that facilitate such narrowing of the probability mass function around the array site occupancy value of an analyte are preferred for the formation of single-analyte arrays.

[0036] Intermediate particles offer a potential approach for controlling the deposition of molecules on surfaces or interfaces. Particularly useful intermediate particles have tunable characteristics that allow them to selectively interact with surfaces or interfaces while desirably presenting analytes and other molecules thereon. Surfaces can be easily patterned using nanofabrication techniques to create uniquely configured sites or addresses for capturing the particles described herein. In this manner, surfaces can be patterned with arrays of sites configured for capturing multiple particles. By using multiple particles, each bound to a different analyte, arrays of different analytes can be formed on a surface in a predetermined pattern appropriate for a desired analytical assay method, such as the analytical method described herein. An exemplary intermediate particle is a structured nucleic acid particle (SNAP), e.g., nucleic acid origami. The tunability of such particles arises from the helical nature of nucleic acid tertiary structures. Over the course of a single helical rotation, a nucleic acid helix can orient attached ligands in virtually any direction across its entire 360° appearance. Consequently, structured nucleic acid particles can be engineered to present bound molecules at specific locations and orientations on the particle, allowing multiple bound molecules to be optimally separated and positioned for best cure. Other nucleic acid nanostructures can be similarly configured as intermediate particles for presenting analytes on surfaces.

[0037] Described herein are structured nucleic acid particles and systems that can be used to facilitate the formation of single-molecule arrays of analytes and other molecules. In certain configurations, the structured nucleic acid particles contain several structural features that increase the specificity of connection interactions on a surface or interface or reduce the particle's sensitivity to defects or irregularities on the surface or interface, thereby enabling the formation of uniform single-molecule arrays. In particular, provided herein are systems including structured nucleic acid particles and solid supports in which complementary chemistry facilitates the controlled deposition of single-analyte arrays. Each structured nucleic acid particle can be connected to one or more analytes of interest, enabling the formation of uniform arrays of analytes on a surface or interface. For example, the analytes of interest can be nucleic acids, proteins, metabolites, or other targets of interest for analytical characterization. In another example, the analytes can be reagents used for synthetic methods, such as the synthesis of nucleic acids, proteins, small molecules, potential therapeutic agents, non-biological polymers, etc.

[0038] Also described herein are complexes that can be formed by connecting multiple structured nucleic acid particles. The complexes can increase the efficiency and control of analyte or molecule presentation at a surface or interface by increasing surface binding sites and binding interactions and / or reducing the likelihood of unwanted analyte or molecule co-deposition at a single location on the surface or array. In some configurations, structured nucleic acid complexes can be configured to form self-assembling or self-patterning arrays for presentation or analytes or other molecules.

[0039] definition As used herein, the terms "nucleic acid nanostructure" or "nucleic acid nanoparticle" refer synonymously to a single- or multi-stranded polynucleotide molecule comprising a compacted three-dimensional structure. The compacted three-dimensional structure can optionally have a distinctive tertiary structure. An exemplary nucleic acid nanostructure is a structured nucleic acid particle (SNAP). SNAPs can be configured to have an increased number of interactions between regions of polynucleotide strands, shorter distances between regions, an increased number of bends in the strands, and / or sharper bends in the strands, compared to the same nucleic acid molecule in a random coil or other unstructured state. Alternatively or additionally, the compacted three-dimensional structure of a nucleic acid nanostructure can optionally have a distinctive quaternary structure. For example, a nucleic acid nanostructure can be configured to have an increased number of interactions between polynucleotide strands or shorter distances between strands, compared to the same nucleic acid molecule in a random coil or other unstructured state. In some configurations, the tertiary structure of a nucleic acid nanostructure (i.e., the helical twist or orientation of the polynucleotide strands) can be configured to be denser than the same nucleic acid molecule in a random coil or other unstructured state. Nucleic acid nanostructures can include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), other nucleic acid analogs, and combinations thereof. Nucleic acid nanostructures can have naturally occurring or engineered secondary, tertiary, or quaternary structures. Structured nucleic acid particles can contain at least one of: i) a moiety configured to connect an analyte to the nucleic acid nanostructure; ii) a moiety configured to connect the nucleic acid nanostructure to another object, such as another SNAP, a solid support, or its surface; iii) a moiety configured to provide a chemical or physical property or characteristic to the nucleic acid nanostructure; or iv) a combination thereof. Exemplary SNAPs can include nanoballs (e.g., DNA nanoballs), nucleic acid nanotubes (e.g., DNA nanotubes), and nucleic acid origami (e.g., DNA origami nucleic acids). SNAPs can be functionalized to include one or more reactive handles or other moieties. SNAPs can include one or more incorporated residues containing reactive handles or other moieties (e.g., modified nucleotides).

[0040] As used herein, the term "primary structure" when used in reference to nucleic acids refers to the residue sequence of a single-stranded nucleic acid. As used herein, the term "secondary structure" when used in reference to nucleic acids refers to the base-pairing interactions within a single nucleic acid polymer or between two polymers. Secondary structures can include multi-stranded nucleic acids formed by the self-complementarity of a single oligonucleotide, such as stems, loops, bulges, and junctions. As used herein, the term "tertiary structure" when used in reference to nucleic acids refers to the three-dimensional conformation of nucleic acids, such as the overall three-dimensional shape of a single-stranded or multi-stranded nucleic acid.

[0041] As used herein, the term "transport" when used in reference to a nucleic acid structure refers to a structure containing two or more structural elements (e.g., single-stranded nucleic acid, double-stranded nucleic acid, nucleic acid chain containing double-stranded and single-stranded nucleic acid, non-nucleic acid moiety, etc.) that have spatial freedom (e.g., translational, rotational, vibrational, bending, etc.) to facilitate contact between the two or more structural elements and another molecule. The other molecule may be, for example, a molecule having a molecular weight greater than 0.5 kilodaltons, 1 kilodalton, 5 kilodaltons, 10 kilodaltons, or more. Optionally, each structural element of the two or more structural elements can move in concert with the movement of the nucleic acid. Optionally, for an unbound nucleic acid containing a transmission structure containing multiple pendant non-interacting moieties, each pendant moiety rotates when the nucleic acid rotates, but the free end of each pendant moiety can move independently of the movement of the other free ends of the other pendant moieties. Spatial freedom can be assessed for structural elements of nucleic acids with respect to natural and / or stochastic spatial variations in the structure of the nucleic acid (e.g., spatial freedom including motion beyond the natural thermal or Brownian motion of the nucleic acid structure). A first structural element of a permeable structure can have spatial freedom relative to a second structural element in one spatial dimension, two spatial dimensions, or three spatial dimensions. Permeable structures can be characterized as containing different chemical features from the compacted structures of nucleic acids shown herein, such as higher or lower mass diffusivity for small molecules or macromolecules, higher or lower hydrophobicity, higher or lower hydrophilicity, higher or lower binding strength or specificity for another nucleic acid, higher or lower likelihood of binding to another nucleic acid, higher or lower likelihood of binding to a solid support, higher or lower binding strength or specificity for a solid support, or a combination thereof. Permeable structures can contain different characteristics or configurations when bound to another entity (e.g., a solid support, a second nucleic acid).In some configurations, when bound to a second entity, the transmission structure may satisfy one or more of the following: i) each structural element of the two or more structural elements moves in coordination with the movement of the nucleic acid; ii) each structural element of the two or more structural elements has a reduced spatial degree of freedom relative to the unbound configuration; and iii) each structural element of the two or more structural elements contains at least one spatial degree of freedom (e.g., translational, rotational, vibrational, bending, etc.) relative to each other structural element of the two or more structural elements. For example, for a nucleic acid connected to a solid support by a transmission structure containing multiple pendant non-interacting moieties, each pendant moiety may be connected to a complementary moiety on the solid support, thereby colocalizing the nucleic acid and the transmission structure on the solid support, but each pendant moiety may possess the independent ability to disrupt existing interactions with complementary surface moieties and form new interactions with different complementary surface moieties.

[0042] As used herein, the term "residue," when used in reference to a polymer, refers to a monomeric unit of the polymer structure. When used in reference to a nucleic acid, a residue can refer to a nucleotide, a nucleoside, or a synthetic, modified, or non-natural analog thereof. When used in reference to a polypeptide, a residue can refer to an amino acid, or a synthetic, modified, or non-natural analog thereof.

[0043] As used herein, the term "type" or "species," when used in reference to a molecule, refers to a molecule having a unique, distinguishable chemical structure. As used herein, the term "SNAP type" refers to a SNAP having a unique, distinguishable primary structure, for example, compared to other SNAPs. Two SNAPs are the same species if they possess the same primary, secondary, or tertiary structure. SNAP variants are species that differ from each other. For example, members of a "SNAP type" may have a unique, distinguishable structure common to them compared to other SNAPs that lack a unique, distinguishable structure. SNAP types can be identified, for example, by a common shape and / or conformation, the number of connection sites, or the type of connection site.

[0044] As used herein, the terms "click reaction," "click-type reaction," or "bioorthogonal reaction" refer to a single-step, thermodynamically favorable conjugation reaction utilizing a biocompatible reagent. Click reactions can be configured to avoid the use of toxic or biologically incompatible reagents (e.g., acids, bases, heavy metals) or the production of toxic or biologically incompatible by-products. Click reactions can utilize aqueous solvents or buffers (e.g., phosphate buffer, Tris buffer, saline buffer, MOPS, etc.). A click reaction may be thermodynamically favorable if it has a negative Gibbs free energy of reaction, e.g., less than about -5 kilojoules / mol (kJ / mol), -10 kJ / mol, -25 kJ / mol, -50 kJ / mol, -100 kJ / mol, -200 kJ / mol, -300 kJ / mol, -400 kJ / mol, or less than -500 kJ / mol. Exemplary bioorthogonal and click reactions are described in detail in WO 2019 / 195633 A1 (incorporated herein by reference in its entirety). Exemplary click reactions can include metal-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, strain-promoted azide-nitrone cycloaddition, strained alkene reaction, thiol-ene reaction, Diels-Alder reaction, inverse electron demand Diels-Alder reaction, [3 + 2] cycloaddition, [4 + 1] cycloaddition, nucleophilic substitution, dihydroxylation, thiol-yne reaction, photoclick, nitrone dipole cycloaddition, norbornene cycloaddition, oxanobornadiene cycloaddition, tetrazine ligation, and tetrazole photoclick reaction. Exemplary functional groups or reactive handles utilized to perform click reactions can include alkenes, alkynes, azides, epoxides, amines, thiols, nitrones, isonitriles, isocyanides, aziridines, activated esters, and tetrazines. Other well-known click conjugation reactions with complementary bioorthogonal reactive species can be used, for example, where a first click component contains a hydrazine moiety and a second click component contains an aldehyde or ketone group, and the product of such a reaction contains a hydrazone functional group or equivalent.

[0045] As used herein, the term "array" refers to a collection of molecules or analytes bound to unique identifiers so that the analytes can be distinguished from one another. As used herein, the term "unique identifier" refers to a solid support (e.g., particle or bead), a spatial address in an array, a tag, a label (e.g., luminophore), or a barcode (e.g., nucleic acid barcode) that is bound to an analyte and distinguished from other identifiers through one or more steps of a process. The process can be an analytical process, e.g., a method for detecting, identifying, characterizing, or quantifying an analyte. Binding to the unique identifier can be covalent or non-covalent (e.g., ionic bonding, hydrogen bonding, van der Waals forces, etc.). The unique identifier can be exogenous to the analyte, e.g., synthetically bound to the analyte. Alternatively, the unique identifier can be endogenous to the analyte, e.g., bound or associated with the analyte in its native environment. An array can include different analytes, each bound to a different unique identifier. For example, an array can include different molecules or analytes, each located at a different address on a solid support. Alternatively, an array can include separate solid supports, each serving as an address bearing a different molecule or analyte, which can be identified according to the position of the solid support on a surface to which the solid support is attached or according to the position of the solid support in a liquid, e.g., fluid flow. The molecules or analytes of the array can be, for example, nucleic acids, e.g., SNAP, polypeptides, proteins, peptides, oligopeptides, enzymes, ligands, or receptors, e.g., antibodies, functional fragments of antibodies, or aptamers. The addresses of the array can optionally be optically observable, and in some configurations, adjacent addresses can be optically distinguishable when detected using the methods or devices described herein. As used herein, the terms "address," "binding site," and "site," when used in reference to an array, refer to the location in the array where a particular molecule or analyte is present.An address may contain only a single molecule or analyte, or it may contain a population of several molecules or analytes of the same species (i.e., a collection of molecules). Alternatively, an address may include populations of molecules or analytes of different species. The addresses of an array are typically separate. Separate addresses may be continuous, or they may have interstitial spaces between them. Arrays useful herein may have addresses separated by, for example, 100 microns, 10 microns, 1 micron, 500 nm, 100 nm, 10 nm or less. Alternatively or additionally, arrays may have addresses separated by at least 10 nm, 100 nm, 500 nm, 1 micron, 5 microns, 10 microns, 50 microns, 100 microns or more. Each address may have an area of ​​less than 1 square millimeter, 500 square microns, 100 square microns, 25 square microns, 1 square micron or less. An array may have at least about 1x10 4 , 1x10 5 , 1x10 6 , 1x10 8 , 1x10 10 , 1x10 12 One or more addresses may be included.

[0046] As used herein, the term "solid support" refers to a substrate that is insoluble in aqueous liquids. Optionally, the substrate may be rigid. The substrate may be non-porous or porous. The substrate may optionally be capable of absorbing liquid (e.g., by being porous), but is typically, but not necessarily, sufficiently rigid so that the substrate does not substantially expand when absorbing liquid and does not substantially shrink when the liquid is removed by drying. Non-porous solid supports are usually impermeable to liquids or gases. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon™, cyclic olefins, polyimides, etc.), nylon, ceramics, resins, Zeonor™, silica or silica-based materials, including silicon and modified silicon, carbon, metals, metal oxides (e.g., zirconia, titania, alumina, etc.), inorganic glass, fiber optic bundles, gels, and polymers.

[0047] As used herein, the terms "group" and "site" are intended to be synonymous when used in reference to the structure of a molecule. The terms refer to a component or portion of a molecule. The terms do not necessarily indicate the relative size of the component or portion compared to the remainder of the molecule, unless otherwise indicated. A group or site may contain one or more atoms. As used herein, the term "presentation site" refers to a component or portion of a molecule configured to attach or allow a molecule to attach to an analyte. As used herein, the term "capture site" refers to a component or portion of a molecule configured to attach or allow a molecule to attach to a solid support, surface, or interface. As used herein, the term "attachment site" refers to a component or portion of a molecule configured to attach or allow a molecule to attach to a second molecule. As used herein, the term "utilization site" refers to a component or portion of a molecule configured to provide or allow a molecule to provide functionality or structure. The functionality or structure can be a new function or structure not provided by the presentation, capture, or connection site of the molecule; or it can be a modification (e.g., inhibition or activation) of the structure or function provided by the presentation, capture, or connection site of the molecule.

[0048] As used herein, the term "surface" refers to a portion of a molecule, particle, or complex (e.g., a SNAP or SNAP complex) containing one or more moieties with substantially similar orientation and / or function. For example, a substantially rectangular or square SNAP may have a connection surface including one or more connection sites, each of which has a substantially similar orientation to each other (e.g., oriented approximately 180° from a presentation site configured to connect to an analyte). In another example, a spherical nanoparticle may have a connection surface including multiple connected connection sites confined to a hemisphere of the particle (i.e., the multiple connection sites have similar functions but different orientations). In some cases, a surface may be defined by a relative imaginary plane along which a site or portion thereof may have spatial proximity or angular orientation when the plane contacts a point or portion of the molecule, particle, or complex. A site or portion thereof can have a spatial separation from an imaginary plane defining the surface of the molecule, particle, or complex of about 100 nanometers (nm), 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 25 nm, 20 nm, 15 nm, 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, 0.5 nm, 0.1 nm or less, or less than 0.1 nm. o , 85 o , 80 o , 75 o , 70 o , 65 o , 60 o , 55 o , 50 o , 45 o , 40 o , 35 o , 30 o ,twenty five o , 20 o , 15 o , 10 o , 5 o , 1 o Less than or equal to 1 o The angle .theta. .gtoreq..times ...

[0049] As used herein, the terms "analyte" and "analyte of interest," when used in reference to a structured nucleic acid particle, refer to a molecule, particle, or complex of molecules or particles connected to a presentation site on the structured nucleic acid particle. An analyte may comprise a target for an analytical method (e.g., sequencing, identification, quantification, etc.) or may comprise a functional element, such as a binding ligand or catalyst. An analyte may comprise a biomolecule, such as a polypeptide, polysaccharide, nucleic acid, lipid, metabolite, enzyme cofactor, or a combination thereof. An analyte may comprise a non-biological molecule, such as a polymer, metal, metal oxide, ceramic, semiconductor, mineral, or a combination thereof. As used herein, the term "sample analyte" refers to an analyte derived from a sample collected from a biological or non-biological system. A sample analyte may be purified or unpurified. As used herein, the term "control analyte" refers to an analyte provided as a positive or negative control for comparison to a sample analyte. A control analyte may be derived from the same source as the sample analyte or from a different source than the sample analyte. As used herein, the term "standard analyte" refers to a known or characterized analyte that serves as a physical or chemical reference for a process. A standard analyte may contain the same type of analyte as the sample analyte, or may be different from the sample analyte. For example, a polypeptide analyte process may utilize a polypeptide standard analyte with known characteristics. In another example, a polypeptide analyte process may utilize a non-polypeptide standard analyte with known characteristics. As used herein, the term "inert analyte" refers to an analyte that does not have an expected function in a process or system.

[0050] As used herein, the terms "linker," "linking group," or "linking moiety" refer to a molecule or molecular chain configured to connect a first molecule to a second molecule. The linker, linking group, or linking moiety can be configured to provide chemical or mechanical properties, such as hydrophobicity, hydrophilicity, charge, polarity, rigidity, or flexibility, to the region separating the first molecule from the second molecule. The linker, linking group, or linking moiety can contain two or more functional groups that facilitate connection of the linker, linking group, or linking moiety to the first and second molecules. The linker, linking group, or linking moiety can include multifunctional linkers, such as homobifunctional linkers, heterobifunctional linkers, homomultifunctional linkers, and heteromultifunctional linkers. Molecular chains may be characterized by a minimum size such as, for example, at least about 100 Daltons (Da), 200 Da, 300 Da, 400 Da, 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1 kilodalton (kDa), 2 kDa, 3 kDa, 4 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa or greater than 20 kDa. Alternatively or additionally, the molecular tethers may be characterized by a minimum size, such as, for example, about 20 kDa, 15 kDa, 10 kDa, 5 kDa, 4 kDa, 3 kDa, 2 kDa, 1 kDa, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, 100 Da or less, or less than 100 Da, etc. Exemplary molecular tethers may include polyethylene glycol (PEG), polyethylene oxide (PEO), alkane chains, fluorinated alkane chains, dextran, and polynucleotides.

[0051] As used herein, the terms "reversible" and "reversibility" refer to a chemical or physical connection between two entities (e.g., molecules, analytes, functional groups, or moieties) that has a substantial potential for disconnection under one or more conditions of use. Reversibility can consist of thermodynamic reversibility, kinetic reversibility, or a combination thereof. A reversible connection of a first entity to a second entity can be characterized by a temporal change to the structure or function of the first and / or second entities when connected to each other. Reversing the connection can optionally return the structure or function of the first and / or second entities to the same state as before the temporal change. Context for determining reversibility can include the possibility of detecting a reversed connection given the specific spatial, temporal, and physical environment in which the two connected molecules are located. For example, in a population of one million streptavidin-biotin conjugated pairs, a detectable number of reversed conjugations can be thermodynamically predicted, but the slow kinetic reversal of the binding reaction may produce such unconjugations that are not detectable above the detection noise when the detection time scale is on the order of seconds or minutes. In this context, streptavidin-biotin conjugations would be described as irreversible. The context of irreversibility can be process-dependent for systems that undergo multiple processes. For example, measurable unconjugation of conjugated molecules may occur during months of storage, while subsequent processes utilizing the conjugated molecules may occur within minutes. In this context, conjugated molecules may be reversibly conjugated for storage but irreversibly conjugated for utilization. Indicators of reversibility can include the use of quantitative indicators, such as equilibrium constants or kinetic on-rates and / or off-rates. Reversibility can be measured directly by equilibrium assays. Reversibility can change with changes in the chemical system, such as changes in temperature or solvent composition. A reversible connection can include a metastable connection that remains connected until the physical environment changes, for example, complementary nucleic acids may remain stably connected at 20°C, but rapidly become unconnected above 75°C.The reversible connection may remain connected for at least about 1 second(s), 1 minute (min), 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour (hr), 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 1 day, 1 week, 1 month, 6 months, 1 year, or for more than 1 year. Alternatively or additionally, the reversible connection may be disconnected in a period of about 1 year, 6 months, 1 month, 1 week, 1 day, 18 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, 1 minute, 1 second or less, or less than 1 second.

[0052] As used herein, the terms "irreversible" and "irreversibility" are used in reference to a chemical or physical connection between two entities (e.g., molecules, analytes, functional groups, or moieties) that has the potential to remain connected under one or more conditions of use. A system determined to be non-reversible as described above may be described as irreversible. For example, irreversible connection of a first entity to a second entity may be characterized by a permanent change to the structure or function of the first and / or second entities after they are connected to one another. Disconnection may cause a substantial change to the structure or function of one or both of the entities compared to the structure or function of the respective entity or entities prior to connection. An irreversible connection may remain connected for a period of at least about 1 second(s), 1 minute (min), 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour (hr), 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 1 day, 1 week, 1 month, 6 months, 1 year, or more than 1 year.

[0053] As used herein, the term "affinity reagent" refers to a molecule or other substance capable of specifically or reproducibly binding to a binding partner or other substance. Binding can optionally be used to identify, track, capture, modify, or affect the binding partner. The binding partner can optionally be larger, smaller, or the same size as the affinity reagent. The affinity reagent can form a reversible or irreversible interaction with the binding partner. The affinity reagent can bind to the binding partner in a covalent or non-covalent manner. The affinity reagent can be configured to perform a chemical modification (e.g., ligation, cleavage, linkage, etc.) that produces a detectable change in the larger molecule, thereby allowing for observation of the resulting interaction. Affinity reagents can include chemically reactive affinity reagents (e.g., kinases, ligases, proteases, nucleases, etc.) and chemically non-reactive affinity reagents (e.g., antibodies, antibody fragments, aptamers, DARPins, peptamers, etc.). Affinity reagents can contain one or more known and / or characterized binding moieties or binding sites (e.g., complementarity-defining regions) that mediate or facilitate binding with a binding partner. Thus, affinity reagents can be monovalent or multivalent (e.g., bivalent, trivalent, tetravalent, etc.). Affinity reagents are typically non-reactive and non-catalytic, thereby not permanently altering the chemical structure of the substances to which they bind in the methods described herein.

[0054] As used herein, the terms "protein" and "polypeptide" are used interchangeably to refer to a molecule or analyte of two or more amino acids joined by peptide bonds. Polypeptide can refer to a peptide (e.g., a polypeptide having less than about 200, 150, 100, 75, 50, 40, 30, 20, 15, 10, or less than about 10 amino acids). Polypeptide can refer to a naturally occurring molecule or an artificial or synthetic molecule. A polypeptide can include one or more non-natural, modified amino acids, or a non-amino acid linker. A polypeptide can contain D-amino acid enantiomers, L-amino acid enantiomers, or both. A polypeptide can be modified naturally or synthetically, for example, by post-translational modification.

[0055] As used herein, the term "detectable label" refers to a moiety on an affinity reagent or other substance that provides a detectable characteristic. The detectable characteristic can be, for example, an optical signal, such as radiation absorbance, luminescence or fluorescence emission, luminescence or fluorescence lifetime, luminescence or fluorescence polarization, etc.; Rayleigh and / or Mie scattering; binding affinity for a ligand or receptor; magnetic properties; electrical properties; charge; mass; radioactivity, etc. The labeling component can be a detectable chemical entity that is conjugated to or can be conjugated to another molecule or substance. Exemplary molecules that can be conjugated to the labeling component include affinity reagents or binding partners. The labeling component can generate a signal (e.g., fluorescence, luminescence, radioactivity) that is detected in real time. The labeling component can generate a signal that is detected offline (e.g., nucleic acid barcode) or in a time-resolved manner (e.g., time-resolved fluorescence). The labeling component can generate a signal with a characteristic frequency, intensity, polarization, duration, wavelength, sequence, or fingerprint. Exemplary labels include, but are not limited to, fluorophores, luminophores, chromophores, nanoparticles (e.g., gold, silver, carbon nanotubes), heavy atoms, radioisotopes, mass labels, charge labels, spin labels, receptors, ligands, nucleic acid barcodes, polypeptide barcodes, polysaccharide barcodes, and the like.

[0056] As used herein, the term "nucleic acid origami" refers to a nucleic acid construct containing engineered secondary, tertiary, or quaternary structures. Nucleic acid origami may include DNA, RNA, PNA, LNA, other nucleic acid analogs, modified or non-natural nucleic acids, or combinations thereof. Nucleic acid origami may include multiple oligonucleotides that hybridize via sequence complementarity to produce engineered structuring of the origami particle. Nucleic acid origami may include sections of single-stranded or double-stranded nucleic acids, or combinations thereof. Nucleic acid origami may include one or more tertiary structures of nucleic acids, such as A-DNA, B-DNA, C-DNA, L-DNA, M-DNA, and Z-DNA. Nucleic acid origami may include single-stranded nucleic acids, double-stranded nucleic acids, multi-stranded nucleic acids, or combinations thereof. Exemplary nucleic acid origami structures may include nanotubes, nanowires, cages, tiles, nanospheres, blocks, and combinations thereof.

[0057] As used herein, the term "nucleic acid nanoball" refers to a spherical or spherical nucleic acid structure. A nucleic acid nanoball may comprise a concatemer of oligonucleotides arranged in a spherical structure. A nucleic acid nanoball may comprise one or more oligonucleotides, including oligonucleotides comprising self-complementary nucleic acid sequences. A nucleic acid nanoball may comprise a palindromic nucleic acid sequence. A nucleic acid nanoball may comprise DNA, RNA, PNA, LNA, other nucleic acid analogs, modified or non-natural nucleic acids, or a combination thereof.

[0058] As used herein, the term "oligonucleotide" refers to a molecule containing two or more nucleotides joined by a phosphodiester bond or its analog. Oligonucleotides can contain DNA, RNA, PNA, LNA, other nucleic acid analogs, modified nucleotides, non-natural nucleotides, or combinations thereof. Oligonucleotides can contain a limited number of linked nucleotides, such as less than about 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, or 5 nucleotides. Oligonucleotides can contain linking groups or linking sites at terminal or intermediate positions. For example, oligonucleotides can contain two nucleic acid strands joined by an intermediate PEG molecule. In another example, oligonucleotides can contain a cleavable linker (e.g., a photocleavable linker, an enzymatically cleavable linker, a restriction site, etc.) that joins the two portions of the oligonucleotide. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein with the term "oligonucleotide."

[0059] As used herein, the term "scaffold" refers to a molecule or complex of molecules with a structure that connects two or more entities to each other. The scaffold can form a structural basis for connecting binding moieties and / or labeling moieties to detectable probes. The scaffold can contain multiple binding sites that allow for the attachment or conjugation of detectable probe moieties to the scaffold. The scaffold binding sites can include functional groups, active sites, binding ligands, binding receptors, nucleic acid sequences, or any other entities capable of forming covalent or non-covalent bonds to binding moieties, labeling moieties, or other detectable probe moieties. The scaffold can include oligonucleotide molecules that serve as the main structural units for nucleic acid origami. The scaffold can include single-stranded nucleic acids, double-stranded nucleic acids, or combinations thereof. The scaffold can be a circular oligonucleotide or a linear (i.e., non-circular) oligonucleotide. The scaffold can be derived from natural sources, such as bacterial or viral genomes (e.g., plasmid DNA or phage genomes). Circular scaffolds can be formed by ligation of non-circular nucleic acids. The backbone can comprise a certain number of nucleotides, for example, at least about 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, or more than 10000 oligonucleotides. The backbone can comprise organic or inorganic particles or nanoparticles. The backbone can include a coating or layer applied to the particles or nanoparticles that allows for attachment of a detectable label moiety.

[0060] As used herein, the term "two-dimensional protrusion" refers to the area or shape that would be occupied by the protrusion of a three-dimensional structure onto a flat two-dimensional surface without substantial geometric or spatial distortion. For example, the two-dimensional protrusion of a sphere onto a flat two-dimensional surface would generate a circular area on the surface with a diameter equal to the diameter of the sphere. Two-dimensional protrusions can be formed from any reference frame, including a reference frame that is perpendicular to any surface of the three-dimensional structure. Many three-dimensional structures can generate protrusions of different sizes or shapes depending on the reference frame. Thus, the maximum two-dimensional protrusion for a three-dimensional structure refers to the largest area or shape generated from all reference frames for the three-dimensional structure; the minimum two-dimensional protrusion for a three-dimensional structure refers to the smallest area or shape generated from all reference frames for the three-dimensional structure; and the average two-dimensional protrusion for a three-dimensional structure refers to the average area or shape generated from all reference frames for the three-dimensional structure.

[0061] As used herein, the term "effective surface area," when used with reference to a nucleic acid, refers to the surface area of ​​the two-dimensional protrusion of a nucleic acid, or a portion thereof, when the nucleic acid is not bound to a surface (e.g., solvated or suspended in a fluid medium). As used herein, the term "footprint," when used with reference to a nucleic acid, refers to the surface area of ​​the two-dimensional protrusion of a nucleic acid, or a portion thereof, when the nucleic acid is bound to a surface (e.g., attached to a solid support). Figure 48 illustrates the difference between the effective surface area and footprint of a nucleic acid. In the unbound configuration, the two-dimensional protrusion of nucleic acid 4810 onto surface 4800 will have a surface area proportional to length 11, which is substantially the same as the distance between the two ends of unbound nucleic acid 4810. In the bound configuration, the attachment of nucleic acid 4810 to surface 4800 increases the distance between the ends of the nucleic acid, thereby increasing the surface area of ​​the two-dimensional protrusion of the nucleic acid onto surface 4800. Thus, the nucleic acid has a footprint that is larger than its effective surface area.

[0062] As used herein, the term "offset" refers to a spatial difference in orientation between two lines (two dimensions) or surfaces (three dimensions). The offset can include a distance offset and / or an angular offset. FIGS. 1A and 1B show examples of angular offsets for different two-dimensional shapes (which may be two-dimensional protrusions of a three-dimensional structure). The isosceles triangle 100 in FIG. 1A has a 120° angular offset between the first face 110 and the second face 120, and their relative orientation is shown by the orthogonal vectors A and A'. The rectangle 130 in FIG. 1B has a 180° angular offset between the first face 110 and the second face 120, and their relative orientation is shown by the orthogonal vectors A and A'.

[0063] As used herein, the term "binding specificity" refers to the tendency of an affinity reagent to preferentially interact with a binding partner, affinity target, or target site compared to other binding partners, affinity targets, or target sites. An affinity reagent can have calculated, observed, known, or predicted binding specificity for any potential binding partner, affinity target, or target site. Binding specificity can refer to the selectivity for a single binding partner, affinity target, or target site in a sample over at least one other analyte in the sample. Moreover, binding specificity can refer to the selectivity for a subset of binding partners, affinity targets, or target sites in a sample over at least one other analyte in the sample.

[0064] As used herein, the term "binding affinity" or "affinity" refers to the strength or degree of binding between an affinity reagent and a binding partner, affinity target, or target site. In some cases, the binding affinity of an affinity reagent for a binding partner, affinity target, or target site may be negligible or virtually zero. The binding affinity of an affinity reagent for a binding partner, affinity target, or target site may be qualified as "high affinity," "medium affinity," or "low affinity." The binding affinity of an affinity reagent for a binding partner, affinity target, or target site may be quantified as "high affinity" when the interaction has a dissociation constant of less than about 100 nM, "medium affinity" when the interaction has a dissociation constant of about 100 nM to 1 mM, and "low affinity" when the interaction has a dissociation constant of more than about 1 mM. Binding affinity can be measured using terms known in the field of biochemistry, such as the equilibrium dissociation constant (K D ), equilibrium association constant (K A ), association rate constant (k on ), dissociation rate constant (k off ), etc. See, e.g., Segel, Enzyme Kinetics, John Wiley and Sons, New York (1975), which is incorporated herein by reference in its entirety.

[0065] As used herein, the term "broad-spectrum," when used in reference to binding, can refer to: 1) binding to multiple binding partners due to the presence of a particular affinity target or target site, regardless of the binding context of the affinity target or target site; or 2) binding to multiple affinity targets or target sites within the same or different binding partners; or 3) a combination of both properties. Regarding the first form of binding broad-spectrum, the "binding context" can refer to the local chemical environment surrounding the affinity target or target site, such as nearby, adjacent, or nearby chemical entities (e.g., in the case of a polypeptide epitope, nearby amino acid sequences, or non-contiguous amino acid sequences adjacent or nearby to the epitope). Regarding the second form of binding broad-spectrum, the definition can refer to affinity reagents or probes that bind to structurally or chemically related affinity targets or target sites, regardless of differences between the affinity targets or target sites. For example, an affinity reagent can be considered broad-spectrum if it possesses binding affinity for a trimeric peptide sequence of the form WXK (W is tryptophan, K is lysine, and X is any possible amino acid). Additional concepts regarding binding broadness are discussed in WO2020106889A1, which is incorporated herein by reference in its entirety.

[0066] As used herein, the term "binding probability" refers to the probability that an affinity reagent may be observed to interact with a binding partner and / or affinity target within a particular binding context. Binding probability may be expressed as a discrete number, e.g., a value N (e.g., 0.4) or a percentage value (e.g., 40%) within the range 0≦N≦1, a matrix of discrete numbers, or a mathematical model (e.g., a theoretical or empirical model). Binding probability may include one or more factors, including binding specificity, the likelihood of finding the affinity target, and the likelihood of binding long enough for a binding interaction to be detected. The overall binding probability may include the binding probability when all factors are weighted against the binding context.

[0067] As used herein, the term "binding context" can refer to the environmental conditions under which affinity reagent-binding partner interactions are observed. The binding context can be constant conditions or conditions that vary within a range. Environmental conditions can include any factor that can affect the interaction between an affinity reagent and a binding partner, such as temperature, fluid properties (e.g., ionic strength, polarity, pH), relative concentration, absolute concentration, fluid composition, binding partner conformation, affinity reagent conformation, and combinations thereof.

[0068] As used herein, the term "tunable," when used in reference to structured nucleic acid particles, refers to a specific, precise, and / or rational location of components or binding sites for components having an assembly or structure. A tunable retention component can refer to the ability to connect or conjugate a probe component at a specific site or within a specific region of the retention component structure, or to generate a binding site for connecting or conjugating a probe component at a specific site or specific region of the retention component structure. As used herein, "tunability" refers to the property of a probe or retention component having a tunable structure or configuration.

[0069] As used herein, the term "functional group" refers to a group of atoms in a molecule that confers a chemical property to the molecule, such as reactivity, polarity, hydrophobicity, hydrophilicity, solubility, etc. A functional group may include organic moieties or may include inorganic atoms. Exemplary functional groups may include alkyl, alkenyl, alkynyl, phenyl, halide, hydroxyl, carbonyl, aldehyde, acyl halide, ester, carboxylate, carboxyl, carboalkoxy, methoxy, hydroperoxyl, ether, hemiacetal, hemiketal, acetal, ketal, orthoester, epoxide, carboxylic anhydride, carboxamide, amine, ketimine, aldimine, imide, azide, azo, cyanate, isocyanate, nitrate, nitrile, isonitrile, nitrosoxy, nitro, nitroso, oxime, pyridyl, carbamate, sulfhydryl, sulfide, disulfide, sulfinyl, sulfonyl, sulfino, sulfo, thiocyanate, isothiocyanate, carbonothioyl, thioester, thioester, phosphino, phosphono, phosphonate, phosphate, borono, boronate, and borinate functional groups.

[0070] As used herein, the term "functionalized" refers to any material or substance that has been modified to include a functional group. The functionalized material or substance can be naturally or synthetically functionalized. For example, a polypeptide can be naturally functionalized with phosphate, oligosaccharide (e.g., glycosyl, glycosylphosphatidylinositol, or phosphoglycosyl), nitrosyl, methyl, acetyl, lipid (e.g., glycosylphosphatidylinositol, myristoyl, or prenyl), ubiquitin, or other naturally occurring post-translational modifications. The functionalized material or substance can be functionalized for any given purpose, including modifying its chemical properties (e.g., modifying its hydrophobicity or surface charge density) or its reactivity (e.g., being capable of reacting with a site or reagent to form a covalent bond to the site or reagent).

[0071] Other than in the examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." As used herein, the term "about," when used in connection with percentages, can mean a variance of up to ±5% of the referenced value. For example, about 90% can mean 85% to 95%. In some cases, "about" can mean a variance of up to ±4%, ±3%, ±2%, ±1%, ±0.5%, or less of the referenced value. As used herein, the term "substantially," when used in reference to a measurable quantity or characteristic, refers to an quantity or characteristic having a value within ±10% of the referenced value. For example, a first value can be substantially the same as a second value if the first value is within ±10% of the second value. In another example, a shape can be substantially rectangular if the ratio of the side lengths of the rectangle is within the range of 0.90 to 1.10, inclusive. In some cases, "substantially" can mean an amount or characteristic having a value within up to ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.5%, or less of a reference value.

[0072] As used herein, the terms "bound" or "connected" refer to the state in which two entities are joined, fastened, adhered, connected, or bonded to one another. Bonds can be covalent or non-covalent. For example, a particle can be bound or connected to a protein by covalent or non-covalent bonds. Similarly, a first nucleic acid can be bound or connected to a second nucleic acid through hybridization or Watson-Crick base pairing. Covalent bonds are characterized by the sharing of electron pairs between atoms. Non-covalent bonds are chemical bonds that do not involve the sharing of electron pairs and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions, adhesion, adsorption, and hydrophobic interactions.

[0073] The term "comprising" is intended herein to be open-ended to include not only the recited elements but also to include any additional elements.

[0074] As used herein, the term "each," when used in reference to a collection of items, is intended to identify an individual item in the collection and not necessarily refer to every item in the collection. Exceptions may occur where express disclosure or context clearly indicates otherwise.

[0075] nucleic acid structure Provided herein are nucleic acids useful for forming arrays of analytes that enable interrogation of the analytes of the array with single-analyte resolution. The nucleic acids provided herein can be characterized as possessing adjustable two- or three-dimensional structures that facilitate one or more features selected from: i) presenting the analyte in an orientation that facilitates interrogation of the analyte with single-analyte resolution; ii) maximizing the likelihood of binding to a solid support or its surface at sites configured for nucleic acid binding; iii) maximizing the likelihood of binding to sites on a solid support or its surface in a controllable and / or non-random manner; iv) minimizing the likelihood of binding to a solid support or its surface at sites already occupied by another nucleic acid; and v) minimizing the likelihood of binding to a solid support or its surface at addresses not configured for nucleic acid binding. In some configurations, the nucleic acids provided herein can possess all of the aforementioned features. In other configurations, two or more nucleic acids can be complexed, in which case the nucleic acid complex possesses all of the aforementioned features.

[0076] Described herein are nucleic acids that are useful for organizing individual sites in single-analyte systems. The nucleic acids described herein can be characterized by one or more of the following features: i) comprising a presentation site configured to connect an analyte to the nucleic acid or contact the analyte with the nucleic acid; ii) comprising a capture site configured to connect the nucleic acid to a solid support or its surface or comprising a capture site that connects the nucleic acid to the solid support or its surface; iii) comprising a connection site configured to connect a second molecule to the nucleic acid or comprising a connection site that connects a second molecule to the nucleic acid; and iv) comprising an utilization site that modifies the physical and / or chemical properties of the nucleic acid. In some cases, the nucleic acid is a nucleic acid nanostructure or structured nucleic acid particle (SNAP).

[0077] Nucleic acids as provided herein can include naturally occurring nucleic acid structures, such as naturally occurring primary structures (e.g., naturally occurring single-stranded nucleotide sequences, single strands of plasmids, etc.), naturally occurring secondary structures (e.g., naturally occurring A-DNA, B-DNA, Z-DNA, or double-stranded helical structures), and naturally occurring tertiary structures (e.g., nucleic acids including origami structured nucleosomes, chromatin, etc.). Nucleic acids as provided herein can also include synthetic, artificial, or engineered nucleic acid structures. In some configurations, nucleic acids include nucleic acid nanostructures, which can include condensed three-dimensional structures. Nucleic acid nanostructures can include one or more structures not known to exist in naturally occurring nucleic acids. Nucleic acid nanostructures may include one or more structures that have a characterizable property that differs from the same characterizable property of naturally occurring nucleic acids (e.g., a longer or shorter average persistence length across a nucleic acid strand comprising N nucleotides, a longer or shorter radius of curvature of a nucleic acid strand comprising at least 75% double-stranded nucleic acids, a shorter or longer distance between two non-contiguous regions of a nucleic acid strand, a temporal variation in any of the aforementioned properties, etc.).

[0078] The compositions and methods provided herein will generally be illustrated with respect to nucleic acid nanostructures or SNAPs; however, it is understood that the methods and compositions exemplified can be extended to other nucleic acids, such as those provided herein.

[0079] It is also understood that nucleic acid structures are described in terms of average spatial and / or temporal configurations. Nucleic acid structures as described herein may be dynamic states with respect to common physical phenomena that cause spatial and / or temporal variations in nucleic acid configuration (e.g., thermal motion, intermolecular collisions, externally applied forces, intramolecular vibrations, intramolecular bending, intramolecular rotation, etc.). Quantitative descriptions of nucleic acid structures may include spatial and / or temporal variations in accordance with the dynamic nature of molecular structure as understood in the art.

[0080] Nucleic acid structure aspects: Nucleic acid nanostructures, such as SNAPs, can contain a variety of structures or structural motifs that result in more ordered structures or geometries. For example, ligated rotating circle amplification (RCA) products can produce spherical nanoball structures (i.e., nanoscale hedgehog-like structures) with spike-like structures at the outer boundary, where the single-stranded ligated nucleic acids form approximately 180° turns. In another example, SNAPs can include DNA origami particles containing a backbone single-stranded nucleic acid hybridized with multiple oligonucleotides that shape the backbone into an overall tertiary structure. Regions of tertiary structure can be connected by a predetermined oligonucleotide of the multiple oligonucleotides to pattern the backbone into regular or irregular shapes, such as tiles, disks, triangles, tori, cubes, pyramids, cylinders, tubes, and other more complex two- or three-dimensional structures.

[0081] Nucleic acid nanostructures, e.g., SNAPs, can include one or more surfaces that provide structural features and / or perform a function for the nucleic acid nanostructure. Nucleic acid nanostructures, e.g., SNAPs, can include one or more of: 1) a presentation surface; 2) a capture surface; 3) a connection surface; and 4) an application surface. The presentation surface can include a capture site that connects or is configured to connect the nucleic acid nanostructure to an analyte. The capture surface can include a capture site that connects or is configured to connect the nucleic acid nanostructure to a surface or interface. The connection surface can include a connection site that connects or is configured to connect a first nucleic acid nanostructure to a second nucleic acid nanostructure. The utilization surface may include utilization moieties that provide additional utilities to the nucleic acid nanostructure (e.g., SNAP), such as providing structure, providing stability, modifying interactions (e.g., attractive or repulsive forces, steric hindrance, etc.) between the nucleic acid nanostructure and another entity (e.g., a second nucleic acid nanostructure, a surface, etc.), or modifying the physical properties of the nucleic acid nanostructure (e.g., utilization moieties may include electrical, magnetic, or optical materials, etc.). A nucleic acid nanostructure, e.g., SNAP, may include surfaces with multiple functions. For example, a connection surface may also include a utilization surface. In another example, a presentation surface may also include a utilization surface or a capture surface. A nucleic acid nanostructure, e.g., SNAP, may include surfaces composed of one or more other types of surfaces. For example, a presentation surface may include a portion or region that is a utilization surface that includes steric blocking groups (e.g., PEG, PEO, dextran, etc.). In some configurations, a multifunctional surface may be counted as a single surface. For example, a cube-like SNAP may include approximately six distinct faces, each of which may include one or more functions on one of the six sides, such as a presentation face and a utilization face.

[0082] Nucleic acid nanostructures, e.g., SNAPs, can include one or more surfaces that provide functionality to the nucleic acid nanostructure. Surfaces can include sides or portions of a nucleic acid nanostructure that have similar orientations or two-dimensional protrusions on a virtual flat surface. Figures 2A-2D show examples of surfaces for simplified structures similar to those that may be encountered on nanostructures, e.g., SNAPs. Figure 2A shows two shorter tertiary structures 210 and 212 (e.g., DNA double helices) connected by a first pivoting linker 215. The two shorter tertiary structures 210 and 212 are connected to longer tertiary structures 220 and 222, which are connected by a third pivoting linker 225. The two shorter tertiary structures 210 and 212 are connected to the two longer tertiary structures 220 and 222 by a second pivoting linker 230. Two shorter tertiary structures 210 and 212 and two longer tertiary structures 220 and 222 are oriented so as to be coplanar. Functional groups R1, R2, R3, and R4 extend outward from the tertiary structures in specific orientations that extend outward from the plane in which the tertiary structures are oriented. An imaginary plane P is positioned orthogonally to and intersects the four tertiary structures. Figure 2B shows a cross-section of the tertiary structures captured in plane P. The relative positions of functional groups R1, R2, R3, and R4 are shown with respect to the tertiary structures in which they are displayed. The structure shown in Figure 2A can be defined by four planes S1, S2, T, and B, as shown in Figure 2B. The planes represent projections of the tertiary structures onto the imaginary plane defined by planes S1, S2, B, and T. Due to some freedom in the location of functional groups and / or moieties that may extend from the tertiary structure, as well as the size and length of the functional groups or moieties, the faces may extend beyond simple orthogonal protrusion of the tertiary structure onto faces S1, S2, B, or T. In some cases, functional groups or moieties extending from a nucleic acid nanostructure may be considered to be located on more than one face of the nucleic acid nanostructure. In other cases, functional groups or moieties extending from a nucleic acid nanostructure may be considered to be located within a single face of the nucleic acid nanostructure. The face to which a functional group or moiety is assigned may be defined by the use or purpose of the functional group or moiety.For example, a site with rigid chains located near two different faces can be assigned to a single face because the orientation induced by the rigid chains makes the site functionally inaccessible to the other faces. Due to the matched coplanar geometry of the tertiary structure, faces S1 and S2, when extended, will meet orthogonally with faces B and T. In some cases (e.g., cylindrical or tubular structures), the faces may be up to 360°. o The total aspect or orientation of the

[0083] Figures 2C-2D illustrate the location of a nucleic acid nanostructure surface for multiple non-coplanar tertiary structures. Figure 2C shows two shorter tertiary structures 210 and 212 (e.g., DNA double helices) connected by a first pivot linker 215. The two shorter tertiary structures 210 and 212 are connected to a longer tertiary structure 220 and 222, which is connected by a third pivot linker 225. The two shorter tertiary structures 210 and 212 are connected to the two longer tertiary structures 220 and 222 by a second pivot linker 230. The two shorter tertiary structures 210 and 212 are positioned below the longer tertiary structures 220 and 222. A virtual reference plane P' schematically defines a plane of mirror symmetry with respect to the tertiary structures. Figure 2D illustrates the projection of the tertiary structures above plane P'. Two planes, D and B, can be defined for the nucleic acid nanostructure shown in Figure 2C. The planes will intersect when extended, but due to their relative geometries, the intersection will not occur orthogonally.

[0084] Nucleic acid nanostructures, such as SNAPs, can have a specific number of faces. Nucleic acid nanostructures can have at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 faces. Additionally or alternatively, nucleic acid nanostructures can have no more than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer than 2 faces. The number of faces of a nucleic acid nanostructure can be selected to match the functionality of the nucleic acid nanostructure. For example, a SNAP configured to connect an analyte to a solid support may require at least two faces (a presentation face and a connection face), with additional faces (e.g., a utilization face) added based on other design considerations.

[0085] Nucleic acid nanostructures, such as SNAPs, can include two or more surfaces, each surface having a different utility. A nucleic acid nanostructure can include one or more utilities selected from the group consisting of: 1) a presentation surface configured to connect or allow the attachment of an analyte; 2) a capture surface configured to connect or allow the attachment of a surface; 3) a connection surface configured to connect or allow the attachment of a first nucleic acid nanostructure to a second nucleic acid nanostructure; and 4) a utility surface providing any additional utility (e.g., a steric block). In some configurations, a nucleic acid nanostructure can include a first utility (e.g., a presentation surface including a presentation site) and a second surface can include a second utility (e.g., a capture surface including a capture site). In other configurations, two or more surfaces can have the same utility (e.g., two or more presentation surfaces), but one of the two or more surfaces can include a different utility (e.g., a capture surface). In some configurations, a nucleic acid nanostructure can include the same two or more utilities on two or more surfaces (e.g., two opposing surfaces that function as a presentation surface and a capture surface).

[0086] A nucleic acid nanostructure, e.g., a SNAP, can have structural symmetry, for example, along an axis of symmetry (i.e., rotational symmetry) or a plane of symmetry (i.e., reflection symmetry). The tertiary structure of a nucleic acid nanostructure can have structural symmetry, for example, along an axis of symmetry (e.g., aligned with the centerline of a helical structure). A plurality of tertiary structures taken as a whole can have structural symmetry, for example, along an axis or plane of symmetry. A plane of a nucleic acid nanostructure can be oriented relative to an axis or plane of symmetry for the nucleic acid nanostructure or the tertiary structure of the plurality of tertiary structures forming the nucleic acid nanostructure. For example, for the cross-section shown in FIG. 2B, the top plane T can be oriented at 0° relative to the axis of symmetry that is coaxial with any of the four tertiary structures, while planes S1, B, and S2 can be oriented at 90°, 180°, and 270°, respectively. For nucleic acid nanostructures (e.g., SNAP) comprising a first tertiary structure and a second tertiary structure, the orientation of the first surface (e.g., a presentation surface, a capture surface, a connection surface, or an utilization surface) or the orientation of the second surface (e.g., a presentation surface, a capture surface, a connection surface, or an utilization surface) can be defined relative to an axis of symmetry for the first tertiary structure or an axis of symmetry for the second tertiary structure. In some configurations, the orientation of the first surface can be the same as the orientation of the second surface (e.g., a surface having presentation and capture surfaces). The orientation of the first surface can be determined relative to the orientation of the second surface based on an angular offset between a first vector perpendicular to a plane defining the average spatial location of the first surface and a second vector perpendicular to a plane defining the average spatial location of the second surface. In other configurations, the orientation of the first surface can be offset by at least about 90° from the orientation of the second surface. In other configurations, the orientation of the first surface can be offset by about 180° from the orientation of the second surface. Nucleic acid nanostructures can be configured to have at least about 10 o , 20 o , 30 o , 40 o , 50 o , 60 o , 70 o , 80 o , 90 o , 100 o , 110 o , 120 o , 130 o , 140o , 150 o , 160 o , 170 o , 180 o , 190 o , 200 o , 210 o , 220 o , 230 o , 240 o , 250 o , 260 o , 270 o , 280 o , 290 o , 300 o , 310 o , 320 o , 330 o , 340 o , 350 o , or 350 o Alternatively or additionally, the nucleic acid nanostructure may include a first surface and a second surface having an angular offset of more than about 360°. o , 350 o , 340 o , 330 o , 320 o , 310 o , 300 o , 290 o , 280 o , 270 o , 260 o , 250 o , 240 o , 230 o , 220 o , 210 o , 200 o , 190 o , 180 o , 170 o , 160 o , 150 o , 140 o , 130 o , 120 o , 110 o , 100 o , 90 o , 80 o , 70 o , 60 o , 50 o , 40 o , 30o , 20 o , 10 o Less than or equal to 10 o The first and second surfaces may have an angular offset of less than .gtoreq. .gtoreq.

[0087] Nucleic acid nanostructures, such as SNAPs, can include multiple tertiary or quaternary structures that at least partially surround or substantially surround an internal volume region. Nucleic acid nanostructures can have three-dimensional structures, such as pyramids, shells, cylinders, disks, spheres, cuboids (e.g., cubes or rectangular cuboids), or blocks, that include the internal volume region. The internal volume region can be a three-dimensional volume within the nucleic acid nanostructure that is large enough to accommodate the analytes or other molecules described herein. Nucleic acid nanostructures can be configured to include an internal volume region, which includes an application surface, such as a presentation surface or capture surface. An application site can be presented within the internal volume region. For example, a presentation site can be presented within the internal volume region of a SNAP such that the analyte is at least partially connected within the internal volume region. In another example, a capture site can be presented within the internal volume region of a SNAP such that a complementary site on the surface must at least partially penetrate the internal volume region to connect with the capture site (see Figures 38A and 38B).

[0088] In some configurations, interior volume regions can be created in nucleic acid nanostructures (e.g., SNAPs) to control interactions between the nucleic acid nanostructure and other entities. The interior volume regions can include one or more sites that modify the chemical properties (e.g., hydrophobicity, hydrophilicity, reactivity, polarity, solubility, etc.) of the interior volume regions to differ from those of the surrounding nucleic acid nanostructures. Figure 39A shows a SNAP 3910 including an interior volume region 3920 containing a capture site comprising a reactive group 3925 and a plurality of hydrophobic molecules 3928 surrounding the reactive group 3925. The SNAP can be contacted with a surface 3930 comprising a plurality of hydrophilic groups 3932 terminated with complementary reactive groups 3935 and a plurality of hydrophobic groups 3938 terminated with complementary reactive groups 3935. As shown in Figure 39B, the hydrophobic properties of the interior volume region 3920 can increase the likelihood that the SNAP 3910 will deposit and attach to the surface 3930 in a region comprising the plurality of hydrophobic groups 3938.

[0089] In some configurations, interior volume regions can be generated in nucleic acid nanostructures (e.g., SNAPs) to control interactions that can involve sites within the interior volume region. The orientation of sites within the interior volume region can be controlled to increase, decrease, or otherwise control the orientations at which interactions can occur. Sites can be presented within the interior volume region in a manner that limits or controls the size of entities that can interact with the site. Figure 38A shows a SNAP 3810 that includes an interior volume region 3820 containing connected multivalent binding sites (e.g., streptavidin, avidin) 3830. The connected multivalent binding sites 3830 are oriented within the interior volume region 3820 such that only one binding site 3835 is available to participate in a binding interaction with an entity 3840 that includes a complementary binding group (e.g., biotin) 3845 configured to connect to the binding site 3835. As shown in Figure 38B, connected multivalent binding site 3830 is rendered substantially monovalent by its orientation within interior volume region 3820, thereby forming only one binding interaction with entity 3840.

[0090] A nucleic acid nanostructure can include a first tertiary structural domain and a second tertiary structural domain oriented relative to each other by one or more nucleic acid strands that form a connecting chain (e.g., a staple oligonucleotide) between the first and second tertiary structural domains. The connecting chain can comprise a single-stranded, double-stranded, partially double-stranded, or multi-stranded nucleic acid. In some configurations, a nucleic acid nanostructure can include a first oligonucleotide having a first nucleic acid sequence and a second nucleic acid sequence that hybridize to a complementary sequence of the second oligonucleotide to form the first and second tertiary structural domains, the first and second nucleic acid sequences of the first oligonucleotide being separated by a connecting nucleic acid sequence that comprises a single-stranded connecting chain between the first and second tertiary structural domains. For example, the first oligonucleotide can be a staple that hybridizes to a backbone nucleic acid to form the first and second tertiary structural domains of the nucleic acid origami structure.

[0091] A nucleic acid nanostructure can include a first tertiary structure domain and a second tertiary structure domain, with the relative angular orientation or spatial separation of the two domains controlled by one or more connecting strands. The angular orientation and / or spatial separation of the first tertiary structure domain and the second tertiary structure domain can be tunable based on the spatial position of nucleotides within the helical structure of the domains. Each complete turn of a double-stranded nucleic acid helix typically contains 10 to 11 nucleotide base pairs. Thus, the initial angle of protrusion of the connecting strand can be adjusted depending on the nucleotide position within the helical structure. The structural tunability of a nucleic acid nanostructure can also be controlled by varying the length of the connecting strand and the separation distance between consecutive connecting strands. Figures 49A-49E illustrate an embodiment of controlling the orientation of the tertiary structure in a nucleic acid nanostructure. Figure 49A shows a top-down view of a portion of a nucleic acid nanostructure comprising a first oligonucleotide 4910 (e.g., a backbone strand) and a second oligonucleotide 4920 (e.g., a staple oligonucleotide) that has hybridized to first oligonucleotide 4910 to form first tertiary structure domain 4930 and second tertiary structure domain 4932 connected by a linking strand comprising the single-stranded nucleic acid sequence of second oligonucleotide 4920. Figures 49B-49C illustrate differences in the initial orientation of the linking strand, determined by the nucleotide position within the helical turn of second oligonucleotide 4920, as viewed relative to the helical axis of first tertiary structure domain 4930 and second tertiary structure domain 4932. Figure 49B illustrates a configuration in which the initial orientation of the linking strands is non-coplanar, while Figure 49C illustrates a configuration in which the initial orientation of the linking strands is coplanar. Furthermore, for a fixed length of the connecting chain, differences in the initial orientation of the connecting chain can affect the separation distance or amount of variation in separation distance between two neighboring tertiary structure domains, as shown, for example, in Figures 49B and 49C. Figures 49D-49E show the possible relative positions of the tertiary structure domains based on the connecting chain orientations shown in Figures 49B-49C, respectively.Figure 49D shows a distorted orientation between the first tertiary structure domain 4930 and the second tertiary structure domain 4932, while Figure 49E shows a coplanar orientation between the first tertiary structure domain 4930 and the second tertiary structure domain 4932, with each orientation of the two tertiary structure domains resulting from the arrangement of nucleotides as the second oligonucleotide 4920 transitions from a component of the double-stranded nucleic acid to the single-stranded nucleic acid of the connecting strand.

[0092] The position of the connecting chain can affect the conformation of the first tertiary structure domain relative to the second tertiary structure domain in the nucleic acid nanostructure. For example, to configure the first tertiary structure domain and the second tertiary structure domain in a substantially coplanar orientation (i.e., the minimum angular offset between the two tertiary structure domains), the continuous connecting chains can be arranged at approximately an odd number of half helix turns (e.g., about 1, 3, 5, 7, 9, etc. half turns or about 6, 16, 27, 37, 48, etc. nucleotides apart). Alternatively, to configure the first tertiary structure domain and the second tertiary structure domain in an inclined orientation (i.e., a measurable angular offset between the two tertiary structures), the continuous connecting chains can be arranged at positions other than half helix turns, or at random or various positions including half helix turns and positions other than half helix turns. For example, successive linking strands can be spaced apart by approximately an even number of half helical turns (e.g., about 2, 4, 6, 8, 10, etc. half turns or about 11, 21, 31, 41, 52, etc. nucleotides) or by fractions of a half helical turn (e.g., ¾ turn, 1 (¾) turn, 2 (¼) turns, etc.). In some configurations, it may be preferable to generate nucleic acid nanostructures that include substantially flat structures, where the flat structures include multiple coplanar tertiary structures. For example, a nucleic acid nanostructure may include a capture surface that is substantially flat to increase electrostatic interactions between the capture surface and the flat surface of a solid support. In other configurations, it may be preferable to generate nucleic acid nanostructures that include non-flat structures that include multiple tertiary structures, such as curved or corrugated surfaces. For example, a nucleic acid nanostructure may include a capture surface that includes a corrugated texture to increase electrostatic interactions between the capture surface and the rough surface of a solid support.

[0093] Nucleic acid nanostructures can include one or more features or configurations that deviate from those of naturally occurring nucleic acids. The nucleic acid nanostructures provided herein can include one or more non-natural nucleic acid structures that increase the tunability of the nanostructure for one or more purposes, e.g., attachment and / or presentation of analytes, and attachment of the nanostructure to a solid support or surface thereof. Nucleic acid nanostructures may be characterized by the presence of one or more non-natural nucleic acid structures, including, but not limited to: i) a greater number of oligonucleotides hybridized to a given nucleic acid strand compared to the number of oligonucleotides hybridized to a natural nucleic acid strand of the same length and sequence; ii) an increased volumetric and / or areal density of nucleotide packaging within the nanostructure or its component structures compared to natural nucleic acids having the same or similar sequence content; iii) an increased sharpness of bends in the nucleic acid strand relative to naturally occurring nucleic acids having the same sequence or length; iv) a decreased separation distance between non-contiguous regions of nucleic acid strands within the nanostructure compared to naturally occurring nucleic acids having the same sequence or length; v) a lower degree of sequence complementarity within the nanostructure relative to the degree of sequence complementarity in naturally occurring nucleic acids occupying a similar volume in solution; vi) a higher mechanical rigidity of nucleic acid strands in the nanostructure compared to the mechanical rigidity of naturally occurring nucleic acids having the same sequence or length; and vii) combinations thereof.

[0094] The nucleic acid nanostructures described herein may contain more complex oligonucleotides or nucleic acid strands known to occur in natural nucleic acid systems, for example, those with the same mass as the nucleic acid nanostructure. Naturally occurring nucleic acids are primarily nucleic acid strands with partial or complete complementarity (e.g., chromosomal DNA, plasmid strands). Naturally occurring nucleic acids can be distinguished by the complete or near-complete complementarity of the hybridized nucleic acid strands. Naturally occurring nucleic acids can further be distinguished by the relatively small number of nucleic acids simultaneously complexed through hybridization between each nucleic acid strand within the nucleic acid complex. For example, naturally occurring Holliday junction structures typically involve the hybridization of four nucleic acid strands, with each strand of the junction complex having a high degree of sequence complementarity to the two other strands in the complex. Naturally occurring nucleic acids often require additional proteins to complex multiple nucleic acid strands (e.g., chromosomal centromeres, ternary nucleic acid complexes formed by RNA polymerase during gene transcription, RNA strands, and two complementary DNA strands, etc.). In contrast, the nucleic acid nanostructures described herein may contain a greater amount of complex nucleic acid oligonucleotides or nucleic acid strands than those known to occur in natural nucleic acid systems. For example, the nucleic acid nanostructures may contain at least 10, 25, 50, 100, 150, 200, or more than 200 complexed oligonucleotides or nucleic acid strands, each of which is hybridized to at least one other oligonucleotide or nucleic acid strand in the nucleic acid nanostructure. In some configurations, the nucleic acid nanostructures may be further characterized by the absence of non-nucleic acid structural elements (e.g., polypeptides, proteins, polymers, nanoparticles) that are configured to connect a first oligonucleotide or nucleic acid strand to a second oligonucleotide or nucleic acid strand.

[0095] The nucleic acid nanostructures provided herein may contain increased volumetric and / or areal density of nucleotide packing within the nanostructure or its component structures compared to naturally occurring nucleic acids, such as naturally occurring nucleic acids having the same mass, nucleotide sequence, or sequence length as the nucleic acid nanostructure. Naturally occurring nucleic acids typically achieve their volumetric nucleotide density through helical coiling of the double-stranded nucleic acid and supercoiling of the helical nucleic acid into a compacted structure. However, to achieve packaging of double-stranded nucleic acids with strand curvature exceeding the persistence length of the double-stranded nucleic acid, naturally occurring nucleic acids are typically complexed with proteins (e.g., histones) that contract the helical nucleic acid into a supercoiled structure. In contrast, nucleic acid nanostructures may contain a volumetric nucleotide density that exceeds that of naturally occurring nucleic acids. Nucleic acid nanostructures may achieve higher volumetric nucleotide density than naturally occurring nucleic acids through increased bending and / or curvature of the nucleic acid structure and / or closer proximity of helical structures within the nucleic acid structure. In some configurations, nucleic acid nanostructures can achieve higher volumetric nucleotide densities than naturally occurring nucleic acids in the absence of non-nucleic acid structural elements (e.g., polypeptides, proteins, polymers, nanoparticles) configured to reduce the nucleic acid structure.

[0096] The nucleic acid nanostructures described herein may include increased sharpness of nucleic acid bends relative to sequence length and / or degree of secondary structure relative to naturally occurring nucleic acids, such as naturally occurring nucleic acids having the same nucleotide sequence or mass as the nucleic acid nanostructure. Naturally occurring double-stranded nucleic acids have a large persistence length, making it unlikely that any portion of the double-stranded nucleic acid can approach within about 10 nanometers of any other portion, for example, in the absence of structural modifying groups (e.g., histones). Even when single-stranded nucleic acids are present in naturally occurring nucleic acids, the two portions of the tertiary structure are unlikely to approach each other, for example, within about 10 nanometers, due to electrostatic repulsion by the negatively charged polynucleotide backbone. Furthermore, in the absence of unifying elements (e.g., histones, linking nucleic acids), the two tertiary structures are unlikely to remain stably oriented in the tight configuration found in naturally occurring nucleic acids. In contrast, the nucleic acid nanostructures described herein may include sharply bent nucleic acid structures that increase the proximity of the helical structure through segmentation of the double-stranded nucleic acid with the sequence of the single-stranded nucleic acid. Neighboring helical structures may be held in close proximity by a linking nucleic acid strand that spatially and / or temporally stabilizes the proximity and orientation of the neighboring helical structures relative to one another. The nucleic acid nanostructures provided herein may be further distinguished from naturally occurring nucleic acids by the presence of a stable (i.e., spatially and / or temporally invariant) bend (e.g., at least a 90° to 180° bend) in the nucleic acid strand comprising the two segmented regions of the helical structure relative to the length (e.g., 50, 40, 30, 25, 20, 15, or 10 nucleotides or less) of the single-stranded nucleic acid segment of the nucleic acid strand separating the two segmented regions of the helical structure. Alternatively or additionally, the nucleic acid nanostructures provided herein may be further distinguished from naturally occurring nucleic acids by the presence of stable (i.e., spatially and / or temporally invariant) bends (e.g., bends of at least 90° to 180°) in the nucleic acid strand comprising two segmented regions of helical structure relative to the degree of secondary structure of the nucleic acid nanostructure (e.g., comprising at least about 80%, 85%, 90%, or 95% base-paired nucleotides relative to the total nucleotide content).

[0097] The nucleic acid nanostructures described herein may include a reduced separation distance between neighboring nucleic acid structures within the nanostructure relative to naturally occurring nucleic acids, e.g., naturally occurring nucleic acids having the same mass, nucleotide sequence, or sequence length as the nucleic acid nanostructure. Adjacent helical (e.g., tertiary) structures may be maintained in a temporally and / or spatially stable configuration, for example, at a distance of less than about 10, 9, 8, 7, 6, 5, 4, 3, or 2 nanometers. The close proximity of adjacent helical structures in a nucleic acid nanostructure is unlikely to be caused by structural distortion introduced by electrostatic repulsion of adjacent polynucleotide strands. Nucleic acid nanostructures may be able to achieve close spatial proximity of helical structures and sharp bending angles of nucleic acid strands due to the presence of one or more connecting nucleic acid strands that stabilize the nucleic acid structure.

[0098] The nucleic acid nanostructures described herein may contain a low degree of sequence complementarity relative to the total amount of nucleic acid present, such as naturally occurring nucleic acids with the same mass or sequence length as the nucleic acid nanostructure. Naturally occurring nucleic acid strands will typically hybridize to complementary nucleic acid strands with the same sequence length. Aside from replication or proofreading errors, the co-hybridized strands can be expected to have near-perfect sequence complementarity, resulting in a nearly fully hybridized structure in a stable configuration. In contrast, the nucleic acid nanostructures described herein may contain multiple single-stranded nucleic acids within the nanostructure. Single-stranded nucleic acids within the nanostructure can be characterized as being spatially and / or temporally stable, in contrast to naturally occurring nucleic acids, where single-stranded nucleic acids are often formed through the structure of the nucleic acid by various biological processes and are not formed primarily. The nucleic acid nanostructures described herein may contain a stable fraction of single-stranded nucleic acids, as measured by the percentage of unpaired nucleotides within the nanostructure. In some configurations, nucleic acid nanostructures can include regions of compaction of predominantly double-stranded nucleic acids and regions of transparency of predominantly single-stranded nucleic acids. In particular configurations, nucleic acid nanostructures can include regions of compaction of predominantly double-stranded nucleic acids and regions of transparency of predominantly single-stranded nucleic acids, the transparency regions containing a greater total amount of nucleotides than the compaction regions. Nucleic acid nanostructures can contain a spatially and / or temporally stable fraction of single-stranded nucleic acids, as measured by unpaired nucleotides, for example, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, or greater than 60% single-stranded nucleic acids.

[0099] The nucleic acid nanostructures described herein may comprise a higher mechanical rigidity relative to the amount of single-stranded nucleic acid within the nanostructure when compared to naturally occurring nucleic acids, such as naturally occurring nucleic acids having the same mass, nucleotide sequence, or sequence length as the nucleic acid nanostructure. For example, a single-stranded nucleic acid strand within a linear double-stranded nucleic acid typically produces reduced rigidity within the double-stranded nucleic acid, as evidenced by increased relative movement between the ends of the nucleic acid. An increased amount of single-stranded nucleic acid within a linear double-stranded nucleic acid would be expected to further reduce the amount of nucleic acid rigidity. In contrast, the nucleic acid nanostructures described herein may comprise a higher rigidity relative to the total single-stranded nucleic acid content on a spatial and / or temporal basis relative to naturally occurring nucleic acids with the same single-stranded nucleic acid content. The increased rigidity may result from the linking chains that stabilize the nucleic acid structures relative to each other within the nucleic acid nanostructure.

[0100] Nucleic Acid Configuration: Nucleic acid nanostructures, e.g., SNAPs, are described herein. Nucleic acid nanostructures can be utilized for multiple purposes, including the presentation of molecules or analytes at a surface or interface, such as a solid support or phase boundary. The described nucleic acid nanostructures, e.g., SNAPs, can include various primary, secondary, tertiary, or quaternary structures, resulting in compacted nucleic acid particles with various geometries that add utility to the nanostructure. Any given nucleic acid nanostructure can serve one or more functions, including presenting a molecule or analyte (presentation SNAP) or providing other nanostructure-related utility (utilization SNAP). A nucleic acid nanostructure, e.g., utilization SNAP, can perform functions such as connecting a molecule or analyte to a surface or interface (capture SNAP), connecting the nucleic acid nanostructure to another nucleic acid nanostructure (connection SNAP), providing other structural utility to the nucleic acid nanostructure or its complex (structural SNAP), or a combination thereof. In some configurations, a nucleic acid nanostructure can include a presentation SNAP, a utilization SNAP, or a combination thereof. For example, a nucleic acid nanostructure (eg, SNAP) can be configured to connect to an analyte and a solid support, thereby making the nucleic acid nanostructure both a presentation nanostructure and an application nanostructure.

[0101] Nucleic acid nanostructures, such as SNAP, may include a presentation surface containing presentation sites. The presentation sites may be configured to attach analytes through suitable interactions, such as covalent bonds, non-covalent interactions, electrostatic interactions, or magnetic interactions. The presentation sites may include one or more functional groups, ligands, or other moieties configured to attach analytes. The presentation sites may include nucleic acid residues or may include functional groups, ligands, or moieties connected thereto. The presentation sites may further include one or more secondary, tertiary, or quaternary structures disposed within the presentation surface. Nucleic acid nanostructures, such as SNAP, may include a capture surface containing capture sites. The capture sites may be configured to attach to a surface through suitable interactions, such as covalent bonds, non-covalent interactions, electrostatic interactions, or magnetic interactions. The capture sites may include one or more functional groups, ligands, or other moieties configured to attach to a surface. The capture sites may further include one or more secondary, tertiary, or quaternary structures disposed within the capture surface.

[0102] A presentation site may include two or more presentation tertiary structures of the plurality of tertiary structures. A capture site may include two or more capture tertiary structures of the plurality of tertiary structures. In some configurations, a presentation tertiary structure of two or more presentation tertiary structures may include a capture tertiary structure of two or more capture tertiary structures. For example, in FIG. 2B, face T may include a presentation site and face B may include a capture site, with four tertiary structures belonging to both sites. In other configurations, a presentation tertiary structure of two or more presentation tertiary structures does not include any capture tertiary structure of two or more capture tertiary structures. For example, in FIG. 2D, a presentation site may include two tertiary structures associated with face D, and a capture site may include two tertiary structures associated with face B. In some configurations, a capture tertiary structure of two or more presentation tertiary structures does not include any presentation tertiary structure of two or more presentation tertiary structures.

[0103] Nucleic acid nanostructures, such as SNAPs, can contain multiple nucleic acid strands, each of which is a molecule separable from another without breaking a covalent bond. For example, a SNAP can contain a backbone strand and a nucleic acid molecule hybridized to the backbone strand to form multiple staple oligonucleotide molecules. In some configurations, the backbone strand can contain a plurality of oligonucleotides, each of which is connected to a greater number of oligonucleotides than any other oligonucleotide of the plurality of oligonucleotides. The backbone strand can comprise a linear, branched, or circular polynucleotide. In some configurations, a nucleic acid nanostructure can contain two or more backbone strands, for example, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more backbone strands, each of which is optionally a molecule separable from the other strand(s) of the nucleic acid nanostructure. A nucleic acid nanostructure having two or more backbone strands can include a first backbone strand connected to a second backbone strand by one or more oligonucleotides of the plurality of oligonucleotides hybridized to the first backbone strand and the second backbone strand. The first backbone strand can be linked to the second backbone strand by a predetermined number of the plurality of oligonucleotides, such as, for example, at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or more than 50% of the oligonucleotides in the plurality of oligonucleotides.Alternatively or additionally, the first backbone strand can be linked to the second backbone strand by about 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the oligonucleotides in the plurality of oligonucleotides.

[0104] The nucleic acid backbone can comprise a continuous strand of nucleic acid, either circular or conjugated (i.e., a backbone without a 5' or 3' end), with or without complementary oligonucleotides. In some configurations, the backbone is derived from a natural source, such as a viral genome or a bacterial plasmid. In other configurations, the backbone can be wholly or partially engineered, rationally designed, or synthetic. The backbone can comprise one or more modified nucleotides. The modified nucleotides can provide conjugation sites for attaching additional components, such as affinity reagents or detectable labels. The modified nucleotides can be utilized as conjugation sites for additional components (e.g., binding components or labeling components) before, during, or after construction of the nucleic acid nanostructure, e.g., SNAP. The modified nucleotides can include linking groups or reactive handles (e.g., functional groups configured to perform a click reaction). In some configurations, the nucleic acid backbone can comprise a single strand of an M13 viral genome. The size of the backbone can vary depending on the desired size of the nucleic acid nanostructure. The backbone may comprise at least about 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5200, 5400, 5600, 5800, 6000, 6200, 6400, 6600, 6800, 7000, 7200, 7400, 7600, 7800, 8000, 8200, 8400, 8600, 8800, 9000, 9500, 10000, or more than 10000 nucleotides in length. Alternatively or additionally, the backbone strand may comprise a length of up to about 10000, 9500, 9000, 8800, 8600, 8400, 8200, 7800, 7600, 7400, 7200, 7000, 6800, 6600, 6400, 6200, 6000, 5800, 5600, 5400, 5200, 5000, 4500, 4000, 3500, 3000, 2500, 3000, 2500, 2000, 1500, 1000 or less than 1000 nucleotides.

[0105] Nucleic acid nanostructures, such as SNAP, can contain multiple staple oligonucleotides. Staple oligonucleotides can include any oligonucleotide hybridized or configured to hybridize with a nucleic acid backbone, other staples, or a combination thereof. Staple oligonucleotides can be modified to include additional chemical entities, such as binding moieties, labeling moieties, chemically reactive groups or handles, or other groups (e.g., polyethylene glycol (PEG) moieties). Staple oligonucleotides can include linear or circular nucleic acids. Staple oligonucleotides can include one or more single-stranded regions, double-stranded regions, or a combination thereof. Staple oligonucleotides can be hybridized or configured to hybridize with a backbone or one or more other staples, for example, via complementary base pair hybridization (e.g., Watson-Crick hybridization). Staple oligonucleotides can hybridize with other nucleic acids by complementary base pair hybridization or ligation. Staple oligonucleotides can be configured to act as primers for complementary nucleic acid strands, and the primer-staples can be extended by an enzyme (e.g., a polymerase) to form extended regions of double-stranded nucleic acid, for example, using the backbone, staple, or other strand as a template. In some cases, the primer does not need to be hybridized to a template when extended. For example, a primer can be extended by the non-templated addition of one or more nucleotides by a terminal transferase enzyme, the non-templated addition of one or more oligonucleotides by a ligase enzyme, or the non-templated addition of nucleotide(s) or oligonucleotide(s) by a non-enzymatic chemical reaction. The staple oligonucleotide can include one or more modified nucleotides. The modified nucleotide can include a linking group or a reactive handle (e.g., a functional group configured to perform a Click-type reaction).

[0106] Stapled oligonucleotides can be of any length depending on the design of the SNAP. Stapled oligonucleotides can be generated using software packages such as caDNAno. 2 Staple oligonucleotides can have a length of at least about 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more than 5000 nucleotides. Alternatively or additionally, the staples can have a length of about 5000, 4500, 4000, 3500, 3000, 2500, 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 25, 10 or less than 10 nucleotides.

[0107] A staple can comprise a first nucleotide sequence and a second nucleotide sequence, where the first nucleotide sequence is hybridized to a first complementary sequence and the second nucleotide sequence is hybridized to a second complementary sequence. In some configurations, a staple can comprise a first nucleotide sequence and a second nucleotide sequence, where the first nucleotide sequence is hybridized to a first complementary sequence and the second nucleotide sequence is hybridized to a second complementary sequence, and the first nucleotide sequence is linked to the second nucleotide sequence by a linking moiety (e.g., a linker as described herein, an intermediate single-stranded nucleotide sequence, an intermediate double-stranded nucleotide sequence, an intermediate nucleotide sequence not configured to connect to a complementary nucleotide sequence, etc.). In some configurations, a staple can comprise a first nucleotide sequence and a second nucleotide sequence, where the first nucleotide sequence is hybridized to a first complementary sequence of a backbone strand and the second nucleotide sequence is hybridized to a second complementary sequence of a backbone strand. In certain configurations, the first complementary sequence and the second complementary sequence of the backbone strand can be non-contiguous, such that the two complementary sequence regions are separated by a third region of the backbone strand. The staple can comprise a first nucleotide sequence and a second nucleotide sequence, where the first nucleotide sequence is hybridized to the first complementary sequence and the second nucleotide sequence is not hybridized to the second complementary sequence (e.g., a pendant moiety). The first nucleotide sequence or the second nucleotide sequence of the staple oligonucleotide can comprise a sequence length of at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides.Alternatively or additionally, the first nucleotide sequence or the second nucleotide sequence of the staple oligonucleotide can comprise a sequence length of about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or less nucleotides. The sequence length of the nucleotide sequence of the staple oligonucleotide can be selected to provide a hybridized nucleic acid containing the staple oligonucleotide with a particular melting temperature as set forth herein.

[0108] The staple oligonucleotide may contain one or more modified nucleotides. The modified nucleotide may provide a conjugation site for attaching additional components, such as binding components or labeling components. The modified nucleotide may increase the stability of the oligonucleotide against chemical degradation, such as locked nucleic acid (LNA). The modified nucleotide may be used as a conjugation site for additional components before, during, or after the construction of a nucleic acid nanostructure, such as SNAP. The staple oligonucleotide may contain at least about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 75, 100, or more than 100 modified nucleotides. Alternatively or additionally, the staple oligonucleotide can include no more than about 100, 75, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer than 2 modified nucleotides.

[0109] The nucleic acid nanostructures provided herein can comprise a plurality of nucleic acids, each nucleic acid of the plurality of nucleic acids hybridized to one or more other nucleic acids of the plurality of nucleic acids. In some configurations, the nucleic acid nanostructures can comprise at least five nucleic acids, each nucleic acid of the at least five nucleic acids connected to one or more other nucleic acids of the at least five nucleic acids. The plurality of nucleic acids of the nucleic acid nanostructure can comprise a backbone strand characterized by one or more of the following features: i) comprising the longest nucleotide sequence of the plurality of nucleic acids; and ii) being configured to hybridize with a greater amount of the other nucleic acids of the plurality of nucleic acids. The plurality of nucleic acids of the nucleic acid nanostructure may further comprise one or more staple oligonucleotides, wherein the staple oligonucleotides are characterized by one or more of the following features: i) comprising two or more non-contiguous nucleotide sequences configured to hybridize to one or more other nucleic acids (e.g., one or more regions of a backbone strand, a backbone strand and a second staple oligonucleotide, a second staple oligonucleotide and a third staple oligonucleotide, etc.); ii) comprising two or more non-contiguous nucleotide sequences configured to form two or more secondary and / or tertiary structures when hybridized to one or more other nucleic acids; ii) comprising one or more nucleotide sequences that are not configured to hybridize to other nucleic acids; and iii) comprising one or more nucleotide sequences configured to constrain the position, orientation, and / or movement of a first secondary and / or tertiary nucleic acid structure relative to a second secondary and / or tertiary nucleic acid structure.

[0110] 51 shows a schematic diagram of a nucleic acid nanostructure comprising a backbone strand 5101 and a plurality of staple oligonucleotides, where the staple oligonucleotides have various structural and / or functional roles. The nucleic acid nanostructure comprises a plurality of structural staple oligonucleotides, each of which has one or more of the following properties: i) binding with the backbone strand 5101 to form one or more tertiary structures, and ii) forming linked single-stranded nucleic acids that position and orient the two or more tertiary structures of the nucleic acid nanostructure relative to each other. The structural staple oligonucleotide includes: 1) nucleic acid 5104 that binds to backbone strand 5101 to form a region of tertiary structure; 2) nucleic acid 5107 that binds to backbone strand 5101 with two nucleotide sequences to form a substantially 180° bend in the nucleic acid nanostructure and connects two tertiary structures formed by the binding of nucleic acid 5107 to backbone strand 5101 with a linking strand comprising the single-stranded nucleotide sequence of nucleic acid 5107; 3) nucleic acid 5108 that binds to backbone strand 5101 with three non-contiguous nucleotide sequences to form at least three tertiary structures and two substantially 180° bends in the nucleic acid nanostructure; and 4) nucleic acid 5109 that each comprises a first sequence that is complementary to backbone strand 5101 and a second sequence that is complementary to another nucleic acid 5109 to form three tertiary structures and one substantially 180° bend in the nucleic acid nanostructure. The nucleic acid nanostructure may also include non-nucleic acid structural elements 5110, such as nucleic acid-binding proteins (e.g., histones) or nanoparticles, which form or stabilize a portion of the two-dimensional and / or three-dimensional structure of the nucleic acid nanostructure. The nucleic acid nanostructure further includes a plurality of functional staple oligonucleotides, each having one or more of the following properties: i) binding with the backbone strand 5101 to form one or more tertiary structures, and ii) modifying the nucleic acid nanostructure to provide additional chemical and / or physical properties to the nucleic acid nanostructure.The functional staple oligonucleotide includes: 1) nucleic acid 5102 that is attached to backbone 5101 to form a tertiary structure and includes moiety 5103 (e.g., terminal ligand, non-terminal ligand, terminal functional group, non-terminal functional group, modified nucleotide, non-nucleic acid polymer, etc.); 2) nucleic acid 5105 that is attached to backbone 5101 to form a tertiary structure and includes detectable label 5106 (e.g., fluorophore, nucleic acid barcode, peptide barcode, etc.); 3) pendant nucleic acid 5111 that is attached to backbone 5101 to form a tertiary structure and includes unattached terminal residues or nucleotide sequences; 4) pendant nucleic acid 5112 that includes two unattached terminal residues or nucleotide sequences and an intermediate nucleotide sequence that is attached to backbone 5101 to form a tertiary structure; and 5) pendant nucleic acid 5113 that includes two terminal nucleotide sequences that are attached to backbone 5101 to form a tertiary structure and an intermediate single-stranded nucleotide sequence that is pendant from the nucleic acid nanostructure (including one or more attached oligonucleotides 5114 that provide tertiary structuring to the pendant portion of nucleic acid 5113).

[0111] Nucleic acid nanostructures, such as SNAP, can include nucleic acid origami. Thus, nucleic acid nanostructures can include one or more nucleic acids having tertiary or quaternary structures, such as spheres, cages, tubes, boxes, tiles, blocks, trees, pyramids, wheels, combinations thereof, and any other possible structures. Examples of such structures formed with DNA origami are shown in Zhao et al. Nano Lett. 11, 2997-3002 (2011) (incorporated herein by reference). In some configurations, nucleic acid nanostructures, such as SNAP, can include a backbone and multiple staple oligonucleotides, where the backbone is a continuous single strand of nucleic acid and the staple oligonucleotides are configured to bind, in whole or in part, to the backbone. Examples of DNA origami structures formed using a continuous backbone strand and several staple strands are shown in Rothemund Nature 440:297-302 (2006) and U.S. Patent Nos. 8,501,923 and 9,340,416 (each of which is incorporated herein by reference). Nucleic acid nanostructures containing one or more nucleic acids (e.g., found in origami or nanoball structures) can include regions of single-stranded nucleic acids, regions of double-stranded nucleic acids, or combinations thereof. In some configurations, nucleic acid nanostructures can include nucleic acid origami and nucleic acid structures other than nucleic acid origami. For example, nucleic acid origami can be connected to one or more single-stranded nucleic acids, and the one or more single-stranded nucleic acids do not form any secondary and / or tertiary structures. In advantageous configurations, nucleic acid origami can include tile structures. A nucleic acid origami tile structure can refer to a structure having an average thickness that is substantially smaller than its characteristic dimensions (e.g., side length, side width, maximum diameter, average diameter, etc.). For example, nucleic acid origami tile structures can have an aspect ratio between a characteristic dimension and an average thickness of at least about 2:1, 3:1, 4:1, 5:1, 10:1, 20:1, or greater than 20:1. Alternatively or additionally, the tile structures can have an aspect ratio between a characteristic dimension and an average thickness of no more than about 20:1, 10:1, 5:1, 4:1, 3:1, 2:1, or less than 2:1.The tile structures may have shapes such as substantially rectangular tiles, substantially square tiles, substantially triangular tiles, substantially circular tiles, substantially oval tiles, or substantially polygonal tiles. The tiles may include one or more faces that are substantially flat. The tiles may include one or more faces that are substantially non-flat (e.g., curved, wavy, etc.).

[0112] A nucleic acid nanostructure, such as a SNAP, may include two or more application surfaces formed by a backbone strand hybridized to multiple staple oligonucleotides. Hybridization of multiple staple oligonucleotides to the backbone strand may form multiple tertiary nucleic acid structures in the nucleic acid nanostructure. In some configurations, the multiple tertiary structures may include a first tertiary structure belonging to a first application surface (e.g., a presentation surface) and a second tertiary structure belonging to a second application surface (e.g., a capture surface). Two tertiary structures in a nucleic acid nanostructure (e.g., a SNAP) may be oriented relative to each other with respect to an axis or plane of symmetry. Two tertiary structures in a nucleic acid nanostructure may be oriented relative to each other with respect to an axis or plane of symmetry of one or both of the tertiary structures, for example, a coaxial axis of symmetry for a nucleic acid double helix. In some configurations having a first and second tertiary structure belonging to different application surfaces, the axis of symmetry of the first tertiary structure and the axis of symmetry of the second tertiary structure are coplanar. In configurations in which the first and second tertiary structures reside in different application planes, the symmetry axis of the first tertiary structure and the symmetry axis of the second tertiary structure may not be coplanar. In some configurations in which the first and second tertiary structures reside in different application planes, the symmetry axis of the first tertiary structure and the symmetry axis of the second tertiary structure may intersect. In some configurations in which the first and second tertiary structures reside in different application planes, the symmetry axis of the first tertiary structure and the symmetry axis of the second tertiary structure may not intersect. The symmetrical features of a nucleic acid nanostructure (e.g., SNAP) can be determined with respect to the average dimensions, shape, or configuration of the nucleic acid nanostructure. For example, slight variations in the arrangement of features due to the helical structure and tertiary structure of the nucleic acid nanostructure or temporal variations due to environmental conditions (e.g., Brownian motion, fluid shear, electromagnetic forces, etc.) can cause small differences between two opposite sides of a nucleic acid nanostructure designed to have a symmetrical structure. A nucleic acid nanostructure can be considered symmetric if two symmetric features fall within about 10% of their expected positions relative to an axis or plane of symmetry.

[0113] A nucleic acid nanostructure composition (e.g., a SNAP composition) may further comprise a molecule or analyte. Optionally, the molecule or analyte is a non-nucleic acid molecule or analyte, respectively. In some configurations, the presentation site of a nucleic acid nanostructure may be connected to a molecule or analyte. For example, a plurality of SNAPs may be deposited on an array after each SNAP of the plurality is connected to a molecule or analyte. In other configurations, the presentation site of a nucleic acid nanostructure need not be connected to a molecule or analyte. For example, a plurality of SNAPs may be deposited on an array before each SNAP of the plurality is connected to a molecule or analyte. In some configurations, the molecule or analyte may comprise a biomolecule selected from the group consisting of a polypeptide, a polysaccharide, a nucleic acid, a lipid, a metabolite, an enzyme cofactor, and combinations thereof. In some configurations, the molecule or analyte may comprise a non-biological particle selected from the group consisting of a polymer, a metal, a metal oxide, a ceramic, a semiconductor, a mineral, and combinations thereof.

[0114] Nucleic acid nanostructure compositions (e.g., SNAP compositions) can include a linker configured to connect an entity (e.g., SNAP, analyte, connecting surface, etc.) to a site (e.g., surface interaction site, presentation site, capture site, surface linkage site, etc.). The linker can have a size of at least about 100 Da, 500 Da, 1 kDa, 5 kDa, 10 kDa, 20 kDa, 25 kDa, 50 kDa, 100 kDa, 250 kDa, 500 kDa, or greater than 500 kDa. Alternatively or additionally, the linker can have a size of about 500 kDa, 250 kDa, 100 kDa, 50 kDa, 25 kDa, 20 kDa, 10 kDa, 5 kDa, 1 kDa, 500 Da, 100 Da or less, or less than about 100 Da. A linker can include chemical and physical properties (e.g., hydrophobicity, hydrophilicity, polarity, steric size, net charge, etc.) that mediate interactions between entities and moieties joined by the linker. For example, a SNAP can include a rigid linker that separates an analyte of interest from a surface by a separation distance and / or prevents contact between the analyte of interest and the surface of the SNAP.

[0115] Nucleic acid nanostructures (e.g., SNAP) may contain functional nucleic acids. Functional nucleic acids may provide additional utility to nucleic acid nanostructures. Functional nucleic acids may include nucleic acid barcodes, which may provide tagging or information coding functions, for example, in the form of identification sequences for analytes colocalized with the functional nucleic acid. As shown in Figures 10A-10D, utilization sites 1040 may include nucleic acid barcode sequences that can be transcribed onto molecules that interact with analyte 1020, or vice versa. Barcode sequences included in utilization sites 1040 or interacting molecules may be sequenced to determine characteristic or prior uses of analyte 1020, such as any interactions that may have occurred with analyte 1020. Functional nucleic acids may include retention sites, which include hybridizable nucleic acid sequences configured to form short-term or weak interactions that temporarily colocalize interacting molecules near the analyte to increase the likelihood of an observed interaction or to decrease the rate at which the interacting molecules dissociate from the analyte. A hybridizing nucleic acid sequence can include a short region of complementarity to another oligonucleotide (e.g., less than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 nucleotides), a nucleic acid sequence with incomplete complementarity to another nucleic acid, a toehold sequence, or any other configuration that promotes a readily reversible nucleic acid hybridization interaction. A functional nucleic acid can include a nucleic acid sequence configured to bind to a labeled nucleic acid (e.g., a fluorescently labeled oligonucleotide) for purposes such as detecting the spatial address of a nucleic acid nanostructure (e.g., on a site on a solid support).

[0116] In another aspect, provided herein is a method of forming a multiplexed array of analytes, the method comprising: a) contacting an array comprising a plurality of sites with a first plurality of nucleic acid nanostructures as described herein, wherein each nucleic acid nanostructure of the first plurality of nucleic acid nanostructures is connected to an analyte of interest of a first plurality of analytes of interest; b) contacting the array comprising a plurality of sites with a second plurality of nucleic acid nanostructures as described herein, wherein each nucleic acid nanostructure of the second plurality of nucleic acid nanostructures is connected to an analyte of interest of a second plurality of analytes of interest; c) depositing the first plurality of nucleic acid nanostructures at a first subset of sites of the plurality of sites; and d) depositing the second plurality of nucleic acid nanostructures at a second subset of sites of the plurality of sites, wherein the first subset of sites and the second subset of sites comprise a random spatial distribution. In some configurations, each nucleic acid nanostructure of a first plurality of nucleic acid nanostructures can comprise a first functional nucleic acid, where the first functional nucleic acid comprises a first nucleotide sequence, and each nucleic acid nanostructure of a second plurality of nucleic acid nanostructures can comprise a second functional nucleic acid, where the second functional nucleic acid comprises a second nucleotide sequence, where the first nucleotide sequence is different from the second nucleotide sequence. In some configurations, a method of forming a multiplexed array can include simultaneously contacting the array with the first plurality of nucleic acid nanostructures and the second plurality of nucleic acid nanostructures. For example, the array can be contacted with a fluid medium containing a mixture of the first plurality of nucleic acid nanostructures and the second plurality of nucleic acid nanostructures. In other configurations, a method of forming a multiplexed array can include sequentially contacting the array with the first plurality of nucleic acid nanostructures and the second plurality of nucleic acid nanostructures. In some configurations, a method of forming a multiplexed array can include simultaneously depositing the first plurality of nucleic acid nanostructures and the second plurality of nucleic acid nanostructures on the array. For example, the array can be contacted with a fluid medium containing a mixture of a first plurality of nucleic acid nanostructures and a second plurality of nucleic acid nanostructures, and then contacted with a second fluid medium that facilitates deposition of the nucleic acid nanostructures on the sites of the array.In other configurations, a method of forming a multiplexed array can include sequentially depositing a first plurality of nucleic acid nanostructures and a second plurality of nucleic acid nanostructures onto an array.

[0117] The method for forming a multiplexed array of analytes may further include contacting the array with a first plurality of detectable nucleic acids, each first detectable nucleic acid of the first plurality of detectable nucleic acids comprising a first complementary nucleotide sequence and a detectable label, wherein the first complementary nucleotide sequence is complementary to the first nucleotide sequence of a first functional nucleic acid of a nucleic acid nanostructure of the first plurality of nucleic acid nanostructures. After connecting the array with the first plurality of detectable nucleic acids, the method for forming a multiplexed array of analytes may further include contacting the first detectable nucleic acid with each first functional nucleic acid. After connecting the first detectable nucleic acid to each first functional nucleic acid, the method may further include detecting each address of the array containing the first detectable nucleic acid described herein. After connecting the first detectable nucleic acid to each first functional nucleic acid, the method may further include removing the first detectable nucleic acid from the first functional nucleic acid. In some configurations, removing the first functional nucleic acid from the first detectable nucleic acid may include heating a nucleic acid nanostructure of the first plurality of nucleic acid nanostructures to at least the melting temperature of the first functional nucleic acid, thereby delinking the first detectable nucleic acid from the first functional nucleic acid. In other configurations, removing the first detectable nucleic acid from the first functional nucleic acid may include contacting the solid support with a fluid medium (e.g., a denaturant, chaotrope, etc.) configured to separate the first detectable nucleic acid from the first functional nucleic acid, optionally in the presence of heat.

[0118] A method for forming a multiplexed array of analytes may include contacting the array with two or more detectable nucleic acids. For example, the method exemplified above may further include contacting the array with a second plurality of detectable nucleic acids, each of which comprises a second complementary nucleotide sequence and a detectable label, and the second complementary nucleotide sequence is complementary to the second nucleotide sequence of the second functional nucleic acid of the nucleic acid nanostructure of the second plurality of nucleic acid nanostructures. After connecting the array with the second plurality of detectable nucleic acids, the method for forming a multiplexed array of analytes may further include contacting the second detectable nucleic acid with each second functional nucleic acid. After connecting the second detectable nucleic acid to each second functional nucleic acid, the method may further include detecting each address of the array containing the second detectable nucleic acid described herein. After connecting the second detectable nucleic acid to each second functional nucleic acid, the method may further include removing the second detectable nucleic acid from the second functional nucleic acid. In some configurations, removing the second functional nucleic acid from the second detectable nucleic acid can include heating nucleic acid nanostructures of the second plurality of nucleic acid nanostructures to at least the melting temperature of the second functional nucleic acid, thereby decoupled the second detectable nucleic acid from the second functional nucleic acid. In other configurations, removing the second detectable nucleic acid from the second functional nucleic acid can include contacting the solid support with a fluid medium configured to separate the second detectable nucleic acid from the second functional nucleic acid (e.g., a denaturant, chaotrope, etc.), optionally in the presence of heat.

[0119] Figures 50A-50F illustrate a method of utilizing functional nucleic acids to form a multiplexed array of analytes of interest. Figure 50A shows an array including a solid support 5000 including a plurality of sites 5001, each site connected to a SNAP 5010. The solid support 5000 is contacted with a plurality of SNAPs 5010. A first subset of the plurality of SNAPs 5010 includes SNAPs 5010 connected to a first analyte of interest 5020 (e.g., a polypeptide from a first sample), and each SNAP 5010 of the first subset includes a first functional nucleic acid 5030 containing a nucleotide sequence of CGT. A second subset of the plurality of SNAPs includes SNAPs 5010 connected to a second analyte of interest 5025 (e.g., a polypeptide from a second sample), and each SNAP 5010 of the second subset includes a second functional nucleic acid 5035 containing a nucleotide sequence of CCA. Figure 50B shows a multiplexed array formed by deposition of a plurality of SNAPs 5010 at a plurality of sites 5001 on a solid support 5010. The first subset of SNAPs 5010 and the second subset of SNAPs 5010 comprise a random spatial distribution at the plurality of sites 5001, and the addresses of the first analyte of interest 5020 and the second analyte of interest 5025 on the array are initially unknown after deposition. Figure 50C shows contacting the solid support 5000 with a first plurality of detectable nucleic acids, each detectable nucleic acid comprising a detectable label 5045 and a complementary nucleic acid 5040 having a nucleotide sequence of GCA. Figure 50D shows a multiplexed array of SNAPs 5010, wherein a first subset of SNAPs 5010 binds a detectable nucleic acid of the first plurality of detectable nucleic acids by base pairing between a first functional nucleic acid 5030 and a complementary nucleic acid 5040. Each site 5001 containing a first analyte of interest 5020 may be detectable at single-analyte resolution by detection of a detectable label 5045 at an address on the array. Figure 50E shows contacting the solid support 5000 with a second plurality of detectable nucleic acids, each detectable nucleic acid comprising a detectable label 5046 and a complementary nucleic acid 5041 having the nucleotide sequence of GGT.Figure 50F shows a multiplexed array of SNAPs 5010, where a second subset of SNAPs 5010 have detectable nucleic acids of a second plurality of detectable nucleic acids connected by base pairing between a second functional nucleic acid 5035 and a complementary nucleic acid 5041. Each site 5001 containing a first analyte of interest 5025 may be detectable at single-analyte resolution by detection of a detectable label 5046 at an address on the array. In some configurations, the addresses of the first analyte of interest 5020 and the second analyte of interest 5025 may be detected simultaneously, for example, by use of detectable labels 5045 and 5046 (e.g., fluorophores) with different detection characteristics (e.g., excitation wavelength, emission wavelength).

[0120] The functional nucleic acids provided herein may comprise a nucleotide sequence configured to hybridize with a complementary nucleotide sequence of a linked moiety (e.g., a detectable label, a nucleic acid barcode, a retention moiety, etc.). The functional nucleic acid may comprise a nucleotide sequence configured to form a double-stranded nucleic acid with the complementary nucleic acid, wherein the double-stranded nucleic acid can be disrupted by melting the double-stranded nucleic acid. The double-stranded functional nucleic acid may comprise a nucleotide sequence configured to hybridize with a complementary nucleotide sequence of at least about 50 o C, 55 o C, 60 o C, 61 o C, 62 o C, 63 o C, 64 o C, 65 o C, 66 o C, 67 o C, 68 o C, 69 o C, 70 o C, 71 o C, 72 o C, 73 o C, 74 o C, 75 o C, 76 o C, 77 o C, 78 o C, 79 o C, 80 o C, 81 o C, 82 o C, 83 o C, 84o C, 85 o C, 86 o C, 87 o C, 88 o C, 89 o C, 90 o C, 91 o C, 92 o C, 93 o C, 94 o C, 95 o C, 96 o C, 97 o C, 98 o C, 99 o C, or 99 o C may have a melting temperature above. Alternatively or additionally, the double-stranded functional nucleic acid is about 99 o C, 98 o C, 97 o C, 96 o C, 95 o C, 94 o C, 93 o C, 92 o C, 91 o C, 90 o C, 89 o C, 88 o C, 87 o C, 86 o C, 85 o C, 84 o C, 83 o C, 82 o C, 81 o C, 80 o C, 79 o C, 78 o C, 77<000017L>C, 76 o C, 75 o C, 74 o C, 73 o C, 72 o C, 71 o C, 70 o C, 69[[ID=9 +]] o C, 68 o C, 67 o C, 66 o C, 65 o C, 64 o C, 63 o C, 62 o C, 61 o C, 60 oC, 55 o C, 50 o C or below, or 50 o C. In some configurations, the melting temperature of a double-stranded functional nucleic acid in a nucleic acid nanostructure can be designed to be lower than the melting temperatures of some or all of the other double-stranded nucleic acids in the nucleic acid nanostructure. In certain configurations, the melting temperature of a double-stranded functional nucleic acid in a nucleic acid nanostructure can be designed to be lower than the melting temperatures of at least 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% of the double-stranded nucleic acids in the nucleic acid nanostructure. For example, a functional nucleic acid can be separated from a complementary nucleic acid at a melting temperature that does not cause loss of component oligonucleotides in the nucleic acid nanostructure containing the functional nucleic acid. In some configurations, the melting temperature of a double-stranded nucleic acid containing a functional nucleic acid can be designed to be lower than the dissociation temperature (e.g., nucleic acid melting temperature, ligand-receptor dissociation temperature, covalent bond degradation temperature, etc.) for a nucleic acid nanostructure connected to a solid support or a connection site bound to a solid support. For example, the melting temperature of the double-stranded functional nucleic acid of the nucleic acid nanostructure is at least 5°C higher than the dissociation temperature of the nucleic acid nanostructure attached to the solid support or the attachment site of the nucleic acid nanostructure bound to the solid support. o C, 6 o C, 7 o C, 8 o C, 9 o C, 10 o C, 11 o C, 12 o C, 13 o C, 14 o C, 15 o C, 16 o C, 17 o C, 18 o C, 19 o C, 20 o C, 21 o C, 22 o C, 23 o C, 24 o C, 25 o C, 26 o C, 27 o C, 28 o C, 29 o C, 30 oC, 35 o C, 40 o C, 45 o C, 50 o C, or 50 o C can be designed to be ultra-low.

[0121] Nucleic acid nanostructures (e.g., SNAP) can include a capture surface or capture site that includes one or more modifying groups that alter the interaction between the nucleic acid nanostructure and the surface. The modified interaction between the nucleic acid nanostructure and the surface can include: 1) increasing the rate or strength of attachment to desired regions of the surface; 2) decreasing the rate or strength of attachment to unwanted regions of the surface; 3) improving the specificity of attachment to the surface; 4) reducing nonspecific attachment to the surface; 5) reducing the strength of interactions (e.g., aggregation, co-binding) between two or more nucleic acid nanostructures; and 6) combinations thereof. In some configurations, the capture site can include a modifying site selected from the group consisting of a charged site (e.g., cationic or anionic site), a polar site, a non-polar site, a ligand site recognized by a receptor, a receptor site recognized by a ligand, a magnetic site, a steric site, an amphipathic site, a hydrophobic site, and a hydrophilic site. In some configurations, the charged site can include a single-stranded nucleic acid or a charged polymer (e.g., a cationic or anionic polymer). In some configurations, the capture site of a nucleic acid nanostructure comprises a plurality of single-stranded nucleic acids, which may comprise a region (e.g., a tail or loop) of a longer oligonucleotide hybridized to the nucleic acid nanostructure. In other configurations, the capture site of a nucleic acid nanostructure may comprise a plurality of single-stranded nucleic acids or charged polymers, where the single-stranded nucleic acids are connected to the oligonucleotides hybridized to the nucleic acid nanostructure by, for example, a covalent linker (e.g., a click reaction product) or a non-covalent linker (e.g., a streptavidin-biotin complex).

[0122] Provided herein are compositions comprising: a) a nucleic acid nanostructure (e.g., a structured nucleic acid particle), the nucleic acid nanostructure comprising: i) a presentation site comprising a connection site connected to or configured to connect with an analyte; and ii) a capture site connected to or configured to connect with a surface, the capture site comprising a plurality of first surface-interaction oligonucleotides, each first surface-interaction oligonucleotide of the plurality of first surface-interaction oligonucleotides comprising a first nucleic acid connected to the structured nucleic acid particle and a first surface-interaction site, the first surface-interaction site being connected to or configured to form a connecting interaction with the surface-interaction site, and the capture site and presentation site comprising different orientations; and b) an analyte comprising a complementary connection group connected to or configured to connect with the presentation site of the structured nucleic acid particle.

[0123] Nucleic acid nanostructure compositions (e.g., SNAP compositions) can include capture sites having multiple pendant groups that mediate connection interactions with a surface (e.g., the connection surface of a solid support). The pendant groups provided herein can be characterized by one or more of the following features: i) comprising unconnected terminal moieties or residues; ii) comprising moieties (e.g., polymer chains) whose spatial degrees of freedom are not constrained by connection interactions with a second moiety on the nucleic acid nanostructure; and iii) comprising moieties whose average temporal variation in position relative to the nucleic acid nanostructure exceeds the average temporal variation in position of moieties incorporated within the nucleic acid nanostructure. Without wishing to be bound by theory, the pendant groups may facilitate multiple properties of nucleic acid nanostructures, including: 1) increased specificity of surface attachment due to interactions between the capture sites and surface attachment sites on the solid support; 2) increased avidity of binding due to the multiplicity of binding interactions between the nucleic acid nanostructure and the connecting surface; 3) tunable binding kinetics based on the pendant groups attached to the nucleic acid nanostructure; 4) tunable binding thermodynamics based on free energy minimization between the capture sites and the connecting surface; 5) reduced interactions between fortuitous nucleic acid nanostructures due to binding incompatibility of the nucleic acid nanostructure capture sites; and 6) combinations thereof.

[0124] Figures 40A-40C show SNAP compositions comprising pendant groups on the SNAP capture sites. Figure 40A shows SNAP 4010 comprising an upwardly oriented presentation surface containing presentation sites 4015 connected to an analyte 4020 (e.g., a polypeptide). The downwardly oriented capture surface of SNAP 4010 comprises a plurality of pendant groups. Each pendant group comprises an optional linker 4017 and a surface-interacting moiety, such as a surface-interacting oligonucleotide 4018 or a surface-interacting connecting group 4019 (e.g., a reactive group, streptavidin, etc.). SNAP 4010 can be contacted with a solid support 4000 comprising a connecting surface 4002 and one or more interstitial regions 4004. Connecting surface 4002 can include a plurality of surface linking groups, each containing an optional linker 4030 (e.g., a passivating molecule, e.g., PEG) and a surface linking moiety, e.g., a complementary oligonucleotide 4038 or a complementary connecting group 4039 (e.g., a complementary reactive group, such as biotin). Optionally, the surface can include a mixture of surface linking groups, where a first plurality of surface linking groups includes a passivating moiety (e.g., PEG chains) and no connecting moiety, and a second plurality of surface linking groups includes a connecting moiety and a passivating moiety (e.g., an oligonucleotide attached to a PEG chain). Figure 40B shows a first connecting configuration of SNAP 4010 to solid support 4000. One or more surface-interacting oligonucleotides 4018 are hybridized to surface-attached complementary oligonucleotides 4038, while one or more other surface-interacting moieties remain unbound. This may suggest that the attached SNAP is not in an energetically favorable binding position. Figure 40C shows a second attachment configuration of SNAP 4010 to solid support 4000, in which each surface interaction site forms a connecting interaction with a complementary surface attachment site. Such a configuration may be the most energetically and / or most stable position for SNAP 4010 on connecting surface 4002.

[0125] A nucleic acid nanostructure (e.g., SNAP) can include a capture site comprising a plurality of oligonucleotides that connect to the nucleic acid nanostructure and provide a plurality of pendant groups, each pendant group comprising a surface interaction site. The surface interaction site can form a connecting interaction with a surface linkage site on a solid support, thereby connecting the nucleic acid nanostructure comprising the surface linkage site to the solid support. The nucleic acid nanostructure can include a plurality of oligonucleotides, and the oligonucleotides of the plurality of oligonucleotides can include a) a first nucleic acid configured to connect to the capture site of the nucleic acid nanostructure, and b) a first surface interaction site. In some configurations, the first surface interaction site can include a second nucleic acid. For example, the oligonucleotides of the plurality of oligonucleotides can include a first nucleic acid sequence configured to connect to SNAP and a second nucleic acid sequence configured to bind to the complementary surface-linked nucleic acid strand of the surface linkage site by base pair hybridization. In some cases, the oligonucleotides containing the first and second nucleic acid sequences can further include a third nucleic acid sequence configured not to hybridize to another nucleic acid, e.g., to provide flexibility or rigidity to the pendant group as needed. In some configurations, the first surface interaction site may comprise, in addition to or instead of the second nucleic acid, a capture group selected from the group consisting of a reactive group, a charged group, a magnetic group, and a component of a binding pair. In some configurations, the binding pair may be selected from the group consisting of streptavidin-biotin, SpyCatcher-Spytag, SnoopCatcher-Snooptag, and SdyCatcher-Sdytag. In some configurations, the reactive group may be configured to undergo a click-type reaction with the surface linkage site. In some configurations, the first surface interaction site may comprise a group configured to form a non-covalent interaction with the surface linkage site, the interaction being selected from the group consisting of an electrostatic interaction, a magnetic interaction, a hydrogen bond, an ionic bond, a van der Waals bond, a hydrophobic interaction, or a hydrophilic interaction.In certain configurations, the first surface interaction site may comprise a nanoparticle selected from the group consisting of an inorganic nanoparticle, a carbon nanoparticle, a polymer nanoparticle, and a biopolymer. In some configurations, the first surface interaction site may further comprise a linker connecting the surface interaction site to the nucleic acid nanostructure. In some configurations, the linker may comprise a hydrophobic linker, a hydrophilic linker, or a cleavable linker.

[0126] An oligonucleotide comprising a surface-interaction moiety can form part of a nucleic acid nanostructure (e.g., a SNAP structure). The nucleic acid nanostructure can comprise a) a backbone nucleic acid strand; and b) a plurality of staple nucleic acid strands connected to the backbone nucleic acid strand. In some configurations, the plurality of staple nucleic acid strands can comprise a first surface-interaction oligonucleotide of a plurality of first surface-interaction oligonucleotides, the first surface-interaction oligonucleotide comprising the surface-interaction moiety. The connection of the first surface-interaction oligonucleotides can form a tertiary structure of the nucleic acid nanostructure (e.g., a SNAP structure). In some configurations, the capture site can comprise a tertiary structure formed by the connection of the first surface-interaction oligonucleotide to the nucleic acid nanostructure (e.g., a SNAP structure). In other configurations, the presentation site can comprise a tertiary structure formed by the connection of the first surface-interaction oligonucleotide to the nucleic acid nanostructure.

[0127] A nucleic acid nanostructure (e.g., SNAP) can include a capture site containing multiple pendant groups, each of which contains a nucleic acid. In some configurations, the pendant groups can include a nucleic acid having a nucleotide sequence that does not contain self-complementarity. In this manner, a surface-interacting oligonucleotide or other nucleic acid can be inhibited from forming a self-hybrid structure under the conditions of the compositions or methods described herein. For example, the nucleotide sequence of the pendant nucleic acid can include a DNA sequence (e.g., ACTACCTACAT) having no more than three deoxyribonucleotide species selected from the group consisting of deoxyadenosine, deoxycytosine, deoxyguanosine, and deoxythymidine. In other configurations, a nucleic acid, e.g., a surface-interacting oligonucleotide or pendant group, can include a nucleotide sequence that contains self-complementarity. For example, the nucleic acid sequence can form a self-hybrid structure, such as a double helix, stem-loop, pseudoknot, hairpin, or G-quadruplex, under some or all of the conditions of the compositions or methods described herein. The methods described herein can be configured to self-hybridize in some steps but not in other steps. For example, in the first step of the method, the first nucleic acid can be in a self-hybridized state to inhibit unwanted hybridization to a second nucleic acid strand, and in the second step, the first nucleic acid can be single-stranded or hybridized to a second nucleic acid strand. In some configurations, the surface-interactive oligonucleotides of the plurality of surface-interactive oligonucleotides can comprise a homopolymer nucleotide sequence selected from the group consisting of poly-deoxyadenosine sequences, poly-deoxycytosine sequences, poly-deoxyguanosine sequences, or poly-deoxythymidine sequences. The first contiguous sequence of the nucleic acid strand configured to form self-complementarity with the second portion of the nucleic acid strand can comprise at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, or more than 50 contiguous nucleotides.Alternatively or additionally, the first contiguous sequence of the nucleic acid strand configured to form self-complementarity with the second portion of the nucleic acid strand can include no more than about 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or fewer than 3 contiguous nucleotides. A first contiguous sequence of the nucleic acid strand configured to form self-complementarity with a second portion of the nucleic acid strand can be separated from the second portion of the nucleic acid strand by at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 750, 1000, or more than 1000 nucleotides. Alternatively or additionally, the first contiguous sequence of the nucleic acid strand configured to form self-complementarity with the second portion of the nucleic acid strand can be separated from the second portion of the nucleic acid strand by no more than about 1000, 750, 500, 400, 300, 200, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or less than 3 contiguous nucleotides.

[0128] The pendant nucleic acid portion of the pendant group of the surface interaction moiety may comprise a certain number of linked nucleotides (e.g., natural nucleotides, modified nucleotides, etc.). In some cases, the nucleic acid portion of the surface interaction moiety may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more than 100 nucleotides. Alternatively or additionally, the nucleic acid portion of the surface interaction site may comprise no more than about 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 2 nucleotides.

[0129] A nucleic acid nanostructure (e.g., SNAP) can include a capture site having a plurality of pendant groups containing surface interaction moieties. The capture site can include at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more than 100 surface interaction moieties. Alternatively or additionally, the capture sites can comprise no more than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than 2 surface interaction sites. Nucleic acid nanostructures (e.g., SNAP) can be configured to have an average surface density of pendant groups comprising surface interaction sites (e.g., surface-interacting oligonucleotides, surface-interacting reactive groups, etc.). The average surface density of surface interaction sites for a nucleic acid nanostructure can be determined by the number of surface interaction sites configured to connect to the connecting surface of a solid support relative to the effective surface area or footprint of the capture sites of the nucleic acid nanostructure that connect to the connecting surface. The effective surface area of ​​a capture site can include the two-dimensional protrusion of the capture site onto an effectively planar surface, and can optionally include additional surface area caused by the maximum extension of one or more pendant groups from the capture site of the nucleic acid nanostructure. The footprint of a nucleic acid nanostructure can include the maximum cross-sectional area of ​​the nucleic acid nanostructure or its capture site when the nucleic acid nanostructure is attached to a surface. The capture site of a nucleic acid nanostructure (e.g., SNAP) is expressed in nanometers per square (nm 2 ) at least 0.0001 surface interaction sites, 0.005 / nm 2 , 0.001 / nm 2 , 0.05 / nm 2 , 0.01 / nm 2 , 0.05 / nm 2 , 0.1 / nm 2 , 0.5 / nm 2 , 1 / nm 2 , 5 / nm 2 , 10 / nm 2, or 10 / nm 2 Alternatively or additionally, the capture sites of the nucleic acid nanostructure may have an average surface interaction site density of greater than about 10 / nm 2 , 5 / nm 2 , 1 / nm 2 , 0.5 / nm 2 , 0.1 / nm 2 , 0.05 / nm 2 , 0.01 / nm 2 , 0.005 / nm 2 , 0.001 / nm 2 , 0.0005 / nm 2 , 0.0001 / nm 2 or less than 0.0001 / nm 2 The average surface interaction site density may be less than 1000 nm.

[0130] The plurality of surface interaction sites may be distributed or spaced apart across the capture site of a nucleic acid nanostructure (e.g., a SNAP). In some configurations, the surface interaction site distribution or density is substantially uniform across the effective surface area or footprint of the capture site (e.g., approximately uniform spacing and / or orientation between adjacent surface interaction sites). In other configurations, the surface interaction site distribution or density is not substantially uniform across the effective surface area or footprint of the capture site. For example, a fraction or all of the plurality of surface interaction sites may be located near the central region of the capture site. In another configuration, a fraction or all of the plurality of surface interaction sites may be located near the outer regions of the capture site. Figures 41A-41B illustrate SNAP configurations with different SNAP distributions. Figure 41A shows a SNAP 4110 coupled to an analyte 4120 and containing a plurality of surface interaction sites 4118 on the capture site, with the plurality of surface interaction sites distributed toward the outer edges of the capture site surface. FIG. 41B shows a SNAP 4110 connected to an analyte 4120 and containing multiple surface interaction sites 4118 on the capture site, the multiple surface interaction sites being distributed toward the central portion of the capture site surface.

[0131] In some configurations, a nucleic acid nanostructure (e.g., a SNAP) may include a capture site that includes multiple types of surface-interaction sites. The capture site may include multiple types of surface-interaction sites to increase the specificity of the binding location for the nucleic acid nanostructure. For example, a SNAP may include multiple surface-interaction oligonucleotides and one or more surface-interaction reactive groups. In certain instances, such a SNAP may be contacted with a connecting surface that includes a high surface density of complementary oligonucleotides and a low surface density of complementary reactive groups, and the binding interactions between the surface-interaction oligonucleotides and the complementary oligonucleotides maintain the connected SNAP near the connecting surface until covalent interactions can form between the surface-interaction reactive groups and the relatively rare surface-linked complementary reactive groups. Nucleic acid nanostructures can interact with surfaces through a combination of interaction types, for example, two different non-covalent interactions (e.g., nucleic acid hybridization and electrostatic interactions), two different covalent interactions (e.g., two bioorthogonal click-type reactions), or a combination of covalent and non-covalent interactions (e.g., covalent interactions and nucleic acid hybridization, covalent interactions and electrostatic interactions, covalent interactions and nucleic acid hybridization and electrostatic interactions, etc.).

[0132] In another aspect, provided herein are compositions comprising: a) a nucleic acid nanostructure (e.g., SNAP) comprising: i) a presentation moiety coupled to or configured to couple with an analyte; and ii) a capture moiety coupled to or configured to couple with a coupling surface, the capture moiety comprising a plurality of oligonucleotides, each oligonucleotide of the plurality of oligonucleotides comprising a surface interaction moiety; b) an analyte coupled to the presentation moiety; and c) a solid support comprising a coupling surface, the surface comprising one or more surface coupling moieties, the surface interaction moieties of the plurality of surface interaction moieties coupled to the surface coupling moieties of one or more surface coupling moieties.

[0133] Nucleic acid nanostructure compositions (e.g., SNAP compositions) provided herein can further include a separation group. The separation group can include a molecule, linker, or nucleic acid nanostructure (e.g., a presentation SNAP or structure SNAP) configured to create a separation or gap between an analyte and a surface or portion of the nucleic acid nanostructure (e.g., a presentation surface or site, a capture surface or site). Figure 29 shows a profile diagram of a SNAP complex containing an analyte with various possible separation gaps indicated. The SNAP complex can include capture-based SNAPs 2910, 2911, and 2912, which connect the complex to solid support 2900. Presentation SNAP 2930 is connected to structure-based SNAP 2920, which is connected to capture-based SNAP 2911. Analyte 2940 is connected to presentation SNAP 2930. The separation gap can be measured from the analyte to the surface or SNAP. Some possible separation gaps include a gap from the center of the analyte 2940 to the solid support 2900 (g1), to the top surface of the capture-based SNAP 2910 (g2), or to the top surface of the display-SNAP 2930 (g3); a gap between the outer surface of the analyte 2940 and the surface of the solid support 2900 (g4); a gap between the outer surface of the analyte 2940 and the surface of the capture-based SNAP 2910 (g5); or a gap between the outer surface of the analyte 2940 and the surface of the display-SNAP 2930 (g6). Figures 3A-3D show a SNAP 300 including a multivalent linker 320 that generates an average separation gap between the analyte 310 and the top surface of the SNAP 300. When the SNAP 300 is connected to the solid support 330, the analyte 310 will also have an average separation gap with the solid support 330. In some configurations, the separation group may include a rigid separation group selected from groups including polymer linkers, nucleic acid linkers, and nanoparticle linkers. In some particular configurations, the nucleic acid linker comprises a tertiary structure (e.g., a DNA double helix). In other configurations, the separation group comprises a flexible linker. The separation gap can have characteristic average, maximum, or minimum dimensions.The average, maximum or minimum dimension of the separation gap can be at least about 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, or more than 100 nm. Alternatively or additionally, the average, maximum or minimum dimension of the separation gap can be about 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30 nm, 25 nm, 20 nm, 19 nm, 18 nm, 17 nm, 16 nm, 15 nm, 14 nm, 13 nm, 12 nm, 11 nm, 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm or less, or less than 1 nm.

[0134] Nucleic acid nanostructures (e.g., SNAP) can contain multiple nucleic acids (e.g., backbone strands, multiple oligonucleotides) that form stable hybridized structures through complementary base pairing. The stability of a particular hybridized structure can be characterized by conventional methods, such as the degree of complementarity or the melting temperature of the estimated or measured secondary structure. Stability (e.g., melting temperature) can be predicted by software packages such as CADNANO, ATHENA, or DAEDALUS. Hybridized nucleic acid structures are characterized by a minimum of about 50 o C, 51 o C, 52 o C, 53 o C, 54 o C, 55 o C, 56 o C, 57 o C, 58 o C, 59 o C, 60 o C, 61 o C, 62 o C, 63 o C, 64 o C, 65 o C, 66 o C, 67 o C, 68 o C, 69o C, 70 o C, 71 o C, 72 o C, 73 o C, 74 o C, 75 o C, 76 o C, 77 o C, 78 o C, 79 o C, 80 o C, 81 o C, 82 o C, 83 o C, 84 o C, 85 o C, 86 o C, 87 o C, 88 o C, 89 o C, 90 o C, or 90 o Alternatively or additionally, the hybridized nucleic acid structure may have a characterized melting temperature greater than about 90°C. o C, 89 o C, 88 o C, 87 o C, 86 o C, 85 o C, 84 o C, 83 o C, 82 o C, 81 o C, 80 o C, 79 o C, 78 o C, 77 o C, 76 o C, 75 o C, 74 o C, 73 o C, 72 o C, 71 o C, 70 o C, 69 o C, 68 o C, 67 o C, 66 o C, 65 o C, 64 o C, 63 o C, 62 o C, 61 o C, 60 o C, 59 o C, 58 o C, 57o C, 56 o C, 55 o C, 54 o C, 53 o C, 52 o C, 51 o C, 50 o C or below, or 50 o C.

[0135] A nucleic acid nanostructure (e.g., SNAP) or a surface of a nucleic acid nanostructure (e.g., a presentation surface, a capture surface) can have a characteristic dimension (e.g., length, width, radius). Characteristic dimensions can include any characterization of a group or probe size, such as length, width, height, radius, circumference, etc. A nucleic acid nanostructure or a surface of a nucleic acid nanostructure can have a characteristic dimension of at least about 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, or greater than 1000 nm. Alternatively or additionally, a nucleic acid nanostructure or a surface of a nucleic acid nanostructure can have a characteristic dimension of about 1000 nm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 450 nm, 400 nm, 350 nm, 300 nm, 250 nm, 200 nm, 180 nm, 160 nm, 140 nm, 120 nm, 100 nm, 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, 50 nm, 45 nm, 40 nm, 35 nm, 30 nm, 25 nm, 20 nm, 15 nm, 10 nm, 5 nm or less, or less than 5 nm.

[0136] Nucleic acid nanostructures (e.g., SNAP) can be connected to or configured to connect to one or more analytes. Nucleic acid nanostructures can include one or more presentation surfaces or presentation sites connected to or configured to connect to one or more analytes. Nucleic acid nanostructures can be connected to one or more analytes. Nucleic acid nanostructures can include one or more presentation surfaces or presentation sites connected to one or more analytes. Nucleic acid nanostructure presentation surfaces or presentation sites can include one or more functional groups or sites configured to connect to analytes. When multiple functional groups are present, the functional groups can be of the same type as each other, or alternatively, different functional groups can be present. Nucleic acid nanostructures can include at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more than 100 functional groups or moieties configured to connect to an analyte. Alternatively or additionally, nucleic acid nanostructures can include no more than about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less than about 2 functional groups or moieties configured to connect to an analyte.

[0137] Multiple nucleic acid nanostructures (e.g., SNAP) and multiple analytes can be connected at a fixed molecular ratio. The ratio of analyte to nucleic acid nanostructure can be calculated as an average ratio. The analyte:nanostructure ratio can follow several quantifiable distributions, such as Poisson, binomial, beta-binomial, hypergeometric, or bimodal. In some configurations, there can be, on average, more than one analyte connected to a nucleic acid nanostructure. In some configurations, there can be, on average, more than one nucleic acid nanostructure connected to an analyte. Nucleic acid nanostructures having multiple analytes attached can have an average analyte:nanostructure ratio of about 100:1, 50:1, 25:1, 20:1, 15:1, 10:1, 5:1, 4:1, 3:1, 2:1, 1.5:1, 1:1, 1:1.5, 1:2, 1:3, 1:4, 1:5, 1:10, 1:15, 1:20, 1:25, 1:50, 1:100 or less, or less than 1:100. Alternatively or additionally, nucleic acid nanostructures having multiple analytes attached thereto can have an average analyte:nanostructure ratio of at least about 1:100, 1:50, 1:25, 1:20, 1:15, 1:10, 1:5, 1:4, 1:3, 1:2, 1:1.5, 1:1, 1.5:1, 2:1, 3:1, 4:1, 5:1, 10:1, 15:1, 20:1, 25:1, 50:1, 100:1, or greater than 100:1.

[0138] Multiple nucleic acid nanostructures (e.g., SNAPs) can be characterized by an occupancy ratio. The occupancy ratio can be defined as the percentage of nucleic acid nanostructures with at least one attached analyte. The nucleic acid nanostructure occupancy ratio can be controlled to provide a desired occupancy (e.g., maximum occupancy) by increasing the relative ratio of analyte to nucleic acid nanostructures in the analyte-attached state. The nucleic acid nanostructure occupancy ratio can be controlled to minimize the number of nucleic acid nanostructures with multiple analytes, for example, by reducing the concentration of analyte relative to the nucleic acid nanostructure in the analyte-attached state. For example, a SNAP composition in which 70% of the SNAPs are attached to one or more analytes would have an occupancy ratio of 0.7. The occupancy ratio can be determined by an appropriate analytical technique, such as fluorescence microscopy or spectroscopy. The plurality of nucleic acid nanostructures can have an occupancy ratio of at least about 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 0.96, 0.97, 0.98, 0.99, or greater than 0.99. Alternatively or additionally, the plurality of nucleic acid nanostructures can have an occupancy ratio of about 0.99, 0.98, 0.97, 0.96, 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.05, 0.01 or less, or less than about 0.01.

[0139] The nucleic acid nanostructures (e.g., SNAP) described herein may further include a capture surface. The capture surface may be configured to facilitate interactions between surfaces or interfaces, such as binding interactions or phase-separation interactions. A surface may be any solid and / or rigid boundary, where the nucleic acid nanostructure is substantially inhibited or unable to move orthogonally through the solid and / or rigid boundary. An interface may refer to a non-solid or deformable boundary, where the nucleic acid nanostructure may move orthogonally through the non-solid or deformable boundary. A surface may include the surface of a solid material, such as a metal, metal oxide, ceramic, glass, polymer, or semiconductor. An interface may include an air / liquid or liquid / liquid phase boundary. Exemplary internal surfaces may include an air / water interface or a water / oil interface, such as an oil-in-water or water-in-oil emulsion. A capture surface or capture site may be configured to form a reversible or irreversible interaction with the surface. For example, the capture surface of a SNAP can include one or more single-stranded nucleic acid strands configured to hybridize to complementary single-stranded nucleic acids displayed on the surface, thereby reversibly attaching the SNAP to the surface. In another example, the capture surface of a SNAP can include one or more click-type reactive groups configured to covalently bind to complementary click-type reactive groups displayed on the surface, thereby irreversibly attaching the SNAP to the surface. In some configurations, a nucleic acid nanostructure (e.g., a SNAP) can include a capture surface including a first site and a second site, where the first site is configured to reversibly attach to the surface and the second site is configured to irreversibly attach to the surface. In some cases, the nucleic acid nanostructure can be configured to provide a transient association with a solid support. For example, a SNAP can be configured to reversibly attach an analyte (e.g., via an oligonucleotide hybridized to the SNAP structure) and then transiently bind to the surface of the solid support, allowing the analyte to migrate to an analyte-attaching site (e.g., a complementary oligonucleotide, a click-type reactive group, etc.) on the surface. After the analyte has been transferred to the surface, the SNAP can be dissociated and optionally reused to transfer a second analyte to the solid support.

[0140] Nucleic acid nanostructures (e.g., SNAP) can interact with a surface or interface through interactions that associate the nucleic acid nanostructure with the surface or interface. Nucleic acid nanostructures can associate with a surface or interface through binding interactions, such as electrostatic interactions, magnetic interactions, covalent bonds, or non-covalent bonds (e.g., hydrogen bonds, nucleic acid base pairing). Nucleic acid nanostructures can include one or more surfaces configured to effect phase separation at a phase boundary. For example, a SNAP can include a first surface including a plurality of hydrophobic moieties and a second surface including a plurality of hydrophilic moieties, and the SNAP can be configured to associate with the phase boundary by segregation of the first surface into the more hydrophobic phase.

[0141] Figures 4A-4G show various configurations of SNAPs interacting with surfaces or interfaces. Figure 4A shows a SNAP 410 connected to an analyte 420 that interacts with a surface 430 via electrostatic interactions. The SNAP can include a negatively charged capture surface 412 that can be attracted to a positively charged surface 430, e.g., a surface 430 functionalized with positively charged functional groups 432. The negative charge of the SNAP can result from either or both negative charges present in the phosphodiester backbone of nucleic acids or negatively charged moieties conjugated to the SNAP. Figure 4B shows a SNAP 410 connected to an analyte 420 (e.g., a polypeptide) that interacts with a surface 430 via magnetic interactions. The SNAP can include a capture surface 412 that includes multiple magnetic groups (e.g., paramagnetic particles conjugated to the SNAP) that can be attracted to the surface 430, e.g., a surface 430 that includes multiple oppositely polarized magnetic groups 438. Figure 4C shows a SNAP 410 connected to an analyte 420 (e.g., a polypeptide) that interacts with a surface 430 through non-covalent bonding interactions between complementary oligonucleotides. The SNAP 410 includes a capture surface 412 that includes a plurality of oligonucleotides 414 that hybridize to a plurality of complementary oligonucleotides 434 attached to the surface 430. Figure 4D shows a SNAP 410 connected to an analyte 420 (e.g., a polypeptide) that is covalently conjugated to the surface 430. A covalent bond 435 can be formed between complementary reactive groups, such as click reactive groups (e.g., methyltetrazine-transcyclooctylene, azido-dibenzocyclooctyne, etc.), on the surface 430 and the capture surface 412 of the SNAP 410. In some configurations, the SNAP 410 can include multiple reactive groups on the capture surface 412 configured to form covalent bonds 430.

[0142] 4E-4F illustrate configurations of a SNAP that interact with an interface (e.g., water / air or water / oil). The SNAP can associate with the interface through phase-separation interactions. FIG. 4E illustrates a SNAP 410 coupled to an analyte 420 that includes a capture surface 412 containing a plurality of hydrophobic groups 417 (e.g., lipids). The presence of the hydrophobic groups 417 causes the SNAP 410 to associate with an interface 440 that forms between a non-aqueous phase 444 and an aqueous phase 448. The hydrophobic groups 417 may preferentially migrate into the non-aqueous phase 444, while the more hydrophilic SNAP 410 and analyte 420 may remain in the aqueous phase 448. FIG. 4F illustrates an alternative configuration of an interface-associated SNAP 410. FIG. 4F illustrates a SNAP 410 coupled to an analyte 420 that includes a capture surface 412 containing a plurality of hydrophobic groups 417. The SNAP is further configured such that the capture surface 412 also serves as the presentation surface for the SNAP. The presence of hydrophobic groups 417 causes SNAP 410 to associate with interface 440, which forms between non-aqueous phase 444 and aqueous phase 448. Hydrophobic groups 417 and analyte 420 may preferentially migrate into non-aqueous phase 444, while the more hydrophilic SNAP 410 may remain in aqueous phase 448. The configuration of Figure 4F may be advantageous for the presentation of hydrophobic analytes (e.g., membrane proteins, inorganic nanoparticles).

[0143] FIG. 4G shows a configuration of a SNAP 410 connected to an analyte 420 that interacts with a surface 430 through an ion-mediated connection interaction. The SNAP may include a negatively charged capture surface 412 that can be attracted to the surface 430, e.g., a surface 430 functionalized with negatively charged functional groups 433. In other configurations, the surface material may possess an inherent negative charge. The negative charge of the SNAP 410 may result from negative charges present in the phosphodiester backbone of nucleic acids or from negatively charged moieties conjugated to the SNAP. The inherent repulsion between the capture surface 412 of the SNAP 410 and the negatively charged functional groups 433 can be overcome by complexing or layering positively charged ions 450 to form an ion-mediated layer between the SNAP 410 and the surface 430. Those skilled in the art will readily recognize that the ion-mediated interaction can be modified for other situations, such as mediating a positive-positive charge interaction or changing the strength of the positive-negative charge interaction. Deposition of SNAP on surfaces through ion-mediated charge interactions involves the interaction of certain monoatomic, polyatomic, monovalent, multivalent, metallic, or non-metallic ions, e.g., H + , Li + , Na + , K. + , Rb + , Cs + , Mg 2+ , Ca 2+ , Sr 2+ , Ba 2+ , Al 3+ , Ag + , Zn 2+ , Fe 2+ , Fe 3+ , Cu + , Cu 2+ , H - , F - , Cl - , Br - , I - , O 2- , S 2- , N 3- , P 3- , B(OH)4 - , C2H5O - , CH3COO - , C6H5COO - , C6H5O73- , CO3 2- , C2O4 2- , C.N. - , CrO4 2- , Cr2O7 2- , HCO3 - , HPO4 2- , H2PO4 - , HSO4 - , MnO4 2- , MnO4 - , NH2 - , O2 2- , O.H. - , SH - , SCN - , SiO4 2- , S2O3 2- , C(NH2) 3+ , NH4 + , PH4 + , H3O + , H2F + , C5H5O + , Hg2 2+ , and combinations thereof. Figure 4H shows a configuration of a SNAP 410 connected to an analyte 420 that interacts with a surface 430 through a particle-mediated connection interaction. The SNAP may include a positively charged capture surface 412 (e.g., including one or more aminated capture sites) that can be essentially repelled by the surface 430, e.g., a surface 430 functionalized with positively charged functional groups 433 (e.g., aminated silanes). Negatively charged intermediate particles 460 may facilitate the interaction between the SNAP 410 and the surface 430 by passivating the surface positive charge and providing a negative charge that electrostatically connects to the positively charged capture surface 412 of the SNAP 410. The negatively charged particles 460 may include carboxylated inorganic nanoparticles (e.g., carboxylated gold nanoparticles, carboxylated silver nanoparticles, etc.) or carboxylated organic nanoparticles (e.g., carboxylated dextran nanoparticles, carboxylated polystyrene particles, etc.).

[0144] In some configurations, nucleic acid nanostructures (e.g., SNAP) can be structured to inhibit or avoid the formation of charge-mediated interactions. Nucleic acid nanostructures can be nonspecifically attracted to areas of the surface where deposition is not expected to occur by charge-mediated interactions, for example, due to ionic components of the deposition buffer. Nucleic acid nanostructures can be configured to present ligands or other groups on the capture surface or capture site that disrupt unwanted interactions. For example, SNAP can include one or more single-stranded nucleic acids (e.g., pendant tails of oligonucleotides that partially hybridize to the SNAP structure) that disrupt the formation of charge-mediated interactions. In another example, SNAP can include a capture site containing one or more oligonucleotides, each of which contains a modified nucleotide configured to disrupt the formation of charge-mediated interactions. The modified nucleotides can be chemically homogeneous (e.g., same charge, same structure, same polarity, etc.) or chemically heterogeneous.

[0145] The capture surface of a nucleic acid nanostructure (e.g., SNAP) can be configured to mediate the association between the nucleic acid nanostructure and a surface or interface. The configuration of the nucleic acid nanostructure can determine the strength of the association between the nucleic acid nanostructure and a surface or interface. The nucleic acid nanostructure can have a reversible or irreversible association with a surface or interface. An irreversible association between a nucleic acid nanostructure and a surface or interface can be formed by a covalent bond or very strong non-covalent interaction(s) (e.g., streptavidin-biotin). A reversible association between a nucleic acid nanostructure and a surface or interface can be formed by a weaker interaction, such as an electrostatic interaction, a magnetic interaction, or a hydrogen bond. A reversible association can be stable until it is disrupted, for example, by the introduction of a denaturant or salt, or by cleavage of a photolinker.

[0146] The size and / or conformation of a nucleic acid nanostructure capture surface can affect the strength of association between a nucleic acid nanostructure and a surface or interface. A smaller interaction area between the capture surface and a surface or interface can facilitate a weaker interaction between the nucleic acid nanostructure and the surface or interface. A capture surface or capture site can include one or more tertiary nucleic acid structures that form interactions, e.g., electrostatic interactions, with the surface. Increasing the size or number of tertiary structures in a capture surface or capture site can increase the strength of the interaction with the surface. For example, increasing the size, amount, or local density of nucleic acid tertiary structures in a capture site can increase the strength of the electrostatic interaction between the capture site and the surface due to the increased number of negatively charged phosphodiester groups in the nucleic acid backbone of each tertiary structure. Figures 5A-5D show various configurations of SNAP with different capture surface sizes and / or conformations. Figures 5A and 5B show tapered SNAP structures with different two-dimensional protrusions between the presenting surface and the capture surface. Figure 5A shows a SNAP 510 bound to a surface 530. The SNAP includes a larger presentation surface 520 that includes presentation sites 522. The SNAP also includes a capture surface 540 that is smaller in area than the area of ​​the presentation surface 520. The capture surface 540 forms a small interaction area 545 with the surface 530, optionally resulting in a weaker association between the SNAP 510 and the surface 530. Figure 5B shows a SNAP 510 bound to the surface 530. The SNAP includes a smaller presentation surface 520 that includes analyte conjugation sites 522. The SNAP also includes a capture surface 540 that is larger in area than the area of ​​the presentation surface 520. The capture surface 540 forms a large interaction area 545 with the surface 530, optionally resulting in a stronger association between the SNAP 510 and the surface 530. Figure 5C shows a SNAP 510 that includes a textured capture surface 540 that associates the SNAP 510 with the surface 530. The SNAP includes a larger presentation surface 520 that contains presentation sites 522. The non-planar capture surface allows the SNAP to form a small interaction area 545 with the surface 530, optionally resulting in a weaker association between the SNAP 510 and the surface 530. Figure 5D shows a SNAP 510 including a non-planar capture surface 540 that allows the SNAP 510 to associate with the non-planar surface 535.The SNAP includes a presentation surface 520 containing capture sites 522. Due to shape complementarity between the capture surface 540 and the non-planar surface 535, the SNAP forms a larger interaction area 545 with the surface 535, potentially resulting in a stronger association between the SNAP 510 and the surface 535. Thus, the size and / or shape of the nucleic acid nanostructure (e.g., SNAP) capture surface can be useful for orienting the nucleic acid nanostructures on the surface. Surfaces can be patterned with interaction areas to provide further control over the position and / or orientation of nucleic acid nanostructures on the surface. For example, hexagonal arrays of nucleic acid nanostructures can be formed by binding nanostructures to a surface having a hexagonal pattern of interaction areas, with the interaction areas separated by interstitial areas that are inert to binding nanostructures. Furthermore, manipulating the size and / or shape of one or both of the surface and the plurality of nucleic acid nanostructures can provide control over the arrangement of nucleic acid nanostructures into an array. Thus, the user can achieve a desired density of nucleic acid nanostructures in the array, an average spacing of nucleic acid nanostructures in the array, a minimum separation between adjacent nucleic acid nanostructures in the array, or a maximum separation between adjacent nucleic acid nanostructures in the array. In this manner, analytes conjugated to the nucleic acid nanostructures will also be positioned accordingly.

[0147] Nucleic acid nanostructures (e.g., SNAPs) can include capture surfaces that form interaction areas smaller than their two-dimensional protrusions. Figure 6 shows views of the bottom and top surfaces of a rectangular-shaped SNAP 600. The edge correspondence between the top and bottom views is indicated by dashed lines. SNAP 600 includes a capture surface 610 configured to contact only a surface or interface (not shown) around the periphery of SNAP 600. SNAP further includes a presentation surface 620 that includes presentation sites 622. Presentation surface 620 occupies a sufficient area of ​​the top surface of SNAP 600. The configuration shown in Figure 6 will limit the size and / or strength of association between SNAP 600 and the surface or interface while maximizing the available area for analyte presentation. Those skilled in the art will readily recognize that the configuration shown in Figure 6 can be reconfigured to increase or decrease the size of capture surface 610 and presentation surface 620 by modifying the structured nucleic acid components that make up SNAP 600.

[0148] The nucleic acid nanostructures (e.g., SNAP) provided herein can include a utilization surface or utilization site that includes one or more modifying moieties. In some configurations, the utilization surface can comprise all or a portion of another surface, such as a presentation surface or capture surface. The modifying moieties can be added to the capture surface or capture site to alter surface characteristics while mediating association between the nucleic acid nanostructure and a surface, a nucleic acid nanostructure and an interface, a first nucleic acid nanostructure and a second nucleic acid nanostructure, or a nucleic acid nanostructure and an encountered molecule (e.g., affinity reagent, fluorophore, etc.). The modifying moieties can be covalently or non-covalently attached. The modifying moieties can be attached to the nucleic acid nanostructure before, during, or after construction of the nanostructure. The utilization surface modifying groups can include charged moieties, magnetic moieties, steric moieties, amphipathic moieties, optical moieties (e.g., reflective materials, absorbing materials), hydrophobic moieties, and hydrophilic moieties. Charged moieties can include functional groups that can carry an inherent positive or negative charge or that can carry a charge under dissociation conditions (e.g., carboxylic acid, nitrate, sulfone, phosphate, phosphonate, etc.). Magnetic moieties can include paramagnetic, diamagnetic, and ferromagnetic particles, e.g., nanoparticles (e.g., gadolinium, manganese, iron oxide, bismuth, gold, silver, cobalt nanoparticles, etc.). Steric moieties can include polymers and biopolymers (e.g., PEG, PEO, dextran, sheared nucleic acids). Amphipathic moieties can include phospholipids (e.g., phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylinositol phosphate, phosphatidylinositol biphosphate, phosphatidylinositol triphosphate, ceramide phosphorylcholine, ceramide phosphorylethanolamine, ceramide phosphoryllipid), glycolipids (e.g., glyceroglycolipids, glycosphingolipids, rhamnolipids, etc.), and sterols (e.g., cholesterol, campesterol, sitosterol, stigmasterol, ergosterol, etc.).Hydrophobic moieties can include steroids (e.g., cholesterol), saturated fatty acids (e.g., caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, etc.), and unsaturated fatty acids (e.g., myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, etc.). Hydrophilic compounds can include charged and polar molecules (e.g., glycol, cyclodextrin, cellulose, polyacrylamide, etc.).

[0149] In some configurations, nucleic acid nanostructures (e.g., SNAPs) can include a utilization surface or utilization site containing one or more extendable nucleic acids (e.g., nucleic acid primers) or extended nucleic acids (e.g., extended nucleic acid primers). Primers or other extendable nucleic acid ends can be hybridized to a template strand to direct polymerase-based extension. However, extension need not involve the addition of nucleotides by a template-guided polymerase; for example, it can instead involve nucleotide addition by terminal deoxynucleotidyl transferase or oligonucleotide addition by a ligase. Optionally, some or all nucleic acid ends in a nucleic acid nanostructure other than the given primer to be extended can be non-extendable, for example, due to the presence of a 5' or 3' extension block site. Thus, extension can occur selectively at a given primer instead of other ends. Exemplary extension block sites include, but are not limited to, those used in nucleic acid sequencing-by-synthesis reactions, such as reversible terminators. Reversible terminator sites can be particularly useful because they can be present in a first nucleic acid to prevent extension of a second nucleic acid end during extension of that end, and then removed from the first end to make it extendable.

[0150] The extended nucleic acid can be configured to occupy the volume surrounding the nucleic acid nanostructure and / or exclude other molecules (e.g., other SNAPs, analytes, etc.) from approaching or contacting the nucleic acid nanostructure. The extended nucleic acid can include a single-stranded nucleic acid strand, a double-stranded nucleic acid strand, or a combination thereof. The extended nucleic acid can include a secondary structure (e.g., a helical structure). The extended nucleic acid can include regions of random or disordered structure. The extended nucleic acid strand can incorporate modified or non-natural nucleotides or other linkage sites. The extended nucleic acid can be formed by methods such as terminal deoxynucleotidyl transferase (TdT) polymerization. Methods for forming extended nucleic acids are described in Angewandte Chemie Int. Ed., 10.1002 / anie.202107829, (2021), which is incorporated herein by reference in its entirety. The extended nucleic acid may have a sequence containing at least about 100, 200, 300, 400, 500, 750, 1000, 1500, 2000, 2500, 3000, 4000, 5000, 10000, 15000, 20000, or more than 20000 nucleotides. Alternatively or additionally, the extended nucleic acid may have a sequence containing no more than about 20000, 15000, 10000, 5000, 4000, 3000, 2500, 2000, 1500, 1000, 750, 500, 400, 300, 200, 100, or less than 100 nucleotides. An extended nucleic acid can have a length in an extended or contracted state (e.g., coiled, self-hybridized, etc.) of at least about 10 nanometers (nm), 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, or greater than 500 nm.Alternatively or additionally, an extended nucleic acid can have a length in an extended or contracted state (e.g., coiled, self-hybridized, etc.) of about 500 nm, 450 nm, 400 nm, 350 nm, 300 nm, 250 nm, 200 nm, 180 nm, 160 nm, 140 nm, 120 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, 20 nm, 10 nm or less, or less than 10 nm.

[0151] The utilization surface or utilization site of a nucleic acid nanostructure (e.g., SNAP) can include one or more modifying sites. The utilization surface of a nucleic acid nanostructure can include at least about 10, 50, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 50,000, 1,000,000, or more than 1,000,000 modifying groups. Alternatively or additionally, the utilization surface of a nucleic acid nanostructure can include no more than about 1,000,000, 500,000, 100,000, 50,000, 10,000, 500, 100, 50, 10, or fewer than 10 modifying groups.

[0152] Nucleic acid nanostructures (e.g., SNAP) can include application surfaces with a characteristic density of modification sites. Modification site density can refer to the average or localized areal density of modification sites on the nucleic acid nanostructure application surface. The application surface of a nucleic acid nanostructure can have a density of about 1 group / nm 2 , 1 group / 10nm 2 , 1 group / 100nm 2 , 1 group / 1000nm 2 , 1 group / 10000nm 2 , 1 group / 100000nm 2 , 1 group / 1000000nm 2 or less than 1 group / 1000000nm 2 Alternatively or additionally, the nucleic acid nanostructure's application surface may have a modification site density of at least about 1 group / 1,000,000 nm 2 , 1 group / 100000nm 2 , 1 group / 10000nm 2 , 1 group / 1000nm 2 , 1 group / 100nm 2, 1 group / 10nm 2 , 1 group / nm 2 , or 1 group / nm 2 The modified molecule may have a density of modification sites greater than 1000.

[0153] The nucleic acid nanostructures (e.g., SNAP) provided herein can include one or more detectable labels, for example, in terms of nanostructure utilization. Detectable labels can include groups configured to provide or transmit a signal. Detectable labels can provide or transmit a signal in real time (e.g., fluorophores, radioactive labels) or at a later time (e.g., barcodes). Detectable labels can include detectable labels selected from the group consisting of fluorescent groups, luminescent groups, radioactive labels, isotopes, and barcodes. Any of a variety of fluorescent labels known in the art can be used to label the probe. In some cases, the fluorescent label can be a small molecule. In some cases, the fluorescent label can be a protein. In some cases, the fluorescent label can be a nanoparticle (e.g., quantum dots, fluorescently labeled polymer nanoparticles, etc.). Fluorescent labels can include labels that emit in the ultraviolet, visible, or infrared spectrum. In some cases, the fluorescent molecule may be selected from the group consisting of FITC, Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 647, Alexa Fluor® 680, Alexa Fluor® 750, Pacific Blue, coumarin, BODIPYFL, Pacific Green, Oregon Green, Cy3, Cy5, Pacific Orange, TRITC, Texas Red, R-phycoerythrin, and allophycocyanin (APC).In some cases, the label can be an Atto dye, such as Atto390, Atto425, Atto430, Atto465, Atto488, Atto490, Atto495, Atto514, Atto520, Atto532, Atto540, Atto550, Atto565, Atto580, Atto590, Atto594, Atto610, Atto611, Atto612, Atto620, Atto633, Atto635, Atto647, Atto655, Atto680, Atto700, Atto725, Atto740, Atto MB2, Atto Oxa12, Atto RholOl, Atto Rhol2, Atto Rhol3, Atto Rhol4, Atto Rho3B, Atto Rho6G, or Atto Thiol2. A wide range of effective fluorescent labeling groups may be commercially available from ThermoFisher Scientific's Molecular Probes division and are generally described in the Molecular Probes Handbook (11th Edition) (incorporated herein by reference). Detectable labels may also include intercalating dyes such as ethidium bromide, propidium bromide, crystal violet, 4',6-diamidino-2-phenylindole (DAPI), 7-aminoactinomycin D (7-AAD), Hoescht 33258, Hoescht 33342, Hoescht 34580, YOYO-1, DiYO-1, TOTO-1, DiTO-1, or combinations thereof.

[0154] The nucleic acid nanostructures (e.g., SNAP) provided herein may comprise three-dimensional structures. Nucleic acid nanostructures may comprise multiple surfaces, including a presentation surface, a binding surface, and an additional utilization surface. In some configurations, the utilization surface may be located on a region of the nucleic acid nanostructure that constitutes the height or depth of the nucleic acid nanostructure. The utilization surface may be utilized for any of a variety of purposes, including connecting the nucleic acid nanostructure to other structures or providing spacing between the nucleic acid nanostructure and other structures or molecules. The utilization surface may comprise one or more modifying groups. The modifying groups on the utilization surface may be covalently or non-covalently attached. The modifying groups on the utilization surface may be attached to the nucleic acid nanostructure before, during, or after construction of the nanostructure. The modifying groups on the utilization surface may include charged moieties, magnetic moieties, steric moieties, hydrophobic moieties, hydrophilic moieties, and linking groups. The linking group may include any group configured to connect the nucleic acid nanostructure to a solid support or another molecule, e.g., another nucleic acid nanostructure. The linking group may include covalent and non-covalent linking groups. Covalent linking groups can include chemically reactive species, such as click reaction groups, and crosslinking molecules. Crosslinking molecules can include chemical crosslinking molecules and photoinitiated crosslinking molecules. Noncovalent linking groups can include binding pairs (e.g., streptavidin-biotin) and nucleic acids configured to base pair with complementary nucleic acids on other molecules. Nucleic acid nanostructures (e.g., SNAP), molecules conjugated to nucleic acid nanostructures, or solid supports conjugated to nucleic acid nanostructures can include any of a variety of linking groups, such as those set forth in U.S. Patent Application Serial No. 17 / 062,405 or WO2019 / 195633A1 (each of which is incorporated herein by reference). The utility of nucleic acid nanostructures can include one or more steric groups that, as determined by the size of the one or more steric groups, prevent other molecules from approaching within the proximity of the nucleic acid nanostructure.

[0155] Nucleic acid nanostructures (e.g., SNAPs) can include one or more connection surfaces or connection sites. The application surface or application site can include one or more functional groups or sites configured to connect a first nucleic acid nanostructure to a second nucleic acid nanostructure. Connection sites can include, for example, those described herein in the context of application surfaces. Connections between nucleic acid nanostructures (e.g., display SNAPs and spacer SNAPs) or between nucleic acid nanostructure complexes can be formed by reversible or irreversible binding of complementary sets of connection sites on each paired nucleic acid nanostructure. Reversible binding of complementary nucleic acid nanostructures can occur through non-covalent bonds (e.g., nucleic acid hybridization, hydrogen bonds) or thermodynamically reversible covalent bonds (e.g., peroxide bonds, disulfide bonds). A nucleic acid nanostructure or complex thereof can include one or more connection groups configured to connect with one or more complementary connection sites on a second nucleic acid nanostructure or complex thereof. A nucleic acid nanostructure or complex thereof may include one or more surfaces containing one or more connection sites configured to connect with one or more complementary connection sites on a surface of a second nucleic acid nanostructure or complex thereof. A nucleic acid nanostructure or complex thereof may include multiple connection sites configured to connect with multiple complementary connection sites on a second nucleic acid nanostructure or complex thereof. In some configurations, a nucleic acid nanostructure or complex thereof may include multiple connection sites to ensure that at least one connection interaction, but preferably multiple connection interactions, are formed with a complementary nucleic acid nanostructure or complex thereof.

[0156] A nucleic acid nanostructure can include multiple connecting surfaces or connection sites configured to connect a nucleic acid nanostructure to multiple nucleic acid nanostructures. For example, a square or rectangular shaped SNAP can include four connecting surfaces, each configured along one of the four edges comprising the square or rectangle. A connecting surface can include one or more functional groups or sites configured to connect a first nucleic acid nanostructure to a second nucleic acid nanostructure. For example, a connecting surface or connection site can include multiple single-stranded nucleic acids configured to hybridize to multiple complementary single-stranded nucleic acids on the second connecting surface or connection site, thereby connecting the first connecting surface to the second connecting surface. In another example, a connecting surface can include a single streptavidin molecule configured to bind to a biotin molecule on the second connecting surface, thereby connecting the first connecting surface to the second connecting surface. In some configurations, the connection of a first nucleic acid nanostructure to a second nucleic acid nanostructure can include an intermediate connecting group that mediates the connection of the first nucleic acid nanostructure to the second nucleic acid nanostructure. For example, multiple SNAPs can be configured to present only streptavidin molecules on one or more connection surfaces, so that the first SNAP cannot directly bind to the second SNAP. An intermediate connector containing only biotin displayed on the surface can enable the first SNAP to connect to the second SNAP. The intermediate connector can include a nucleic acid nanostructure or a non-nucleic acid particle or molecule (e.g., organic or inorganic nanoparticle). The connection of the first nucleic acid nanostructure to the second nucleic acid nanostructure can be reversible (e.g., nucleic acid hybridization) or irreversible (e.g., click reaction).

[0157] The nucleic acid nanostructures (e.g., SNAP) provided herein may contain one or more sites that allow for controlled degradation of the nucleic acid nanostructure. The nucleic acid nanostructure may contain one or more photocleavable linkers. The photocleavable linker may be located within any portion of the nucleic acid nanostructure, including the backbone chain and any of the multiple oligonucleotides that may be connected within the nucleic acid nanostructure. In some cases, the nucleic acid nanostructure may contain multiple photocleavable linkers. The photocleavable linker may be located within the nucleic acid structure to allow for controlled degradation of the nucleic acid nanostructure, for example, for programmed removal of SNAP or programmed release of SNAP and an analyte from a surface. In the case of nucleic acid nanostructure compositions that include a multifunctional moiety hybridized to a portion of the nucleic acid nanostructure, the multifunctional moiety may include a photocleavable linker. In some configurations, the multifunctional moiety may not include a photocleavable linker. The photocleavable linker may be included in the multifunctional moiety to allow for programmable release of an analyte from the nucleic acid nanostructure or solid support to which the analyte is connected. The photocleavable linker can include any suitable photocleavable linker, such as a nitrobenzyl, carbonyl, or benzyl-based photocleavable linker. The photocleavable linker can be configured to cleave at a specific wavelength or within a specific frequency range, such as far-infrared, near-infrared, visible, near-ultraviolet, far-ultraviolet, or a combination thereof. The photocleavable linker can be selected so that it has a peak cleavage wavelength, such as the absorption or emission wavelength of a fluorophore, that does not interfere with other biological or chemical processes. The nucleic acid nanostructure (e.g., SNAP) can include one or more degradation sites that are substrates for enzymatic degradation by, for example, restriction enzymes, proteases, kinases, or other suitable enzymes. Nucleic acid nanostructures may incorporate sites that are substrates for enzymatic degradation, such as uracil nucleotides, which are degraded by uracil DNA glycosylase and endonuclease VIII (commercially available as USER® enzyme by New England Biolabs, Beverley MA), 8-oxoguanine nucleotides, which are degraded by DNA glycosylase OGG1, or peptides, which are degraded by proteases.In the case of nucleic acid nanostructure compositions that include a multifunctional moiety hybridized to a portion of a nucleic acid nanostructure, the multifunctional moiety can include a degradation site that is a target for enzymatic degradation. In some configurations, the multifunctional moiety may not include a degradation site that is a target for enzymatic degradation.

[0158] The nucleic acid nanostructures (e.g., SNAP) provided herein may contain one or more sites or groups incorporated into the nucleic acid nanostructure to promote stability of the nucleic acid nanostructure. The nucleic acid nanostructures (e.g., SNAP) may contain modified or non-natural nucleotides (e.g., PNA, locked nucleic acids, etc.) that are resistant to degradation via endonucleases or other enzymes. The nucleic acid nanostructures may contain one or more crosslinking groups that connect nucleic acid nanostructure components to each other (e.g., oligonucleotides to a backbone chain) and / or one or more crosslinking groups that connect the nucleic acid nanostructure to another entity (e.g., a solid support, a second nucleic acid nanostructure, etc.).

[0159] The nucleic acid nanostructures (e.g., SNAP) described herein may contain one or more linkers. A linker can comprise, for example, a molecular chain or moiety that connects two portions of any nucleic acid component of a nucleic acid nanostructure, such as an oligonucleotide, including a backbone, an oligonucleotide hybridized to the backbone, or a multifunctional oligonucleotide hybridized to the nucleic acid nanostructure. A linker can comprise a rigid linker or a flexible linker. A linker can comprise a polymeric moiety, such as a polyethylene glycol (PEG) or polyethylene oxide (PEO) moiety, or a polynucleotide. A linker can introduce desired chemical properties, such as hydrophobicity, hydrophilicity, polarity, or charge. A linker can include a moiety configured to link one or more additional moieties or molecules together, such as multiple multifunctional moieties. A linker can include one or more modified nucleotides, such as PNA, LNA, and / or nucleotides modified with functional groups configured to perform click-type reactions. Figures 3A-3D illustrate methods for connecting analytes to solid supports using multifunctional moieties containing linking groups. As shown in FIG. 3A, a SNAP 300 connected to an analyte 310 by a polyvalent linker 320 is contacted with a solid support 330 containing a plurality of surface linkage connection sites 335. The polyvalent linker is connected to four arms (321, 322, 323, 324) of a polyfunctional moiety that is hybridized to the SNAP and contains functional groups 325 configured to connect to the surface linkage connection sites 335. FIG. 3B shows a close-up view of the polyvalent linker 320, which contains five functional groups R1, R2, R3, R4, and R5, respectively. Functional groups R1, R2, R3, and R4 are connected to the four arms 321, 322, 323, and 324 of the polyfunctional moiety, respectively. Functional group R5 is connected to the analyte 310. FIG. 3C shows the connection of the SNAP 300 and analyte 310 to the solid support 330 by connecting functional groups 325 to the surface linkage connection sites 335. FIG. 3D shows the composition after the SNAP 300 structure has been decomposed, thereby leaving the analyte 310 attached to the solid support 330 by the four arms (321, 322, 323, 324) of the multifunctional site.Such a configuration may have the advantage of increasing the chemical stability of the analyte attachment, since multiple connecting multifunctional sites provide redundancy against disconnection of any single strand. The configuration may also be advantageous because multiple connecting multifunctional sites may stabilize the spatial location of the analyte, while only a single connecting multifunctional site may have more translational freedom.

[0160] The nucleic acid nanostructures (e.g., SNAP) provided herein may contain one or more crosslinking groups. Crosslinking groups can include chemical, enzymatic, and photochemical crosslinking groups. The crosslinking groups can stabilize or prevent dissociation of one or more nucleic acid structures in the nucleic acid nanostructure. An oligonucleotide of a plurality of oligonucleotides can be crosslinked to the backbone of the nucleic acid nanostructure. A first oligonucleotide of a plurality of oligonucleotides can be crosslinked to a second oligonucleotide of the plurality of oligonucleotides in the nucleic acid nanostructure. Oligonucleotides containing important structural features, such as utilization moieties (e.g., presentation moieties, capture moieties), can be crosslinked to the nucleic acid nanostructure to improve the stability of the oligonucleotide or prevent its dissociation. Multifunctional moieties containing two or more utilization moieties (e.g., presentation moieties and capture moieties) can contain one or more crosslinking groups to the nucleic acid nanostructure.

[0161] Nucleic acid nanostructures (e.g., SNAP) can include fully structured and / or partially structured portions. A fully structured portion of a nucleic acid nanostructure can be defined as a region of the nucleic acid nanostructure that maintains its primary, secondary, and tertiary structure during use. A partially structured portion of a nucleic acid nanostructure can be defined as a region of the nucleic acid nanostructure that includes a primary structure but does not maintain a specific secondary and / or tertiary structure during use. In some configurations, a partially structured portion of a nucleic acid nanostructure can include a single-stranded nucleic acid. A single-stranded nucleic acid can be located between regions of double-stranded nucleic acid or can include a pendant or terminal strand of nucleic acid. A single-stranded nucleic acid can have a specific length, such as at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more than 50 nucleotides. Alternatively or additionally, a single-stranded nucleic acid can have a length of about 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less than 5 nucleotides. In some configurations, the partially structured portion of the nucleic acid nanostructure may include non-nucleic acid moieties, molecular groups, or chains, such as PEG or polymer chains. In some configurations, the partially structured portion of the nucleic acid nanostructure may include amorphous structures, such as spherical structures (e.g., nanoballs, dendrimers, etc.). Figure 37A shows a SNAP 3710 having partially structured regions 3730 (e.g., single-stranded nucleic acids, polymers, dendrimers, etc.). The SNAP 3710 is connected to an analyte 3720. The partially structured regions 3730 may be located on multiple SNAP surfaces (e.g., capture surfaces, presentation surfaces). The partially structured regions 3730 may provide one or more functionality to the SNAP 3710, such as increasing binding strength to targeted binding surfaces or decreasing binding strength to non-targeted surfaces, preventing nonspecific binding of other molecules to the SNAP surface or the connected analyte.

[0162] Multifunctional moieties: In aspects, described herein are compositions comprising a nucleic acid nanostructure (e.g., SNAP) and a multifunctional moiety, where the multifunctional moiety can be configured to be connected to the nucleic acid nanostructure and can be configured to form two or more additional interactions. In some configurations, the multifunctional moiety can be configured to be connected to the nucleic acid nanostructure, allowing the surface to be continuously connected to the analyte. Continuous connection of the surface to the analyte can include a connection where the surface is directly connected to the analyte by the multifunctional moiety without any other intervening groups or moieties. For example, if SNAP is connected to the surface by the multifunctional moiety and the analyte is connected to the SNAP but not to the multifunctional moiety, the analyte will not be continuously connected to the surface by the multifunctional moiety. The multifunctional moiety can include a first functional group and a second functional group. In some configurations, the first functional group can be connected to or configured to connect to the surface, and the second functional group can be connected to or configured to connect to the analyte. In some configurations, a multifunctional site can be connected, or configured to be connected, to a nucleic acid nanostructure and can form two or more connecting interactions with a surface. A multifunctional site can include a presentation site and a surface interaction site.

[0163] The polyfunctional moieties described herein can include multiple functional groups. The polyfunctional moieties can include at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 functional groups. Alternatively or additionally, the polyfunctional moieties can include no more than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or fewer than 3 functional groups.

[0164] The multifunctional sites provided herein may comprise one or more molecular chains. The molecular chains may comprise multimeric compounds, such as oligonucleotides or polymer chains (e.g., polyethylene, polypropylene, polyethylene glycol, polyethylene oxide, etc.). In other configurations, the multifunctional site may not comprise a nucleic acid. In some configurations, the multifunctional site may comprise multiple molecular chains. The multifunctional site may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 molecular chains. Alternatively or additionally, the multifunctional site may comprise no more than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer than two molecular chains. Two or more molecular chains of a multifunctional site may be joined, connected, or linked by a linking moiety. Figures 7A-7B illustrate exemplary configurations of linking moieties. Figure 7A illustrates the formation of a multifunctional site comprising an alkyl linking moiety. The linking site includes an alkyl linking group 710 containing four reactive functional groups, including three methyltetrazine (mTz) groups 720 and one dibenzocyclooctene (DBCO) group 730. The linking site can be contacted with a tether 740 containing an azide functional group, thereby linking the azide-functionalized tether 740 to the DBCO group 730 via an azide-DBCO click reaction. The linking site can also be contacted with a tether 750 containing a transcyclooctene (TCO) functional group, thereby linking the TCO-functionalized tether 750 to the mTz functional group 720 via an mTz-TCO click reaction. Figure 7B shows a multifunctional site containing modified nucleotide groups in a longer oligonucleotide tether. The linking site containing the modified nucleotides is shown in a dashed box. The linking site contains four modified thymine nucleotides, including two mTz-functionalized thymines 760 and two DBCO-functionalized thymines 770. The multifunctional moiety can be contacted with azide-functionalized tethers 740 and / or TCO-functionalized tethers 750 to attach one or more tethers via a click reaction.

[0165] Multifunctional sites can be configured to connect to nucleic acid nanostructures (e.g., SNAP). The connection of the nucleic acid nanostructure can depend on how the nucleic acid nanostructure is utilized. For example, in some configurations, the multifunctional site can facilitate the placement and connection of the SNAP on a surface. In other configurations, the SNAP can facilitate the placement and connection of the multifunctional site to a surface. Figures 8A-8D show various configurations of multifunctional sites connected to a SNAP. Figure 8A shows a multifunctional site 810 having functional groups R1 and R2 comprising oligonucleotides that connect to the SNAP 800 to form a region of hybridized nucleic acid 830. The functional groups R1 and R2 are presented via the top surface (e.g., presentation surface) and the bottom surface (e.g., capture surface), respectively. Figure 8B shows a multifunctional site 810 having functional groups R1 and R2 comprising oligonucleotides that connect to the SNAP 800 to form a region of hybridized nucleic acid 830. The functional groups R1 and R2 are presented on the bottom surface (e.g., capture surface). Figure 8C shows a multifunctional site 840 having functional groups R1 and R2 comprising molecular chains (e.g., polymers, oligonucleotides) that connect to SNAP 800 through functional groups or moieties 850 that connect to complementary functional groups or moieties 860 on SNAP 800 (e.g., by Click reaction, by nucleic acid hybridization). Functional groups R1 and R2 are presented via the top (e.g., presentation) and bottom (e.g., capture) surfaces, respectively. Figure 8D shows a multifunctional site 840 having functional groups R1 and R2 comprising molecular chains (e.g., polymers, oligonucleotides) that connect to SNAP 800 through functional groups or moieties 850 that connect to complementary functional groups or moieties 860 on the exterior surface of SNAP 800 (e.g., by Click reaction, by nucleic acid hybridization). Multifunctional site 810 is configured to connect to SNAP 800 but be completely external to the SNAP 800 structure.

[0166] In some configurations, a nucleic acid nanostructure composition (e.g., a SNAP composition) may comprise a nucleic acid nanostructure and a multifunctional moiety configured to be connected to the nucleic acid nanostructure. In other configurations, a nucleic acid nanostructure composition may comprise a multifunctional moiety connected to the nucleic acid nanostructure. For example, a SNAP composition may, in a first configuration, contain a plurality of partially formed SNAP molecules in contact with a plurality of multifunctional moieties, and in a second configuration, contain a fluid medium containing a plurality of fully formed SNAP molecules, with a multifunctional moiety connected to each SNAP molecule. In some configurations, a nucleic acid nanostructure composition may further comprise an analyte configured to be connected to a multifunctional moiety. For example, a SNAP composition may comprise a fluid medium containing a plurality of SNAP molecules containing multifunctional moieties and a plurality of analytes configured to be connected to the multifunctional moieties. In some configurations, a nucleic acid nanostructure composition may further comprise an analyte connected to a multifunctional moiety. For example, a SNAP composition may comprise a plurality of partially formed SNAP molecules in contact with a plurality of multifunctional moieties, each multifunctional moiety connected to an analyte. In another example, a SNAP composition may comprise a plurality of SNAP molecules containing multifunctional moieties, each multifunctional moiety connected to an analyte. In some configurations, the nucleic acid nanostructure composition may further comprise a surface configured to be connected to a multifunctional moiety. For example, a SNAP composition may comprise a solid support comprising a plurality of surface linkage sites, the solid support being contacted with a plurality of SNAPs containing multifunctional sites, each multifunctional site comprising a surface interaction site configured to connect to the surface linkage site. In some configurations, the nucleic acid nanostructure composition may further comprise a surface connected to a multifunctional site. For example, a SNAP composition may comprise a solid support comprising a plurality of surface linkage sites, one or more surface linkage sites being connected to the surface interaction sites of a plurality of SNAPs containing multifunctional sites, the solid support being contacted with a fluid medium containing a plurality of analytes, each analyte being configured to connect to a presentation site of a multifunctional site.Those skilled in the art will readily recognize numerous variations in nucleic acid nanostructure compositions based on the ordering in which different components (e.g., SNAP, multifunctional moieties, analytes, solid supports, etc.) are introduced into the systems presented herein.

[0167] In some configurations, provided herein are compositions comprising nucleic acid nanostructures (e.g., SNAP) including a presentation site configured to connect to an analyte and a capture site configured to connect to a surface, and a multifunctional site comprising a first functional group and a second functional group, wherein the multifunctional site is hybridized to the nanostructure site, the presentation site comprising the first functional group, and the capture site comprising the second functional group. Such nucleic acid nanostructures can be configured to utilize the first functional group to connect to an analyte and the second functional group to connect to a surface or interface. The nanostructure site can be configured to occupy a given area of ​​the surface to prevent other nucleic acid nanostructures from occupying the same area. This can occur, for example, by steric exclusion, charge repulsion, or other mechanisms. Such configurations can offer surprising advantages, such as a linking connection between the analyte and the surface by the multifunctional site, and the prevention of multiple analytes from occupying a given area of ​​the surface due to the presence of the nanostructure site. The nanostructured moieties can be intentionally or unintentionally removed (e.g., degraded) so that the analyte can remain attached to the surface. Thus, the nanostructured moieties can beneficially inhibit interaction of the analyte with other analytes, reagents, or objects during surface deposition, and the nanostructured moieties can then be removed to facilitate interaction of the analyte with other analytes, reagents, or objects useful for on-surface detection or manipulation of the analyte.

[0168] 9A-9F illustrate a method for attaching an analyte to a surface using a SNAP having a multifunctional oligonucleotide. FIG. 9A shows a schematic diagram of a SNAP 910 comprising an oligonucleotide 940 having a first terminal functional group 920 comprising dibenzocyclooctyne (DBCO) and a second terminal functional group 930 comprising methyltetrazine (mTz). The oligonucleotide 940 is configured to hybridize to a portion of the SNAP, thereby forming a localized region of secondary or tertiary structure 945 (e.g., a double helix), thereby stabilizing the oligonucleotide 940 within the SNAP structure 910. The SNAP 910 is contacted with a solid support 950 comprising a non-reactive region 952 and a region comprising a reactive third functional group 955 comprising an azide moiety configured to react with the first terminal functional group 920. As shown in Figure 9B, first terminal functional group 920 can react with third functional group 955 to form a covalent bond connecting SNAP 910 to solid support 950 near where third functional group 955 is connected to solid support 950. As shown in Figure 9C, the connected SNAP can be contacted with analyte 960, which includes fourth functional group 970 comprising trans-cyclooctene configured to react with second terminal functional group 930. As shown in Figure 9D, second terminal functional group 930 can react with fourth functional group 970 to form a covalent bond connecting analyte 960 to solid support 950. It is understood that functional groups 920, 955, 930, and 970 are exemplary and can be replaced with other connection moieties, such as those shown herein or known in the art. As shown in Figure 9E, the SNAP-analyte composition can be subjected to a degradation event, such as a light source 980, that disrupts the structure of SNAP 910, thereby disintegrating SNAP 910. Degradation can be achieved using other means, such as endonuclease digestion of one or more nucleic acid strands in the SNAP, thermal or chemical denaturation of nucleic acid strand interactions, or chemical dissolution of cleavable linkages in the SNAP. As shown in Figure 9F, after degradation of SNAP 910, analyte 960 can remain connected to solid support 950 by oligonucleotide 940.

[0169] Nucleic acid nanostructures (e.g., SNAP) containing multifunctional sites, such as the configurations shown in Figures 9A-9F, can be configured to form hybridization regions with multifunctional sites consisting of multiple nucleic acid base pairs. In some configurations, the multifunctional sites can form hybridization regions with nucleic acid nanostructures containing at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, or more than 200 nucleotides. Alternatively or additionally, the multifunctional sites can form hybridization regions with nucleic acid nanostructures containing no more than about 200, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or less than about 10 nucleotides. The hybridization region formed between a nucleic acid nanostructure and a multifunctional site can be characterized by the specific number of helical turns formed (a single turn typically contains 10-11 base pairs). In some configurations, a multifunctional site may form a hybridization region that includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 helical turns. Alternatively or additionally, a multifunctional site may form a hybridization region that includes no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less than 1 helical turn.

[0170] A nucleic acid nanostructure (e.g., SNAP) can include multiple tertiary structures that collectively form a quaternary or other higher-order structure in the nucleic acid nanostructure. A particular tertiary structure can include a site or structure belonging to a particular surface of the nucleic acid nanostructure. A nucleic acid nanostructure can include multiple tertiary structures, with the presentation surface including a first tertiary structure of the multiple tertiary structures and the capture surface including a second tertiary structure of the multiple tertiary structures. In some configurations, the first tertiary structure can be the same as the second tertiary structure. In other configurations, the first tertiary structure is different from the second tertiary structure. In nucleic acid nanostructure configurations including a multifunctional site having a first functional group and a second functional group, the multifunctional site can hybridize to the nucleic acid nanostructure, thereby forming part of the first tertiary structure or part of the second tertiary structure. In other configurations, the multifunctional site can hybridize to the nucleic acid nanostructure, thereby forming both part of the first tertiary structure and part of the second tertiary structure.

[0171] A nucleic acid nanostructure (e.g., SNAP) comprising a first multifunctional site can further comprise a second multifunctional site comprising a third functional group and a fourth functional group. In some configurations, the utilization site (e.g., presentation site) can comprise the third functional group, and the second utilization site (e.g., capture site) can comprise the fourth functional group. In some configurations, the third or fourth functional group can be configured to connect to a surface. In some specific configurations, the third or fourth functional group can be connected to a surface. In some configurations, the third or fourth functional group can be configured to connect to a second analyte. In some specific configurations, the third or fourth functional group can be configured to connect to the analyte to which the first multifunctional site is connected. In some specific configurations, the third or fourth functional group can be connected to the analyte to which the first multifunctional site is connected.

[0172] 10A-10D illustrate methods for attaching a SNAP containing two multifunctional moieties to a surface. FIG. 10A shows a SNAP 1000 containing a first multifunctional moiety 1010 connected to an analyte 1020 and containing a first functional group 1015. The SNAP 1000 also contains a second multifunctional moiety 1030 connected to an utilization moiety 1040 and containing a second functional group 1035. The SNAP 1000 can include a capture surface including capture sites containing a first functional group and a second functional group. The SNAP 1000 can be contacted with a solid support 1050 containing multiple functional groups or moieties, including a surface-linked non-connecting group 1060 and a surface-linked connecting group 1065 configured to connect to one or more capture sites. As shown in Figure 10B, the first and / or second functional groups can be connected to a surface linkage connecting group 1065, thereby connecting the SNAP 1010 to the solid support 1050 by at least one of the two functional groups comprising the capture site. As shown in Figure 10C, the SNAP 1000 connected to the solid support 1050 can be exposed to a degradation event, e.g., a light source 1070, that causes disassembly of the SNAP 1000 structure. Disassembly can also be achieved using other means, e.g., endonuclease digestion of one or more nucleic acid strands in the SNAP, heat, a pH change, chemical dissolution of cleavable linkages in the SNAP, or any other suitable degradation method. As shown in Figure 10D, after disassembly of the SNAP structure, the first multifunctional site 1010 connected to the analyte 1020 and the second multifunctional site 1030 connected to the utilization site 1040 are co-localized on the solid support 1050.

[0173] A multifunctional moiety hybridized to a nucleic acid nanostructure (e.g., SNAP) can be configured to connect the nucleic acid nanostructure or a moiety thereof to a surface. In some configurations, the surface can include a surface functional group configured to connect to a functional group contained on the multifunctional moiety. In some configurations, the surface functional group can include a functional group configured to form a covalent bond with a functional group contained on the multifunctional moiety. In some particular configurations, the surface functional group and the functional group contained on the multifunctional moiety can form a covalent bond by, for example, a click-type reaction, a substitution reaction, an elimination reaction, or any other suitable conjugation chemistry.

[0174] Nucleic acid nanostructures (e.g., SNAP) containing multifunctional moieties can be formed before or after attachment of the nucleic acid nanostructure to a surface. Figures 11A-11D illustrate methods for hybridizing multifunctional moieties to SNAP after the SNAP is attached to a surface. Figure 11A shows SNAP 1110 being contacted with surface 1100, thereby allowing the SNAP to attach to the surface, for example, through electrostatic, magnetic, or covalent interactions. Figure 11B illustrates contact between SNAP 1110 attached to surface 1100 and multifunctional moiety 1120 attached to analyte 1130. As shown in Figure 11C, multifunctional moiety 1120 hybridizes to SNAP 1110, forming a region of tertiary structure 1150. Multifunctional moiety 1120 may further attach to surface 1100. Figure 11D illustrates subsequent linkage of analyte 1130 to surface 1100 after SNAP 1110 is optionally removed.

[0175] Partially Compacted Nucleic Acids: Nucleic acids useful for forming arrays of analytes may comprise structures having one or more of the following characteristics: i) allowing attachment of analytes at adjustable and / or controllable locations on the surface of the nucleic acid; ii) inhibiting unwanted attachment of analytes or other moieties at portions of the nucleic acid where attachment is not intended; iii) comprising a structure or surface configured to form a specific binding interaction with a solid support or its surface; iv) comprising a structure or surface configured to form a specific binding interaction with a solid support or its surface that is more likely to occur than a non-specific binding interaction between an analyte attached to the nucleic acid and the solid support or its surface; v) comprising a structure or surface configured to inhibit contact between an analyte attached to the nucleic acid and the solid support or its surface; vi) inhibiting unwanted binding interactions (e.g., aggregation, co-localization, etc.) with other nucleic acids or analytes attached thereto.

[0176] Useful configurations of nucleic acids, e.g., nucleic acid nanostructures, can include nucleic acids containing compressed and transparent structures. The compressed structure of a nucleic acid can provide spatial and orientational adjustability for sites connected to or emerging from the nucleic acid structure. For example, a nucleic acid origami containing a compressed structure can be designed to orient a presentation site at a substantially 180° angle from one or more capture sites, thereby increasing the likelihood that the nucleic acid origami will be connected to the solid support by one or more capture sites and not by an analyte connected to the presentation site. The adjustability of the compressed structure can arise from several aspects of the nucleic acid structure, including multiple tertiary structures that provide substantially 360° of rotational freedom for the orientation of sites connected to the nucleic acid, and one or more linking chains that connect the tertiary structures within the nucleic acid structure, thereby providing a degree of rigidity to the nucleic acid structure and fixing the separation distance and orientation of the tertiary structures relative to each other in the nucleic acid structure. A nucleic acid transmission structure can provide the nucleic acid with additional chemical and / or physical properties that facilitate desired interactions with other entities (e.g., analytes, unbound sites, reagents, other nucleic acids, solid supports, fluid media, etc.) or inhibit unwanted interactions with other entities. For example, a nucleic acid can include multiple pendant single-stranded nucleic acid moieties comprising homopolymeric repeats (e.g., poly-T repeats, poly-A repeats, poly-C repeats, poly-G repeats), where the pendant single-stranded nucleic acid moieties are configured to inhibit colocalization of two or more nucleic acids on a solid support (e.g., at the same address in an array of addresses on a solid support). By connecting the transmission structure to a nucleic acid tunable compaction structure, the position and orientation of the transmission structure can be controlled to generate more specific and localized interactions between the nucleic acid and other entities.

[0177] The nucleic acid nanostructures provided herein may include at least one compacted region or structure. A compacted region of a nucleic acid nanostructure may refer to a region or structure having average characteristics closer to those of a multistranded nucleic acid (e.g., double-stranded DNA, triple-stranded DNA, etc.) than those of a single-stranded nucleic acid. The nucleic acid nanostructures provided herein may include at least one transparent region or structure. A transparent region of a nucleic acid nanostructure may refer to a region or structure having average characteristics closer to those of a single-stranded nucleic acid than those of a multistranded nucleic acid. The nucleic acid nanostructures provided herein are not required to include a transparent region or structure. The condensed region or structure of the nucleic acid nanostructure i) comprises a backbone strand; ii) comprises a plurality of nucleic acids connected to the backbone strand, wherein at least 50%, and optionally at least 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the nucleotides of the backbone strand are base-pair hybridized to nucleotides of the plurality of nucleic acids; iii) comprises a plurality of connected nucleic acids, wherein at least 50%, and optionally at least 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the nucleotides of the plurality of nucleic acids are base-pair hybridized to other nucleotides of the plurality of nucleic acids; iv) comprises a plurality of secondary and / or tertiary nucleic acid structures, wherein the position, orientation, and / or movement of a first secondary and / or tertiary nucleic acid structure relative to a second secondary and / or tertiary nucleic acid structure is not constrained. v) comprising a first helical nucleic acid structure and a second helical nucleic acid structure, the first helical nucleic acid structure and the second helical nucleic acid structure being linked by a single-stranded nucleic acid, the first helical nucleic acid structure and the second helical nucleic acid structure each comprising an axis of helical symmetry oriented parallel to the single-stranded nucleic acid in a 3' to 5' direction, and the orientation of the axis of helical symmetry of the first helical nucleic acid structure relative to the axis of helical symmetry of the second helical nucleic acid structure having an angle of about 90° to 180°; vi) comprising a single-stranded nucleic acid that constrains the position, orientation, and / or movement of the first secondary and / or tertiary nucleic acid structure relative to the second secondary and / or tertiary nucleic acid structure; vii) comprising a moiety (e.g., a polypeptide, a polysaccharide,viii) comprising a volume surrounding each nucleotide of a compressed region or structure, wherein a characteristic dimension (e.g., length, depth, diameter, etc.) of the volume does not vary by more than 10%, and optionally by 5% or 1% or less, due to intermolecular or extramolecular motions (e.g., Brownian motion, fluid shear, electromagnetic forces, etc.) or due to intramolecular motions (e.g., translational, vibrational, bending, rotational, etc.); ix) comprising a first nucleotide having a first adjustable position and a second nucleotide having a second adjustable position, wherein the first adjustable position x) comprising a first nucleotide having a first adjustable position and a second nucleotide having a second adjustable position, wherein the first adjustable position comprises a distance from or orientation relative to the second adjustable position that varies by 10% or less; xi) comprising a volume surrounding each nucleotide of a compressed region or structure, wherein a characteristic dimension (e.g., length, depth, diameter, etc.) of the volume does not vary by more than 10%, and optionally by 5%, or 1%, or less, and wherein the compressed region or structure xii) comprising two-dimensional protrusions of the compressed region or region of the structure surrounding each nucleotide of the compressed region or structure, wherein the two-dimensional protrusions do not vary by more than 10%, and optionally 5%, or 1% or less, when the nucleic acid nanostructure comprising the compressed region or structure forms a binding interaction with a molecule, site, structure, or solid support; xiii) comprising a plurality of single-stranded nucleic acids, wherein each single-stranded nucleic acid is less than about 20 nucleotides in length, and optionally less than about 15 nucleotides in length. xiv) comprising a first tertiary structure and a second tertiary structure, wherein the second tertiary structure is adjacent to the first tertiary structure, and the average separation distance between the first tertiary structure and the secondary structure is about 20 nanometers (nm) or less, and optionally about 10 nm or 5 nm or less, as measured by the average separation distance between the axis of symmetry for the first tertiary structure and the axis of symmetry for the second tertiary structure; xv) comprising a first tertiary structure and a second tertiary structure, wherein the second tertiary structure is adjacent to the first tertiary structure;and xvi) a first tertiary structure and a second tertiary structure, each comprising a common nucleic acid, and optionally two common nucleic acids, wherein the common nucleic acid comprises a bend of at least about 90°, the bend having a radius of curvature of 10 nanometers (nm) or less, and optionally 5 nm or 2.5 nm or less; and xvi) a first tertiary structure and a second tertiary structure, wherein the second tertiary structure is adjacent to the first tertiary structure, wherein the first tertiary structure and the second tertiary structure each comprise a common nucleic acid, and optionally two common nucleic acids, wherein the common nucleic acid comprises a bend of at least about 90°, the bend having a radius of curvature of 10 nanometers (nm) or less, and optionally 5 nm or 2.5 nm or less, and the first tertiary structure is not positioned adjacent to the second tertiary structure by a nucleic acid binding entity (e.g., a nucleic acid binding protein, a nanoparticle, etc.).

[0178] The nucleic acid nanostructures provided herein can include at least one transparent region or structure. The permeable region or structure of the nucleic acid nanostructure may i) be free of a backbone strand, ii) comprise one or more nucleic acids, each nucleic acid of the one or more nucleic acids comprising a first nucleotide sequence configured to hybridize to a backbone strand of the compressed region or structure and a second nucleotide sequence not configured to hybridize to a nucleic acid of the nucleic acid nanostructure, iii) comprise one or more nucleic acids, each nucleic acid of the one or more nucleic acids comprising a single-stranded nucleic acid of at least about 20 nucleotides in length, and optionally comprising at least about 25, 50, 100, 500, 1000, or more than 1000 nucleotides in length, iv) comprise one or more nucleic acids, each nucleic acid of the one or more nucleic acids comprising an unconnected terminal nucleotide (e.g., a 3' terminal nucleotide, a 5' terminal nucleotide), or v) comprise a plurality of pendant moieties (e.g., single-stranded nucleic acid, partially double-stranded nucleic acid, polymeric strand, etc.), each pendant moiety being capable of binding to a target molecule via intramolecular or intrastructural binding interactions (e.g., base pair hybridization, hydrogen bonding, van der Waals interactions, etc.). vi) comprising a plurality of pendant moieties, each pendant moiety comprising a position, orientation, or movement that is not constrained by non-bonded interactions (e.g., steric occlusion, electrostatic repulsion, magnetic repulsion, hydrophobic interactions, hydrophilic interactions); vii) comprising one or more connected nucleic acids, wherein less than 50%, and optionally less than 40%, 30%, 20%, 10%, 5%, or 1% of the nucleotides of the plurality of nucleic acids hybridize to other nucleotides of the plurality of nucleic acids by base pair hybridization. ix) comprising one or more nucleic acids, wherein the one or more nucleic acids comprise a first single-stranded nucleic acid and a second single-stranded nucleic acid, and the first single-stranded nucleic acid is not configured to hybridize to the second single-stranded nucleic acid; x) comprising one or more nucleic acids, wherein the one or more nucleic acids comprise single-stranded nucleic acids comprising polynucleotide repeats (e.g., poly-A, poly-C, poly-G, poly-T), optionally the polynucleotide repeats are at least about 10 nucleotides, or at least about 20, 30, 40, 50,comprising 100, 200, 500, 1000, or more than 1000 nucleotides; xi) comprising a volume surrounding each nucleotide of a permeable region or structure, wherein a characteristic dimension of the volume (e.g., length, depth, diameter, etc.) changes by more than 10%, and optionally more than 15% or 20%, due to intermolecular or extramolecular motion (e.g., Brownian motion, fluid shear, electromagnetic forces, etc.) or due to intramolecular motion (e.g., translational, vibrational, bending, rotational, etc.); xii) comprising a volume surrounding each nucleotide of a permeable region or structure, wherein a characteristic dimension of the volume (e.g., length, depth, diameter, etc.) changes by more than 10%, and optionally more than 15% or 20%, when the nucleic acid nanostructure, including the compressed region or structure, forms a binding interaction with a molecule, moiety, structure, or solid support; xiii xiv) comprising a nucleic acid nanostructure, wherein a first nucleotide sequence of the nucleic acid is connected to a compacted structure and a second nucleotide sequence of the nucleic acid is not connected to the compacted structure, and wherein the nucleotides of the second nucleotide sequence comprise a spatial and / or temporal variation in distance relative to the compacted structure relative to the nucleotides of the first nucleotide sequence.

[0179] In an aspect, provided herein is a nucleic acid nanostructure comprising at least 10 connected nucleic acids, the nucleic acid nanostructure comprising: a) a compressed region comprising high internal complementarity, wherein the high internal complementarity comprises at least 50% double-stranded nucleic acids and at least 1% single-stranded nucleic acids, the compressed region comprising a presentation site, the presentation site being connected or configured to connect to an analyte of interest; and b) a transparent region comprising low internal complementarity, wherein the low internal complementarity comprises at least about 50% single-stranded nucleic acids, the transparent region comprising a connection site, the connection site forming or configured to form a connecting interaction with a solid support.

[0180] In another aspect, provided herein are nucleic acid nanostructures comprising: a) a compressed structure, the compressed structure comprising a backbone strand and a first plurality of staple oligonucleotides, at least 80% of the nucleotides of the backbone strand are hybridized to the nucleotides of the first plurality of staple oligonucleotides, the first plurality of staple oligonucleotides hybridizing to the backbone strand to form a plurality of tertiary structures, the plurality of tertiary structures including adjacent tertiary structures connected by single-stranded regions of the backbone strand, the relative positions of the adjacent tertiary structures being positionally constrained; and b) a transmission structure, the transmission structure comprising a second plurality of staple oligonucleotides, the staple oligonucleotides being connected to the backbone strand of the compressed structure, the transmission structure comprising at least 50% single-stranded nucleic acids, the transmission structure having an anisotropic three-dimensional distribution around at least a portion of the compressed structure.

[0181] In another aspect, provided herein are nucleic acid nanostructures comprising: a) a compressed structure, the compressed structure comprising a backbone strand and a first plurality of staple oligonucleotides, at least 80% of the nucleotides of the backbone strand are hybridized to the nucleotides of the first plurality of staple oligonucleotides, the first plurality of staple oligonucleotides hybridizing to the backbone strand to form a plurality of tertiary structures, the plurality of tertiary structures including adjacent tertiary structures connected by single-stranded regions of the backbone, the relative positions of the adjacent tertiary structures being positionally constrained, and the compressed structure comprising an effective surface area; and b) a permeable structure, the permeable structure comprising a second plurality of staple oligonucleotides, the staple oligonucleotides being connected to the backbone strand of the compressed structure, the permeable structure comprising at least 50% single-stranded nucleic acids; and (i) the effective surface area of ​​the nucleic acid nanostructure is greater than the effective surface area of ​​the compressed structure, or ii) the ratio of the effective surface area to the volume of the nucleic acid nanostructure is greater than the ratio of the effective surface area to the volume of the compressed structure.

[0182] In another aspect, provided herein is a nucleic acid nanostructure comprising a plurality of nucleic acid strands, wherein each strand of the plurality of strands is hybridized to another strand of the plurality of strands to form a plurality of tertiary structures, wherein a strand of the plurality of strands comprises a first nucleotide sequence hybridized to a second strand of the plurality of strands, wherein the strand of the plurality of strands further comprises a second nucleotide sequence of at least 100 contiguous nucleotides, and wherein at least 50 nucleotides of the second nucleotide sequence are single-stranded.

[0183] Figures 52A-52H show various configurations of nucleic acid nanostructures, including compressed and transparent structures. Figure 52A shows a cross-sectional view of a nucleic acid nanostructure including a SNAP 5210 (e.g., nucleic acid origami) connected to an analyte 5220 by a presentation moiety 5215 on the presentation surface of the SNAP 5210. The nucleic acid nanostructure further includes a capture surface opposite (e.g., oriented approximately 180° from) the presentation surface of the SNAP 5210. The capture surface includes a transparent structure including multiple pendant moieties 5212 (e.g., single-stranded nucleic acids, polymer chains, etc.) connected to the capture surface of the SNAP 5210, the pendant moieties 5212 including dangling ends. Depending on the density of the multiple pendant moieties 5212 and the rigidity of the connection points of the SNAP 5210 to the compressed structure, the multiple pendant moieties may be arranged in an outwardly fanned configuration. Volume 5230 encompasses the average space occupied by the transparent structure including the multiple pendant moieties. Pendant moieties 5212 within volume 5230 have an anisotropic spatial distribution with respect to the compressed structure of SNAP 5210 due to the adjustable placement and orientation of the pendant moieties on the capture surface of SNAP 5210. Figure 52B shows a top-down view of the nucleic acid nanostructure in Figure 52A. Line 5241 depicts the effective surface area of ​​the compressed structure of SNAP 5210, and line 5240 depicts the effective surface area of ​​the complete nucleic acid nanostructure (i.e., including the compressed and permeated structures), which is greater than the effective surface area of ​​the compressed structure due to the outward fanning of pendant moieties 5212.

[0184] Figure 52C shows a cross-sectional view of a nucleic acid nanostructure including a SNAP 5210 (e.g., nucleic acid origami) connected to an analyte 5220 by a presentation moiety 5215 on the presentation surface of the SNAP 5210. The nucleic acid nanostructure further includes one or more utilization surfaces adjacent to and orthogonal to (e.g., oriented approximately 90° from) the presentation surface of the SNAP 5210. Each utilization surface includes a permeable structure including a plurality of pendant moieties 5212 (e.g., single-stranded nucleic acids, polymer chains, etc.) connected to the utilization surface of the SNAP 5210. Depending on the density of the plurality of pendant moieties 5212, the flexibility of the pendant moieties 5212, and the rigidity of the connection points to the compressed structure of the SNAP 5210, the plurality of pendant moieties 5212 may be arranged in an outwardly fanned configuration. Lines 5230 and 5231 enclose the average cross-sectional area of ​​the space occupied by the permeable structure including the plurality of pendant moieties. Pendant moieties 5212 in the space indicated by lines 5230 and 5231 comprise a substantially isotropic spatial distribution relative to the centerline of the compressed structure of SNAP 5210 and an anisotropic spatial distribution relative to analyte 5220 due to the adjustable placement and orientation of the pendant moieties on the capture surface of SNAP 5210. Figure 52D shows a top-down view of the nucleic acid nanostructure. Line 5241 depicts the effective surface area of ​​the compressed structure of SNAP 5210, and line 5240 depicts the effective surface area of ​​the complete nucleic acid nanostructure (i.e., including the compressed and permeated structures), which is greater than the effective surface area of ​​the compressed structure due to the outward orientation of pendant moieties 5212.

[0185] Figure 52E shows a cross-sectional view of a nucleic acid nanostructure including a SNAP 5210 (e.g., nucleic acid origami) connected to an analyte 5220 by a presentation moiety 5215 on the presentation surface of the SNAP 5210. The nucleic acid nanostructure further includes a capture surface opposite (e.g., oriented approximately 180° from) the presentation surface of the SNAP 5210. The capture surface includes a permeable structure including a plurality of pendant moieties 5213 (e.g., single-stranded nucleic acids, polymer chains, etc.) connected to the capture surface of the SNAP 5210, the pendant moieties 5213 including both ends connected to the compressed structure of the SNAP 5210. Depending on the density of the plurality of pendant moieties 5213, their flexibility, and the rigidity of the connection points to the compressed structure of the SNAP 5210, the plurality of pendant moieties can occupy a volume directly below the capture surface of the SNAP 5210. Line 5230 encloses the average cross-sectional area of ​​the space occupied by the permeable structure including the plurality of pendant moieties 5213. Pendant moieties 5213 in the space indicated by line 5230 have an anisotropic spatial distribution with respect to the compressed structure of SNAP 5210 due to the tunable placement and orientation of the pendant moieties on the capture surface of SNAP 5210. Figure 52F shows a top-down view of the nucleic acid nanostructure. Line 5241 depicts the effective surface area of ​​the compressed structure of SNAP 5210, and line 5240 depicts the effective surface area of ​​the complete nucleic acid nanostructure (i.e., including the compressed and permeated structures), which is smaller than the effective surface area of ​​the compressed structure of SNAP 5210.

[0186] Figure 52G shows a cross-sectional view of a nucleic acid nanostructure including a SNAP 5210 (e.g., nucleic acid origami) connected to an analyte 5220 by a presentation moiety 5215 on the presentation surface of the SNAP 5210. The nucleic acid nanostructure further includes a plurality of pendant moieties 5212 (e.g., single-stranded nucleic acids, polymer chains, etc.) connected to substantially all orientations of the SNAP 5210 except for the volume occupied by the analyte 5220. Depending on the density of the plurality of pendant moieties 5212, their flexibility, and the rigidity of the connection points to the compressed structure of the SNAP 5210, the plurality of pendant moieties may be arranged in an outwardly fanned configuration. Line 5230 encloses the average cross-sectional area of ​​the space occupied by the transmission structure including the plurality of pendant moieties 5212. The pendant moieties 5212 within the space indicated by line 5230 comprise an anisotropic spatial distribution with respect to the compressed structure of the SNAP 5210, but may be an isotropic spatial distribution except for the volume occupied by the analyte 5220. Figure 52H shows a top view of the nucleic acid nanostructure. Line 5241 depicts the effective surface area of ​​the compressed structure of SNAP 5210, and line 5240 depicts the effective surface area of ​​the complete nucleic acid nanostructure (i.e., including the compressed and permeated structures), which is greater than the effective surface area of ​​the compressed structure due to the outward fanning of pendant moieties 5212.

[0187] Figures 53A-53E show cross-sectional views of various nucleic acid nanostructure configurations, each nucleic acid nanostructure comprising a transmission structure, each transmission structure comprising multiple pendant moieties configured for different interactions with other entities (e.g., analytes, other nucleic acid nanostructures, solid supports, reagents, etc.). Figure 53A shows a condensed structure 5310 (e.g., SNAP) connected to a transmission structure comprising multiple pendant oligonucleotides 5320, each comprising a homopolymer. The homopolymers of each pendant oligonucleotide 5320 may inhibit binding interactions with other nucleic acid nanostructures having the same or similar pendant oligonucleotide sequence. Figure 53B shows a condensed structure 5310 (e.g., SNAP) connected to a transmission structure comprising multiple pendant oligonucleotides 5321, each comprising a homopolymer sequence, some of which are interrupted by random substitutions of nucleotides other than those in the homopolymer sequence (e.g., a poly-T sequence with randomly substituted A, C, or G nucleotides). Figure 53C shows a compressed structure 5310 (e.g., SNAP) connected to a transmission structure containing multiple pendant oligonucleotides 5320, each containing a homopolymer sequence region and a sequence region complementary to the homopolymer sequence region. As shown, the complementary region may form a double-stranded region 5322 to form a loop structure. Figure 53D shows a compressed structure 5310 (e.g., SNAP) connected to a transmission structure containing multiple pendant oligonucleotides 5323, each containing a nucleotide sequence with some degree of self-complementarity (e.g., forming a stem, loop, hairpin, or bulge structure). Figure 53E shows a compressed structure 5310 (e.g., SNAP) connected to a transmission structure containing multiple pendant oligonucleotides 5324, each containing a second oligonucleotide 5325 that hybridizes to the pendant oligonucleotide 5324.The configurations shown in Figures 53A-53E (e.g., polynucleotide repeats, random nucleotide substitutions, self-complementarity, intermittent secondary structures) can facilitate rearrangement of the orientation of nucleic acid nanostructures on the connecting surface, thereby facilitating the arrangement of nucleic acid nanostructures in stable configurations on the connecting surface.

[0188] Figures 54A-54C show schematics for methods of producing nucleic acid nanostructures (e.g., the nucleic acid nanostructures shown in Figures 53A-53E) according to some embodiments provided herein. Figure 54A illustrates a method of forming a nucleic acid nanostructure comprising a plurality of pendant moieties comprising polynucleotide repeats. In a first step, a backbone strand 5410 can be combined, optionally at an elevated temperature, with a plurality of staple oligonucleotides 5420 that hybridize to the backbone strand 5410 to form a condensed structure, and a plurality of oligonucleotides 5421 comprising pendant nucleotide sequences 5422. After cooling the oligonucleotide mixture, a nucleic acid nanostructure comprising a condensed structure 5430 and a plurality of pendant moieties comprising pendant nucleotide sequences 5422 is formed. In a second step, the nucleic acid nanostructure is then contacted with a nucleic acid extending enzyme (e.g., terminal deoxynucleotide transferase, or TdT, shown) in the presence of a homogeneous plurality of nucleotides (e.g., deoxythymidine) to produce a plurality of pendant homopolymeric polynucleotides 5423 (e.g., poly-T repeats). Optionally, the nucleotides provided to the enzyme can include small amounts of other nucleotides to generate randomly incorporated nucleotides in the polynucleotide repeats. Figure 54B illustrates a method for forming a nucleic acid nanostructure comprising multiple pendant moieties comprising a homopolymer polynucleotide, where the position of each pendant moiety is controlled. In a first step, backbone strand 5410 can be combined, optionally at elevated temperature, with multiple staple oligonucleotides 5420 that hybridize to backbone strand 5410 to form a condensed structure, and multiple oligonucleotides 5421 comprising pendant nucleotide sequences 5422, and multiple oligonucleotides 5424 comprising cap moieties 5425 (e.g., dideoxynucleotides, terminator nucleotides, phosphorylated nucleotides, terminal residues buried within condensed structure 5430, etc.), where the cap moieties are configured to inhibit the activity of a nu...

Claims

1. A method for identifying a polypeptide of interest, comprising: a) providing a solid support comprising a plurality of sites, wherein each individual site of the plurality of sites comprises: i) a unique nucleic acid nanostructure connected to each individual site; ii) a unique polypeptide of interest bound to said unique nucleic acid nanostructure; Including, b) delivering a binding reagent to said solid support, thereby attaching the binding reagent to a polypeptide of interest at a site of said plurality of sites; c) repeating step b) using a second binding reagent in place of said binding reagent, wherein said second binding reagent is different from said binding reagent; d) detecting for each individual site the presence or absence of binding to said polypeptide of interest by each individual binding reagent and a second binding reagent; e) identifying the polypeptide of interest at each individual site of the plurality of sites based on the presence or absence of binding to the polypeptide of interest by each individual binding reagent and a second binding reagent. A method comprising:

2. The method of claim 1, wherein the step of preparing a solid support comprises preparing a solid support contacted with a fluid medium containing the denaturing species.

3. The method of claim 1, wherein the step of delivering the binding reagent to the solid support comprises delivering a fluid medium comprising the binding reagent and the modified species.

4. The method of claim 1, wherein the binding reagent comprises an affinity reagent.

5. The method of claim 4, wherein the affinity reagent of the affinity reagents comprises an aptamer.

6. The method of claim 4, wherein the affinity reagent of the affinity reagents comprises an antibody.

7. The method of claim 4, wherein the step of attaching a binding reagent to the target polypeptide comprises attaching an affinity reagent to an epitope of the target polypeptide.

8. The method of claim 4, wherein the step of attaching a binding reagent to the target polypeptide comprises attaching an affinity reagent to a modified N-terminal amino acid of the target polypeptide.

9. The method of claim 8, further comprising the step of cleaving the modified N-terminal amino acid of the target polypeptide after attaching an affinity reagent to the modified N-terminal amino acid.

10. The method of claim 1, wherein the step of delivering a binding reagent to a solid support comprises delivering an N-terminal modifier.

11. The method of claim 1, wherein the target polypeptide has a full-length primary amino acid structure.

12. The method of claim 1, wherein the target polypeptide is a fragment of the full-length primary amino acid structure.

13. The method of claim 1, wherein the step of preparing a solid support comprises providing a solid support comprising a first species of polypeptide and a second species of polypeptide.

14. The method of claim 13, wherein the first species polypeptide and the second species polypeptide have a dynamic range of at least 10 4 .

15. The method of claim 14, wherein the step of identifying a polypeptide of interest at each individual site of the plurality of sites comprises identifying a first species of polypeptide and a second species of polypeptide.

16. The method of claim 1, wherein the step of preparing a solid support comprises preparing a solid support comprising at least 1,000 unique polypeptide species of interest.

17. The method of claim 16, wherein the step of identifying a polypeptide of interest at each individual site of the plurality of sites comprises identifying 1,000 unique species of polypeptide of interest.

18. The method of claim 1, wherein the step of preparing a solid support includes preparing a solid support comprising at least 25% of the polypeptide species of the proteome.

19. The method of claim 18, wherein the step of identifying a polypeptide of interest at each individual site of the plurality of sites comprises identifying at least 25% of the polypeptide species in the proteome.

20. The method of claim 1, wherein the step of detecting the presence or absence of connection by each individual binding reagent to a polypeptide of interest comprises detecting the presence or absence of a fluorescent signal at the address of a site containing the polypeptide of interest.

21. The method of claim 20, wherein the step of detecting the presence or absence of a fluorescent signal at the address of the site containing the target polypeptide occurs for each instance of delivery of the binding reagent to the solid support.

22. The method of claim 1, wherein the step of detecting the presence or absence of connection by each individual binding reagent to a polypeptide of interest comprises detecting the presence or absence of a nucleic acid tag.

23. The method of claim 1, wherein the step of identifying the target polypeptide comprises identifying the species of the target polypeptide.

24. The method of claim 1, wherein the step of identifying the target polypeptide comprises identifying the amino acid sequence of the target polypeptide.

25. The method of claim 1, wherein the step of identifying the polypeptide of interest comprises identifying the proteome of the polypeptide of interest.

26. The method of claim 1, wherein the target polypeptide is attached to the nucleic acid nanostructure by a linking site.

27. The method of claim 26, wherein the linking site creates a separation gap of at least 10 nanometers (nm) between the target polypeptide and the solid support.

28. The method of claim 1, further comprising the step of removing the binding reagent from the target polypeptide.

29. The method of claim 28, wherein a step of removing the binding reagent occurs after step b).

30. The method of claim 28, wherein a step of removing the binding reagent occurs after step c).

31. A system for identifying a polypeptide of interest, comprising: a) a solid support comprising a plurality of sites, wherein each individual site of the plurality of sites comprises: i) a unique nucleic acid nanostructure connected to each individual site; ii) a unique polypeptide of interest bound to said unique nucleic acid nanostructure; Including, b) a first plurality of binding reagents in a first fluid medium, wherein said first fluid medium is deliverable to said solid support; c) a second plurality of binding reagents in a second fluid medium, said second fluid medium being deliverable to said solid support; and d) an optical detector configured to detect the presence or absence of a binding reagent of the first plurality of binding reagents or the second plurality of binding reagents at each site of the plurality of sites with single analyte resolution. Including, the system.

32. The system of claim 31, wherein the optical detector is configured to detect the presence or absence of a binding reagent of the first plurality of binding reagents or the second plurality of binding reagents at each site of the plurality of sites with single analyte resolution.

33. An array composition comprising: a) a solid support comprising a plurality of sites, wherein each individual site of the plurality of sites comprises a unique nucleic acid nanostructure connected to said individual site; b) a plurality of polypeptides of interest, wherein each nucleic acid nanostructure is bound to a unique polypeptide of interest among said plurality of polypeptides of interest; and c) a plurality of binding reagents that bind to polypeptides of interest of said plurality of polypeptides of interest at sites of the plurality of sites; Array composition comprising:

34. The array composition of claim 33, wherein the plurality of polypeptides of interest comprises a first species of polypeptide of interest and a second species of polypeptide of interest, the first species of polypeptide of interest and the second species of polypeptide of interest having a dynamic range of at least 104.

35. The array composition of claim 34, wherein the first species of target polypeptide and the second species of target polypeptide each comprise a common epitope.

36. The array composition of claim 35, wherein a first binding reagent of the plurality of binding reagents is bound to a polypeptide of interest of a first species of polypeptides of interest, and a second binding reagent of the plurality of binding reagents is bound to a polypeptide of interest of a second species of polypeptides of interest.

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