WO2025231225A1

WO2025231225A1 - Probe-based analysis of biological molecules

Info

Publication number: WO2025231225A1
Application number: PCT/US2025/027269
Authority: WO
Inventors: Tuval BEN YEHEZKEL; Alan Shaw
Original assignee: Place Genomics Corp
Current assignee: Place Genomics Corp
Priority date: 2024-05-03
Filing date: 2025-05-01
Publication date: 2025-11-06
Anticipated expiration: 2026-11-03

Abstract

Provided herein are systems and methods for analyzing analytes (e.g., biomolecules). A method can include providing: (i) a sample comprising an analyte, and (ii) a nucleic acid scaffolded structure, wherein the nucleic acid scaffolded structure is coupled to a probe comprising (I) a sensing moiety configured to bind to the analyte, and (II) a barcode sequence corresponding to a spatial position of the probe on the nucleic acid scaffolded structure; (b) contacting the sample with the nucleic acid scaffolded structure, wherein the sensing moiety of the probe on the nucleic acid scaffolded structure binds to the analyte in the sample, thereby coupling the probe and its barcode sequence to the analyte; (c) identifying the barcode sequence, thereby identifying the spatial position of the probe on the nucleic acid scaffolded structure; (d) identifying the analyte in the sample coupled to the probe; and (e) associating the spatial position of the probe on the nucleic acid scaffolded structure with the analyte in the sample.

Description

PROBE-BASED ANALYSIS OF BIOLOGICAL MOLECULES

CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/642,554, filed May 3, 2024, which is incorporated by reference herein in its entirety for all purposes.

BACKGROUND

[0002] Biological samples may be analyzed for various purposes, such as visualization of cellular architecture or for detection of disease. Spatial biology can provide insight into the cellular and molecular complexities that underpin health and disease. Analytes, such as nucleic acids and proteins, within biological samples can be detected and/or analyzed by probes.

SUMMARY

[0003] In one aspect, the present disclosure provides a method comprising: (a) providing: (i) a sample comprising an analyte, and (ii) a nucleic acid scaffolded structure, wherein the nucleic acid scaffolded structure is coupled to a probe comprising (I) a sensing moiety configured to bind to the analyte, and (II) a barcode sequence corresponding to a spatial position of the probe on the nucleic acid scaffolded structure; (b) contacting the sample with the nucleic acid scaffolded structure, wherein the sensing moiety of the probe on the nucleic acid scaffolded structure binds to the analyte in the sample, thereby coupling the probe and its barcode sequence to the analyte; (c) identifying the barcode sequence, thereby identifying the spatial position of the probe on the nucleic acid scaffolded structure; (d) identifying the analyte in the sample coupled to the probe; and (e) associating the spatial position of the probe on the nucleic acid scaffolded structure with the analyte in the sample.

[0004] In some embodiments, the nucleic acid scaffolded structure is further coupled to a plurality of probes. In some embodiments, the plurality of probes comprises probes configured to bind to different analytes in the sample. In some embodiments, the plurality of probes comprises at least 100 probes. In some embodiments, the plurality of probes comprises at least 1000 probes. In some embodiments, the probe is located from 1 to 50 nm away from an additional probe of the plurality of probes. In some embodiments, the sample comprises a plurality of analytes, and, in (b), probes of the plurality of probes on the nucleic acid scaffolded structure couple to the plurality of analytes.

[0005] In some embodiments, (c) comprises detecting the barcode sequence using sequencing. In some embodiments, (c) comprises detecting the barcode sequence using in situ sequencing. In some embodiments, the probe further comprises (III) an additional barcode sequence. In some embodiments, the additional barcode sequence identifies the nucleic acid scaffolded structure, and wherein the method further comprises identifying the analyte as associated with the nucleic acid scaffolded structure.

[0006] In some embodiments, the analyte comprises a protein or a peptide. In some embodiments, the analyte further comprises a nucleic acid coupled to the protein or the peptide. In some embodiments, the analyte comprises a small molecule. In some embodiments, the analyte further comprises a nucleic acid coupled to the small molecule. In some embodiments, the analyte comprises a nucleic acid. In some embodiments, the analyte comprises an RNA transcript. In some embodiments, the analyte comprises genomic DNA. In some embodiments, the analyte comprises an adapter sequence. In some embodiments, the method comprises, prior to (a), generating the analyte by coupling the adapter sequence to a nucleic acid molecule. In some embodiments, coupling the adapter sequence to the nucleic acid molecule comprises a transposition reaction. In some embodiments, the sensing moiety of the probe comprises a sequence that hybridizes to a portion of the adapter sequence. In some embodiments, the sensing moiety of the probe comprises a sensing sequence that hybridizes to a portion of the analyte. In some embodiments, the sensing sequence comprises a poly-T sequence. In some embodiments, the sensing moiety of the probe comprises a protein or a peptide. In some embodiments, the sensing moiety of the probe comprises an enzyme. In some embodiments, the enzyme is a DNA processing enzyme. In some embodiments, the enzyme comprises a transposase domain.

[0007] In some embodiments, the nucleic acid of the analyte comprises a target sequence, and the method further comprises generating a barcoded nucleic acid strand comprising (i) the barcode sequence or a complement thereof, and (ii) the target sequence or complement thereof. In some embodiments, the probe further comprises (III) an additional barcode sequence that identifies the nucleic acid scaffolded structure, and wherein the barcoded nucleic acid strand further comprises (iii) the additional barcode sequence or a complement thereof. In some embodiments, the method comprises generating the barcoded nucleic acid strand using a nucleic acid ligation reaction. The method can further comprise generating the barcoded nucleic acid strand using a nucleic acid extension reaction. In some embodiments, the nucleic acid of the analyte comprises a target sequence, and the method further comprises, prior to (d), amplifying the target sequence using the probe in a nucleic acid amplification reaction to yield an amplification product. In some embodiments, (d) further comprises detecting the amplification product or a derivative thereof. In some embodiments, (d) further comprises sequencing the amplification product or a derivative thereof, thereby obtaining sequencing reads. In some embodiments, (e) further comprises associating the sequencing reads with the spatial position of the probe in the nucleic acid scaffolded structure.

[0008] In some embodiments, the nucleic acid scaffolded structure comprises a dimension of from 100 to 2000 nm. In some embodiments, the nucleic acid scaffolded structure comprises a crisscross assembly. In some embodiments, in (a), the nucleic acid scaffolded structure is coupled to a surface. In some embodiments, the method further comprises, prior to (a), coupling the nucleic acid scaffolded structure to the surface. In some embodiments, the coupling comprises orienting the nucleic acid scaffolded structure on the surface based on a pattern on the surface. In some embodiments, the method further comprises, prior to (a), generating the pattern using electron beam lithography or atomic force lithography. In some embodiments, the nucleic acid scaffold structure is coupled to a bead. In some embodiments, the surface is coupled to a plurality of nucleic acid scaffolded structures. In some embodiments, the sample comprises a cell. In some embodiments, the method further comprises, prior to (a), coupling the probe to the nucleic acid scaffolded structure. In some embodiments, the coupling the probe to the nucleic acid scaffolded structure comprises hybridizing a portion of the probe to a binding segment of the nucleic acid scaffolded structure. In some embodiments, the binding segment of the nucleic acid scaffolded structure comprises a distinct sequence corresponding to a distinct spatial position of the binding segment in the nucleic acid scaffolded structure. In some embodiments, the nucleic acid scaffolded structure further comprises a plurality of binding segments, wherein each binding segment of the plurality of binding segments comprises a distinct sequence corresponding to a distinct spatial position of the binding segment in the nucleic acid scaffolded structure.

[0009] In some embodiments, the identifying in (d) identifies information about the analyte, and wherein the method further comprises (f) constructing an image using: (i) the information about the analyte identified in (d), and (ii) the spatial position of the probe on the nucleic acid scaffolded structure identified in (c). In some embodiments, the method further comprises, after (b), identifying a location of the nucleic acid scaffolded structure in the sample. In some embodiments, constructing the image in (f) further comprises using (iii) the location of the nucleic acid scaffolded structure in the sample. In some embodiments, identifying the location of the nucleic acid scaffolded structure comprises imaging. In some embodiments, (a) further comprises providing a plurality of nucleic acid scaffolded structures, wherein each nucleic acid scaffolded structure is coupled to a probe comprising (I) a sensing moiety configured to bind to an analyte, and (II) a barcode sequence corresponding to a spatial position of the probe on the nucleic acid scaffolded structure to which the probe is coupled; and wherein (b) further comprises contacting the sample with the plurality of nucleic acid scaffolded structures. In some embodiments, the method further comprises, after (b), identifying a location of each nucleic acid scaffolded structure of the plurality of nucleic acid scaffolded structures in the sample. In some embodiments, identifying the location of each nucleic acid scaffolded structure comprises imaging.

[0010] In another aspect, the present disclosure provides a composition comprising a nucleic acid scaffolded structure, wherein the nucleic acid scaffolded structure is coupled to a plurality of probes, wherein each probe of the plurality of probes comprises (I) a sensing moiety configured to bind to an analyte, and (II) a first barcode sequence corresponding to a spatial position of the probe on the nucleic acid scaffolded structure, and (III) a second barcode sequence identifying the nucleic acid scaffolded structure. In some embodiments, the sensing moiety of the probe comprises a nucleic acid sequence. In some embodiments, the sensing moiety of the probe comprises a protein or a peptide. In some embodiments, the composition further comprises the analyte, wherein the analyte is bound to the sensing moiety of a probe of the plurality of probes. In some embodiments, the analyte comprises an adapter sequence that is bound to the sensing moiety of the probe of the plurality of probes. In some embodiments, the nucleic acid scaffolded structure is coupled to an additional nucleic acid scaffolded structure.

[0011] In some embodiments, the nucleic acid scaffolded structure is coupled to a surface. In some embodiments, the surface is coupled to a plurality of nucleic acid scaffolded structures. In some embodiments, nucleic acid scaffolded structures of the plurality of nucleic acid scaffolded structures are aligned on the surface based on a pattern on the surface. In some embodiments, a probe of the plurality of probes is located from 1 to 50 nm away from an additional probe of the plurality of probe. [0012] In another aspect, the present disclosure provides a kit comprising: (a) a plurality of distinct nucleic acid scaffolded structures, wherein each distinct nucleic acid scaffolded structure is coupled to: (i) a plurality of probes, wherein each probe of the plurality of probes comprises (I) a sensing moiety configured to bind to the analyte, (II) a barcode sequence corresponding to a spatial position of the probe on the distinct nucleic acid scaffolded structure; and (III) an additional barcode sequence identifying its corresponding distinct nucleic acid scaffolded structure; and (b) instructions for using a nucleic acid scaffolded structure of the plurality of distinct nucleic acid scaffolded structures to detect an analyte.

[0013] In another aspect, the present disclosure provides a method comprising: (a) providing: (i) a surface; (ii) a first nucleic acid scaffolded structure, wherein the first nucleic acid scaffolded structure is coupled to a first probe, wherein the first probe comprises (I) a first sensing moiety configured to bind to a first analyte in a sample, and (II) a first barcode sequence corresponding to a first spatial position of the first probe on the first nucleic acid scaffolded structure; and (iii) a second nucleic acid scaffolded structure, wherein the second nucleic acid scaffolded structure is coupled to a second probe, wherein the second probe comprises (I) a second sensing moiety configured to bind to a second analyte in a sample, and (II) a second barcode sequence corresponding to a second spatial position of the second probe on the second nucleic acid scaffolded structure; and (b) coupling the first nucleic acid scaffolded structure to the surface and coupling the second nucleic acid scaffolded structure to the surface.

[0014] In some embodiments, the method comprises orienting the first nucleic acid scaffolded structure and the second nucleic acid scaffolded structure on the surface based on a pattern on the surface. In some embodiments, the method further comprises, prior to (a), generating the pattern using electron beam lithography or atomic force lithography. In some embodiments, the first nucleic acid scaffolded structure comprises a handle segment, and (b) comprises hybridizing the handle segment to a binding sequence on the surface. In some embodiments, the method further comprises directly coupling the first nucleic acid scaffolded structure to the second nucleic acid scaffolded structure. In some embodiments, the surface is a solid surface.

[0015] In another aspect, the present disclosure provides a method comprising: (a) providing: (i) a surface coupled to a first nucleic acid primer and a second nucleic acid primer; and (ii) a nucleic acid scaffolded structure, wherein the nucleic acid scaffolded structure is coupled to: (A) a first probe comprising: (I) a first sensing sequence, wherein the first sensing sequence or a reverse complement thereof is configured to bind to a first analyte in a sample, and (II) a first barcode sequence corresponding to a first spatial position of the first probe on the nucleic acid scaffolded structure; and (B) a second probe comprising: (I) a second sensing sequence, wherein the second sensing sequence or a reverse complement thereof is configured to bind to a second analyte in a sample, and (II) a second barcode sequence corresponding to a second spatial position of the second probe on the nucleic acid scaffolded structure; (b) contacting the first nucleic acid primer coupled to the surface and the second nucleic acid primer coupled to the surface with the nucleic acid scaffolded structure, (c) generating a first nucleic acid extension product using the first nucleic acid primer coupled to the surface and the first probe coupled to the nucleic acid scaffolded structure; wherein the first nucleic acid extension product is coupled to the surface, and wherein the first nucleic acid extension product comprises (i) the first sensing sequence or reverse complement thereof, and (ii) the first barcode sequence or reverse complement thereof; and (d) generating a second nucleic acid extension product using the second nucleic acid primer coupled to the surface and the second probe coupled to the nucleic acid scaffolded structure; wherein the second nucleic acid extension product is coupled to the surface, and wherein the second nucleic acid extension product comprises (i) the second sensing sequence or reverse complement thereof, and (ii) the second barcode sequence or reverse complement thereof.

[0016] In some embodiments, (a) further comprises providing (iii) an additional nucleic acid scaffolded structure comprising a third probe, and wherein the method further comprises contacting a third nucleic acid primer coupled to the surface with the additional nucleic acid scaffolded structure. In some embodiments, the method further comprises generating a third nucleic acid extension product using the third nucleic acid primer coupled to the surface and the third probe in the nucleic acid scaffolded structure. In some embodiments, the third probe comprises: (A) a third sensing sequence, wherein the third sensing sequence or a reverse complement thereof is configured to bind to a third analyte in a sample, and (B) a third barcode sequence corresponding to a third spatial position of the third probe on the additional nucleic acid scaffolded structure; and the third nucleic acid extension product comprises the third sensing sequence or reverse complement thereof, and the third barcode sequence or reverse complement thereof.

[0017] In some embodiments, the method further comprises, after generating the first nucleic acid extension product coupled to the surface and generating the second nucleic acid extension product coupled to the surface, (e) contacting the surface with a sample comprising the first analyte and the second analyte. In some embodiments, in (e), the first sensing sequence or reverse complement thereof in the first nucleic acid extension product couples to the first analyte in the sample; and wherein the second sensing sequence or reverse complement thereof in the second nucleic acid extension product couples to the second analyte in the sample. In some embodiments, the method further comprises, after (e), detecting the first barcode sequence or reverse complement thereof in the sample and detecting the second barcode sequence or reverse complement thereof in the sample. In some embodiments, the method further comprises, after (e), identifying the first analyte in the sample using the first nucleic acid extension product coupled to the surface and identifying the second analyte in the sample using the second nucleic acid extension product coupled to the surface. In some embodiments, the method further comprises associating the first barcode sequence with the first analyte and associating the second barcode sequence with the second analyte. In some embodiments, the surface comprises a gel.

[0018] In another aspect, the present disclosure provides a method of barcoding a nucleic acid scaffolded structure, the method comprising: (a) providing: (i) a nucleic acid scaffolded structure, wherein the nucleic acid scaffolded structure comprises a first oligonucleotide and a second oligonucleotide; (ii) a barcoded nucleic acid template comprising a plurality of barcode sequences, wherein the plurality of barcode sequences comprises a first barcode sequence and a second barcode sequence; and (b) generating a barcoded nucleic acid scaffolded structure using the first oligonucleotide, the second oligonucleotide, the first barcode sequence, and the second barcode sequence, wherein the barcoded nucleic acid scaffolded structure comprises (i) a first barcoded oligonucleotide comprising a sequence corresponding to the first barcode sequence, and (ii) a second barcoded oligonucleotide comprising a sequence corresponding to the second barcode sequence.

[0019] In some embodiments, (b) comprises performing nucleic acid extension reactions. In some embodiments, (b) comprises coupling the nucleic acid scaffolded structure to the barcoded nucleic acid template, wherein a 3’ end of the first oligonucleotide of the nucleic acid scaffolded structure hybridizes to a first portion of the barcoded nucleic acid template, and wherein a 3’ end of the second oligonucleotide of the nucleic acid scaffolded structure hybridizes to a second portion of the barcoded nucleic acid template. In some embodiments, (b) further comprises performing (i) a first nucleic acid extension reaction using the first barcode sequence of the barcoded nucleic acid template and the first oligonucleotide of the nucleic acid scaffolded structure, thereby generating the first barcoded oligonucleotide, and (ii) a second nucleic acid extension reaction using the second barcode sequence of the barcoded nucleic acid template and the second oligonucleotide of the nucleic acid scaffolded structure, thereby generating the second barcoded oligonucleotide. In some embodiments, the plurality of barcode sequences are identical.

[0020] In some embodiments, the method further comprises, prior to (a), generating the barcoded nucleic acid template by performing a nucleic acid amplification reaction on a nucleic acid molecule comprising a barcode sequence of the plurality of barcode sequences. In some embodiments, the nucleic acid amplification reaction comprises rolling circle amplification.

[0021] In another aspect, the present disclosure provides a method of generating a plurality of different nucleic acid scaffolded structures, comprising: (a) providing a plurality of nucleic acid scaffolded structures, wherein each nucleic acid scaffolded structure of the plurality of nucleic acid scaffolded structures comprises an oligonucleotide; and (b) combinatorially assembling a barcode sequence on the oligonucleotide of each nucleic acid scaffolded structure. In some embodiments, (b) comprises assembling a barcode sequence on a nucleic acid scaffolded structure that distinguishes the nucleic acid scaffolded structure from other nucleic acid scaffolded structures of the plurality of nucleic acid scaffolded structures. In some embodiments, the method further comprises, prior to or during (b), partitioning the plurality of nucleic acid scaffolded structures into a plurality of partitions, and (b) further comprises appending one or more nucleotides onto the oligonucleotide of each nucleic acid scaffolded structure within a partition of the plurality of partitions, thereby generating an extended oligonucleotide on each nucleic acid scaffolded structure.

[0022] In another aspect, the present disclosure provides a method of generating barcoded nucleic acid scaffolded structures, comprising: (a) partitioning a plurality of nucleic acid scaffolded structures into a plurality of partitions, wherein each nucleic acid scaffolded structure of the plurality of nucleic acid scaffolded structures comprises an oligonucleotide; and (b) appending one or more nucleotides onto the oligonucleotide of each nucleic acid scaffolded structure within a partition of the plurality of partitions, thereby generating an extended oligonucleotide on each nucleic acid scaffolded structure.

[0023] In some aspects, each partition of the plurality of partitions comprises a pool of discrete nucleotide monomers, and (b) comprises appending a discrete nucleotide monomer of the pool of discrete nucleotide monomers onto the oligonucleotide within each partition. In some embodiments, the plurality of partitions comprise different partitions comprising different pools of discrete nucleotide monomers, and wherein (b) generates different extended oligonucleotides in the different partitions.

[0024] In some aspects, each partition of the plurality of partitions comprises a pool of polynucleotides, and (b) comprises appending a polynucleotide of the pool of polynucleotides onto the oligonucleotide within each partition. In some embodiments, the plurality of partitions comprise different partitions comprising different pools of polynucleotides, and (b) generates different extended oligonucleotides in the different partitions.

[0025] In some aspects, after generating an extended oligonucleotide on each nucleic acid scaffolded structure, pooling the plurality of nucleic acid scaffold structures. In some aspects, the method further comprises, after pooling the plurality of nucleic acid scaffold structures, partitioning the plurality of nucleic acid scaffold structures into an additional plurality of partitions. In some aspects, the method further comprises, within each additional partition of the additional plurality of partitions, appending one or more nucleotides onto the extended oligonucleotide of each nucleic acid scaffolded structure.

INCORPORATION BY REFERENCE

[0026] 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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:

[0028] FIG. 1 shows an example flowchart for analyzing an analyte.

[0029] FIG. 2 shows an example workflow for analyzing an analyte.

[0030] FIG. 3 shows an example workflow for analyzing a plurality of analytes. [0031] FIG. 4 shows an example nucleic acid scaffolded structure coupled to a probe.

[0032] FIGs. 5A-5D show examples of nucleic acid scaffolded structures coupled to a probe bound to an analyte. FIG. 5A shows an example of a nucleic acid scaffolded structure coupled to a probe bound to an RNA transcript. FIG. 5B shows an example of a nucleic acid scaffolded structure coupled to a probe bound to an analyte comprising a nucleic acid coupled to an antibody bound to a target. FIG. 5C shows an example of a nucleic acid scaffolded structure coupled to a plurality of probes each bound to an RNA transcript. FIG. 5D shows an example of a nucleic acid scaffolded structure coupled to a probe bound to an adapter sequence of a tagmented nucleic acid.

[0033] FIGs. 6A and 6B shows an example workflows for analyzing an analyte. FIG. 6A shows an example of nucleic acid extension of a probe bound to an analyte. FIG. 6B shows an example of nucleic acid ligation and extension of a probe bound to an analyte.

[0034] FIG. 7 shows an example flowchart for coupling nucleic acid scaffolded structures to a surface.

[0035] FIG. 8 shows an example workflow for coupling nucleic acid scaffolded structures to a surface.

[0036] FIG. 9 shows an example schematic of barcoding a nucleic acid scaffolded structure.

[0037] FIG. 10 shows an example of arraying and aligning nucleic acid scaffolded structures comprising probes to a surface.

[0038] FIG. 11A shows an example workflow of barcoding a nucleic acid scaffolded structure using a barcoded template. FIG. 11B shows an example workflow of generating a barcoded template.

[0039] FIG. 12A shows an example workflow of barcoding a nucleic acid scaffolded structure using a split-pool method using discrete nucleotide monomers. FIG. 12B shows another example workflow of barcoding a nucleic acid scaffolded structure using a splitpool method using polynucleotides.

[0040] FIG. 13A shows an example of nucleic acid scaffolded structures on a surface prior to alignment.

[0041] FIG. 13B shows an example of nucleic acid scaffolded structures on a surface after alignment.

[0042] FIG. 14 shows an example flowchart for generating nucleic acid extension products coupled to a surface from probes coupled to a nucleic acid scaffolded structure. [0043] FIGs. 15A-15C show an example workflow for generating nucleic acid extension products coupled to a surface from probes coupled to a nucleic acid scaffolded structure and analyzing analytes using the nucleic acid extension products. FIG. 15A shows an example of nucleic acid primers binding to probes coupled to the nucleic acid scaffolded structure and nucleic acid extension reactions. FIG. 15B shows an example of nucleic acid extension products coupled to the surface. FIG. 15C shows an example of analyzing analytes using the nucleic acid extension products coupled to the surface.

[0044] FIG. 16 shows an example workflow for generating nucleic acid extension products coupled to a surface from probes coupled to a plurality of nucleic acid scaffolded structures.

[0045] FIG. 17A and 17B show an example workflow for coupling nucleic acid scaffolded structures and information sharing between nucleic acid scaffolded structures. FIG. 17A shows an example of two coupled nucleic acid scaffolded structures. FIG. 17B shows an example of nucleic acid extension to generate products comprising information on the relative positioning and interaction of the nucleic acid scaffolded structures.

DETAILED DESCRIPTION

[0046] Recognized herein is a need for improved methods in spatial biology for detecting and analyzing analytes. Current technologies face limits in resolution and are insufficient for decoding the spatial heterogeneity of analytes within cells, a critical factor in deciphering complex biological processes and disease. For example, traditional light microscopy techniques are limited by the diffraction limit of light, which restricts resolution to roughly half the wavelength used, blurring details closer than 300 nanometers. As another example, molecular sensors comprising beads coupled to oligonucleotides are also limited to ~ 1 pm in resolution for spatial analysis.

[0047] In some aspects, the present disclosure provides compositions and methods for analyte detection and analysis that can overcome current limitations in spatial biology, bypass the diffraction limit, and achieve a resolution as low as single digit nanometer resolution, which is 1000 times finer than conventional methods. In some aspects, the compositions and methods described herein leverage nucleic acid origami structures to provide scaffolds decorated with nanometer-placed, uniquely barcoded probes. The barcoded probes can interact with analytes, and the barcode information and analyte information can be detected, for example, by sequencing. These origami structures can act as miniature sensors with each probe configured to generate a single data point or “molecular pixel” to construct a spatial map of the analytes in a cell or tissue. These approaches offer single-molecule sensitivity, providing data on individual molecule and their spatial positions and interactions, enabling the visualization of new dimensions of cellular architecture. In some cases, multiple nucleic acid scaffolded structures are arrayed and oriented on surfaces using specialized alignment techniques for use in spatial analysis of a larger area (e.g., for spatial analysis of an entire cell or multiple cells in a tissue). In further aspects, the present disclosure provides compositions and methods for mass production of miniature sensors that enable nanometer resolution. The compositions and methods can utilize a scalable technology to copy unique information from one or more nucleic acid scaffolded structures onto probes coupled to a surface, such as a gel that can then be used as miniature sensors for spatial analysis of analytes. The compositions and methods described herein have applications in diagnostics and new target discovery for therapeutics by improving understanding of the cellular and molecular complexities that underpin health and disease.

A. Compositions and systems

[0048] In one aspect, the present disclosure provides composition comprising a nucleic acid scaffolded structure. The nucleic acid scaffolded structure can be coupled to a plurality of probes. A probe of the plurality of probes can comprise a sensing moiety configured to bind to an analyte in a sample. In some cases, multiple probes of the plurality of probes each comprise a sensing moiety configured to bind to an analyte in a sample. One or more probes can comprise a barcode sequence corresponding to a spatial position of the probe on the nucleic acid scaffolded structure. In some cases, the probe comprises an additional barcode sequence identifying the nucleic acid scaffolded structure.

[0049] The nucleic acid scaffolded structure can be a nucleic acid structure having a non- naturally two dimensional or three dimensional architecture. The nucleic acid scaffolded structure can comprise a crossover or a Holliday junction. The nucleic acid scaffolded structure can comprise a parallel crossover, an antiparallel crossover, or a combination thereof. In some cases, the nucleic acid scaffolded structure comprises at least one, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 nucleic acid strands.

[0050] The nucleic acid scaffolded structure can comprise a DNA origami structure or an alternative structured nucleic acid assembly of distinct oligonucleotide modular components through local binding interactions. In some cases, the nucleic acid scaffolded structure comprises one or more scaffold strands and one or more shorter oligonucleotides that interact with different portions of one or more scaffold strands to bring the different portions into proximity with each other and aid in defining the architecture of the nucleic acid scaffolded structure. In other cases, the nucleic acid scaffolded structure comprises multiple oligonucleotide modular components assembled together via local binding interactions. In some cases, a molecular scaffold comprising DNA strands are arranged in a criss-cross origami design. In some cases, the molecular scaffold is further decorated with one or more probes. The one or more probes can be arranged on the nucleic acid scaffolded structure in a particular configuration. Examples of a nucleic acid scaffolded structure design, fabrication, and decoration are described in Minev, D., Wintersinger, C. M., Ershova, A. & Shih, W. M. Robust nucleation control via crisscross polymerization of highly coordinated DNA slats. Nat. Commun. 12, 1741 (2021); Wintersinger, C.M., Minev, D., Ershova, A. etal. Multi-micron crisscross structures grown from DNA-origami slats. Nat. Nanotechnol. 18, 281-289 (2023); Ashwin Gopinath, Chris Thachuk, Anya Mitskovets, Harry A. Atwater, David Kirkpatrick, Paul W. K. Rothemund, Science 371, (2021); International Patent Publication No. W02018026880A2; U.S. Pat. No. US1 1162192B2; Yan, H et al., Science, 301, 1882-1884 (2003); Winfree, E et al., Nature, 394, 539-544 (1998); and Ke, Y et al., Science, 338, 1177-1183 (2012) which are incorporated by reference herein in their entireties. In some cases, a criss-cross origami design is combined with a probe decoration design to generate the nucleic acid scaffolded structure.

[0051] The nucleic acid scaffolded structure can comprise a dimension of from 100 to 10,000 nm, 100 to 5000 nm, 100 to 4000 nm, 100 to 3000 nm, 100 to 2000 nm, 100 to 1500 nm, 100 to 1000 nm, 100 to 800 nm, or 200 to 10,000 nm, 200 to 5000 nm, 200 to 4000 nm, 200 to 3000 nm, 200 to 2000 nm, 200 to 1500 nm, 200 to 1000 nm, or 200 to 800 nm. The nucleic acid scaffolded structure can be coupled to a surface, for example, a bead.

[0052] In some cases, the nucleic acid scaffolded structure is coupled to a probe. The probe can comprise a sensing moiety capable of binding to or configured to bind to the analyte. The sensing moiety can comprise nucleic acid (e.g., DNA or RNA). In some cases, the sensing moiety comprises a sensing sequence configured to hybridize to a nucleic acid in the analyte. For example, the sensing sequence can comprise a poly-T sequence or sequence configured to hybridize to a target sequence in a target nucleic acid (e.g., a gene sequence in an RNA transcript). As another example, the sensing moiety can comprise a nucleic acid aptamer capable of interacting with a specific target. In some cases, the sensing moiety comprises a protein or a peptide. For example, the sensing moiety can comprise an antibody, an antibody fragment (e.g., a fragment antigen-binding (Fab)), a nucleic acid binding domain, or a protein receptor. The sensing moiety can comprise an enzyme. For example, the enzyme can be a DNA processing enzyme, such as a nuclease or a ligase. In some cases, the enzyme comprises a transposase domain. In other cases, a sensing moiety comprises a small molecule, a lipid, or carbohydrate. The sensing moiety of the probe can be configured to bind to a protein, peptide, nucleic acid, lipid, or carbohydrate. FIG. 5A shows an example of a probe 0520a bound to a segment 0542a of a nucleic acid analyte. In some cases, the probe comprises a nucleic acid coupled to a protein, peptide, or small molecule. FIG. 5B shows another example of a probe comprising a nucleic acid strand 0520b coupled to another nucleic acid strand that is coupled to an antibody 0560b. The antibody 0560b is capable of binding to a target molecule 0570b. In some cases, the sensing moiety is configured to bind to an adapter sequence of an analyte. FIG. 5D shows an example of a probe 0580 bound to an adapter sequence 0583RC of an analyte 0590. In this example, the analyte 0590 is a transposase- fragmented nucleic acid product comprising adapter sequences appended to a genomic DNA fragment comprising a genomic sequence 0591.

[0053] In some cases, the probe comprises a barcode sequence corresponding to a spatial position of the probe on the nucleic acid scaffolded structure. For example, the spatial position can be a position on the nucleic acid scaffolded structure at which the probe is coupled to the nucleic acid scaffolded structure. In some cases, the probe is located on the surface of the molecular scaffold. FIG. 4 schematically shows a nucleic acid scaffolded structure 0410, as described herein. The nucleic acid scaffolded structure comprises a probe 0420. The probe 0420 can comprise a barcode sequence 0422 and a sensing moiety comprising sensing sequence 0421 in FIG. 4.

[0054] In some cases, the probe further comprises an additional barcode sequence (e.g., a UMI). The additional barcode sequence can identify the nucleic acid scaffolded structure. In some cases, the probe further comprises a barcode sequence identifying a cell to which the nucleic acid scaffolded structure is provided. In other cases, the probe further comprises a barcode sequence identifying a sample from which the analyte is derived. The probe can further comprise one or more additional functional sequences (e.g., staple segment for anchoring a probe to the nucleic acid scaffolded structure, a hybridizing sequence for a primer for use in generating the nucleic acid scaffolded structure or a sequence for use in generating a sequencing library). In some cases, the nucleic acid scaffolded structure comprises a probe comprising a position barcode sequence and an additional probe comprising a UMI identifying the nucleic acid scaffolded structure. In some cases, the nucleic acid scaffolded structure comprises a probe comprising a position barcode sequence and the UMI identifying the nucleic acid scaffolded structure. FIG. 6B provides an example of a nucleic acid scaffolded structure 0670 that is coupled to a probe 0680. The probe 0680 comprises a bottom staple section 0681 that anchors the probe to the nucleic acid scaffolded structure, a priming site 0682 for next-generation sequencing (NGS) library preparation via PCR, a position barcode 0683 encoding the probe’ s location on the nucleic acid scaffolded structure 0670, and a nucleic acid scaffolded structurespecific unique molecular identifier (UMI) 0685. The probe 0680 further comprises a hybridization segment 0684 used to add the UMI 0685 to the probe in a barcoding reaction, such as, for example, described in Example 3. The probe 0680 further comprises a sensing moiety 0686 configured to bind to an adapter sequence 0686RC in analyte 0690.

[0055] In some cases, the nucleic acid scaffolded structure is coupled to a plurality of probes, for example, as illustrated in FIG. 5C. The plurality of probes can comprise probes configured to bind to different analytes in the sample. In some cases, the plurality of probes are configured to bind to a same analyte. The plurality of probes can comprise at least 2, at least 5, at least 10, at least 20, at least 50, at least 75, at least 100, at least 150, at least 200, at least 500, at least 750, at least 1000, at least 2000, at least 3000, at least 5000, or at least 10000 probes. The plurality of probes can comprise at most 2, at most 5, at most 10, at most 20, at most 50, at most 75, at most 100, at most 150, at most 200, at most 500, at most 750, at most 1000, at most 2000, at most 3000, at most 5000, or at most 10000 probes. The plurality of probes can comprise from 10 to 100 probes, from 10 to 500 probes, from 10 to 1000 probes, or from 100 to 1000 probes. In some cases, a probe of the plurality of probes is located from 0.5 to 10 nm, 0.5 to 20 nm, from 0.5 to 40 nm, from 0.5 to 60 mm, from 0.5 to 100 mm, or from 0.5 to 150 mm away from an additional probe of the plurality of probes. In some embodiments, a probe of the plurality of probes is located at least 0.5 nm, 1 nm, 1.5 nm, 2 nm, 2.5 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, 22 nm, 24 nm, 26 nm, 28 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, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 250 nm, or 300 nm away from an additional probe of the plurality of probes. The probe can be from 0.5 to 50 nm, from 1 to 50 nm, from 1.5 to 50 nm, from 2 to 50 nm, from 2.5 to 50 nm, from 3 to 50 nm, from 4 to 50 nm, from 5 to 50 nm, from 6 to 50 nm, from 7 to 50 nm, from 8 to 50 nm, from 9 to 50 nm, from 10 to 50 nm, from 11 to 50 nm, from 12 to 50 nm, from 13 to 50 nm, from 14 to 50 nm, from 15 to 50 nm, from 16 to 50 nm, from 17 to 50 nm, from 18 to 50 nm, from 19 to 50 nm, from 20 to 50 nm, from 22 to 50 nm, from 24 to 50 nm, from 26 to 50 nm, from 28 to 50 nm, or from 30 to 50 nm from an additional probe of the plurality of probes.

[0056] In some cases, the composition further comprises the sample comprising the analyte. In some cases, the sample comprises a tissue or a cell. The cell can be a prokaryotic or eukaryotic cell. The cell can be a bacterial, plant, animal, or fungal cell. In some cases, the analyte is provided in the cell or on the cell surface. The analyte can be an extracellular or intracellular analyte. The cell can be a lysed or permeabilized cell. In other cases, the sample can comprise a viral nucleic acid sample.

[0057] The analyte can comprise a protein, peptide, nucleic acid, lipid, carbohydrate, or a combination thereof. The analyte can be an intracellular or an extracellular analyte. In some cases, the analyte is localized in a specific organelle. The analyte can be located in a mitochondria, cell nucleus, cytoplasm, or ribosome. In some cases, the analyte is a cell membrane analyte. In some cases, the analyte comprises a nucleic acid (e.g., DNA or RNA). The nucleic acid can comprise a target sequence. In some cases, the nucleic acid can comprise an RNA transcript. In some cases, the RNA transcript can comprise a polyA sequence. In other cases, the nucleic acid comprises genomic DNA. The nucleic acid can comprise an adapter sequence. In some cases, the analyte comprises a transposase- fragmented nucleic acid product comprising adapter sequences appended to a genomic DNA fragment. The nucleic acid can comprise single-stranded nucleic acid or doublestranded nucleic acid. In some cases, the nucleic acid analyte comprises a stem-loop structure. In some cases, the analyte comprises a protein or a peptide. For example, the analyte can be a cell surface protein receptor or an intracellular protein. The protein or peptide can directly interact with the probe or can be coupled to a nucleic acid that interacts with the probe. In further cases, the analyte comprises a small molecule. The small molecule can directly interact with the probe or can be coupled to a nucleic acid that interacts with the probe.

[0058] The nucleic acid scaffolded structure can be coupled to an additional nucleic acid scaffolded structure via binding oligonucleotides or other binding moieties. The nucleic acid scaffolded structure can be coupled to a binding oligonucleotide or a binding moiety (e.g., an antibody, peptide, or small molecule) configured to couple to an additional binding oligonucleotide or binding moiety coupled to an additional nucleic acid scaffolded structure. A binding oligonucleotide can further comprise one or more barcodes (e.g., a position barcode identifying the spatial position of the binding oligonucleotide on the nucleic acid scaffolded structure and/or a barcode identifying the nucleic acid scaffolded structure to which the binding oligonucleotide is coupled). For example, FIG. 17A shows nucleic acid scaffolded structures coupled to a plurality of barcoded oligonucleotides that can be generated by any of the barcoding methods described elsewhere herein. The barcoded oligonucleotides can comprise one or more probes (e.g., 17P1-17P6) configured to bind to an analyte, and/or one or more binding oligonucleotides (e.g., 17B1-17B4) configured to bind to another binding oligonucleotide. The probes and binding oligonucleotides can each comprise a position barcode (e.g., 1700A-1700J) and a UMI that identifies the corresponding nucleic acid scaffolded structure to which the probe/binding oligonucleotide is coupled. The probes can each comprise a sensing sequence (e.g., 17SA, 17SB, 17SC, 17SF, 17SG, or 17SH) configured to bind to an analyte in a sample. The binding oligonucleotides can comprise a binding sequence (e.g., 17SE or 17SI) configured to hybridize to an additional binding oligonucleotide that is coupled to an additional nucleic acid scaffolded structure.

[0059] The one or more binding oligonucleotides can further comprise one or more additional functional sequences (e.g., staple segment for anchoring a probe to the nucleic acid scaffolded structure, a hybridizing sequence for a primer for use in generating the nucleic acid scaffolded structure or a sequence for use in generating a sequencing library). FIG. 17A and 17B show an example of two nucleic acid scaffolded structures 1701 and 1702 coupled together via binding oligonucleotides 17B2 and 17B3. FIG. 17B shows that binding oligonucleotide 17B2 is coupled to nucleic acid scaffolded structure 1701 via a bottom staple section 1711 that anchors the binding oligonucleotide 17B2 to the nucleic acid scaffolded structure 1701. Binding oligonucleotide 17B3 is coupled to nucleic acid scaffolded structure 1702 via a bottom staple section 1721 that anchors the binding oligonucleotide 17B3 to the nucleic acid scaffolded structure 1702. Binding oligonucleotide 17B2 further comprises binding sequence 17SE that hybridizes to binding sequence 17SI of the binding oligonucleotide 17B3, thereby coupling nucleic acid scaffolded structures 1701 and 1702 together. Binding oligonucleotide 17B2 further comprises a priming sequence 1712 for next-generation sequencing (NGS) library preparation via PCR, a position barcode 1700E encoding the binding oligonucleotide’s position on the nucleic acid scaffolded structure 1701, and a nucleic acid scaffolded structure-specific unique molecular identifier UMI l . Binding oligonucleotide 17B2 also comprises a hybridization segment 1714 used to add the UMI l to the binding oligonucleotide in a barcoding reaction, such as, for example, described in Example 3. Binding oligonucleotide 17B3 further comprises a priming sequence 1722 for nextgeneration sequencing (NGS) library preparation via PCR, a position barcode 17001 encoding the binding oligonucleotide’s position on the nucleic acid scaffolded structure 1702, and a nucleic acid scaffolded structure-specific unique molecular identifier UMI 2. Binding oligonucleotide 1720 also comprises a hybridization segment 1724 used to add the UMI 2 to the binding oligonucleotide in a barcoding reaction.

[0060] The one or more nucleic acid scaffolded structures can be coupled to a surface (e.g., a bead surface or a gel surface). A nucleic acid scaffolded structure can be covalently or noncovalently coupled to a surface. In some cases, the nucleic acid scaffolded structure is coupled to the surface via a primer. For example, in FIG. 15A, nucleic acid scaffolded structure 1510 is coupled to surface 1560 via hybridization of surface oligonucleotides 1540 and 1550 to respective oligonucleotides 1520 and 1530 coupled to the nucleic acid scaffolded structure. In some cases, nucleic acid scaffolded structures of the plurality of nucleic acid scaffolded structures are aligned on the surface based on a pattern on the surface.

B. Methods

Methods of analyte analysis using a nucleic acid scaffolded structure

[0061] In one aspect, the present disclosure provides a method for analyzing an analyte. The method can comprise: providing: (i) a sample comprising an analyte, and (ii) a nucleic acid scaffolded structure (e.g., a DNA origami structure). In some cases, the nucleic acid scaffolded structure is coupled to a probe. The probe can comprise a sensing moiety configured to bind to the analyte. The probe can further comprise a barcode sequence. In some cases, the barcode sequence corresponds to a spatial position of the probe on the nucleic acid scaffolded structure (e.g., a position identifier). For example, the spatial position can be a position on the nucleic acid scaffolded structure at which the probe is coupled to the nucleic acid scaffolded structure. In some cases, the barcode sequence can be a unique identifier (e.g., a UMI) that identifies the nucleic acid scaffolded structure. In some cases, the probe comprises a spatial position barcode sequence and an additional barcode sequence, for example, a unique identifier that identifies the nucleic acid scaffolded structure.

[0062] In some cases, the nucleic acid scaffolded structure is coupled to a plurality of probes. The plurality of probes can comprise probes configured to bind to different analytes in the sample. In some cases, a probe of the plurality of probes is located from 0.5 to 10 nm, 0.5 to 20 nm, from 0.5 to 40 nm, from 0.5 to 60 mm, from 0.5 to 100 mm, or from 0.5 to 150 mm away from an additional probe of the plurality of probes. In some embodiments, a probe of the plurality of probes is located at least 0.5 nm, 1 nm, 1.5 nm, 2 nm, 2.5 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, 22 nm, 24 nm, 26 nm, 28 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, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 250 nm, or 300 nm away from an additional probe of the plurality of probes. The probe can be from 0.5 to 50 nm, from 1 to 50 nm, from 1.5 to 50 nm, from 2 to 50 nm, from 2.5 to 50 nm, from 3 to 50 nm, from 4 to 50 nm, from 5 to 50 nm, from 6 to 50 nm, from 7 to 50 nm, from 8 to 50 nm, from 9 to 50 nm, from 10 to 50 nm, from 11 to 50 nm, from 12 to 50 nm, from 13 to 50 nm, from 14 to 50 nm, from 15 to 50 nm, from 16 to 50 nm, from 17 to 50 nm, from 18 to 50 nm, from 19 to 50 nm, from 20 to 50 nm, from 22 to 50 nm, from 24 to 50 nm, from 26 to 50 nm, from 28 to 50 nm, or from 30 to 50 nm from an additional probe of the plurality of probes.

[0063] The nucleic acid scaffolded structure can comprise a DNA origami structure. In some cases, the nucleic scaffolded structure comprise a crisscross assembly. In some cases, the nucleic acid scaffolded structure is further decorated with probes in a particular configuration or pattern. The nucleic acid scaffolded structure can comprise a dimension of from 0.5 to 1 mm, from 0.5 to 2 mm, from 0.5 to 5 mm, or from 0.5 to 10 mm. The nucleic acid scaffolded structure can be coupled to a surface, for example, a bead. In some cases, the method further comprises, prior to (a), coupling the nucleic acid scaffolded structure to the surface. In some cases, the coupling comprises orienting the nucleic acid scaffolded structure on the surface based on a pattern on the surface. The method can further comprise, prior to (a), generating the pattern using electron beam lithography or atomic force lithography. In some cases, the surface is coupled to a plurality of nucleic acid scaffolded structures. In some cases, the nucleic acid scaffolded structure is coupled to another nucleic acid scaffolded structure via one or more binding oligonucleotides or other binding moieties. The method can further comprise coupling the nucleic acid scaffolded structure to an additional nucleic acid scaffolded structure. In some cases, the method comprises hybridizing a binding oligonucleotide of one nucleic acid scaffolded structure to a binding oligonucleotide of the additional nucleic acid scaffolded structure. FIG. 17B shows an example where two nucleic acid scaffolded structures 1701 and 1702 are coupled together via hybridization of the binding oligonucleotides 1710 and 1720. In other cases, the method comprises ligating an oligonucleotide of one nucleic acid scaffolded structure to an oligonucleotide of the additional nucleic acid scaffolded structure. The ligation can be performed, for example, via chemical ligation or a ligase. In some cases, the ligation is performed using one or more splint oligonucleotides. In some cases, the coupling occurs prior to contacting the sample with the nucleic acid scaffolded structure. In other cases, the coupling occurs during or after contacting the sample with the nucleic acid scaffolded structure.

[0064] In some cases, the method further comprises, prior to (a), coupling the probe to the nucleic acid scaffolded structure. Coupling the probe to the nucleic acid scaffolded structure can comprise hybridizing a portion of the probe to a binding segment of the nucleic acid scaffolded structure. In some cases, the binding segment of the nucleic acid scaffolded structure comprises a distinct sequence corresponding to a distinct spatial position of the binding segment in the nucleic acid scaffolded structure. In some cases, the nucleic acid scaffolded structure further comprises a plurality of binding segments, wherein each binding segment of the plurality of binding segments comprises a distinct sequence corresponding to a distinct spatial position of the binding segment in the nucleic acid scaffolded structure.

[0065] In some cases, the sample comprises a tissue or a cell. The cell can be a prokaryotic or eukaryotic cell. The cell can be a bacterial, plant, animal, or fungal cell. In some cases, the analyte is provided in the cell or on the cell surface. The analyte can be an extracellular or intracellular analyte. The cell can be a lysed or permeabilized cell. The cell can be a fixed cell. In some cases, the method further comprises lysing or permeabilizing the cell during any operation disclosed herein. In other cases, the sample can comprise a viral nucleic acid sample. In some cases, the biological sample comprises a tissue section.

[0066] The analyte can comprise a protein, peptide, nucleic acid, lipid, carbohydrate, or a combination thereof. The analyte can be an intracellular or an extracellular analyte. In some cases, the analyte is localized in a specific organelle. The analyte can be located in a mitochondria, cell nucleus, cytoplasm, or ribosome. In some cases, the analyte is a cell membrane analyte. In some cases, the analyte comprises a nucleic acid (e.g., DNA or RNA). The nucleic acid can comprise a target sequence. In some cases, the nucleic acid can comprise an RNA transcript. In some cases, the RNA transcript can comprise a polyA sequence. In other cases, the nucleic acid comprises genomic DNA. The nucleic acid can comprise an adapter sequence. In some cases, the analyte comprises a transposase- fragmented nucleic acid product comprising adapter sequences appended to a genomic DNA fragment. The nucleic acid can comprise single-stranded nucleic acid or doublestranded nucleic acid. In some cases, the nucleic acid analyte comprises a stem-loop structure. In some cases, the analyte comprises a protein or a peptide. For example, the analyte can be a cell surface protein receptor or an intracellular protein. The protein or peptide can directly interact with the probe or can be coupled to a nucleic acid. In further cases, the analyte comprises a small molecule. The small molecule can directly interact with the probe or can be coupled to a nucleic acid. The analyte can be capable of binding to the sensing moiety of the probe, as described elsewhere herein

[0067] The method can further comprise (b) contacting the sample with the nucleic acid scaffolded structure, wherein the sensing moiety of the probe on the nucleic acid scaffolded structure binds to the analyte in the sample, thereby coupling the probe and its barcode sequence to the analyte. In some cases, the sensing moiety comprise comprises a nucleic acid sensing sequence that hybridizes to a nucleic acid of the analyte. The nucleic acid sensing sequence can, for example, hybridize to an adapter sequence of the analyte. In some cases, the sample comprises a plurality of analytes, and wherein, in (b), probes of the plurality of probes on the nucleic acid scaffolded structure couple to the plurality of analytes. In some cases, the method comprises, during or after (b), identifying a location of the nucleic acid scaffolded structure in the sample, for example by imaging using microscopy. The location can be a location at which the nucleic acid scaffolded structure interacts with the sample. In some cases, the analyte is located on a cell surface and the method further comprises contacting the cell surface with the nucleic acid scaffolded structure. In other cases, the analyte is located inside a cell, and the method further comprises introducing the nucleic acid scaffolded structure into the cell. The method can further comprise permeabilizing the cell. In some cases, the cell is permeabilized prior to contacting the sample with the nucleic acid scaffolded structure. In other cases, the cell is permeabilized during or after contacting the sample with the nucleic acid scaffolded structure. In one example, the nucleic acid scaffolded structure can diffuse inside the cell to detect one or more analytes inside the cell. Alternatively, analytes of a cell can diffuse outside the cell to contact the nucleic acid scaffolded structure. In some cases, the method further comprises fixing the cell.

[0068] In some cases, the method further comprises generating the analyte provided in (a). For example, the analyte can be generated by adding an adapter to a cell component (e.g., genomic DNA). The adapter can be, for example, a nucleic acid adapter, which can be coupled to the analyte via hybridization or a ligation reaction. In some cases, the nucleic acid adapter sequence is generated via a nucleic acid extension reaction using a template. In some cases, the adapter is appended on to an analyte via a transposition reaction, for example, by a transposase. A transposase-mediated fragmentation reaction can be performed on nucleic acid, for example, genomic DNA, to generate the analyte. In some cases, the sensing moiety of probe couples to the adapter in the analyte. An example is shown in FIG. 6B, where sensing sequence 0686 of the probe 0680 hybridizes to adapter sequence 0686RC of the analyte.

[0069] The method can further comprise performing a nucleic acid extension reaction and/or a ligation reaction on the probe coupled to the analyte. For example, as shown in FIG. 6A, the probe is extended using the analyte 0640 as a template to generate an extension product comprising the probe and a sequence 0651 that is complementary to and corresponds with a sequence of the analyte (0641). The extension reaction can be performed, for example, using a DNA polymerase or reverse transcriptase. In another example, the method can comprise ligating the probe to the analyte, for example, via a ligase or chemical ligation. In some cases, the ligation reaction comprises splint ligation. In another example, the method can comprise performing a nucleic acid extension reaction and a ligation reaction on the probe coupled to the analyte. FIG. 6B shows a probe that hybridizes to an adapter sequence 0686RC of an analyte. In this example, both a ligation reaction and an extension reaction are performed to generate a product comprising the probe and the analyte sequence 0691. The analyte can comprise a target sequence, the method can further comprise generating a barcoded nucleic acid strand comprising (i) the barcode sequence of the probe (e.g., position barcode identifying the spatial position) or a complement thereof, and (ii) the target sequence or complement thereof. In some cases, the probe further comprises (III) an additional barcode sequence that identifies the nucleic acid scaffolded structure, and the barcoded nucleic acid strand further comprises (iii) the additional barcode sequence or a complement thereof.

[0070] The method can further comprise (c) identifying the barcode sequence. In some cases, (c) comprises detecting the barcode sequence using sequencing. In some cases, comprises detecting the barcode sequence using in situ sequencing. For example, identifying a position barcode sequence can identify the spatial position of the probe on the nucleic acid scaffolded structure. Identifying a barcode sequence that is a nucleic acid scaffolded structure identifier can identify the nucleic acid scaffolded structure.

[0071] The method can further comprise (d) identifying the analyte in the sample coupled to the probe. In some cases, the method further comprises, prior to (d) amplifying the target sequence using the probe in a nucleic acid amplification reaction to yield an amplification product. In some cases, identifying the analyte further comprises detecting the amplification product or a derivative thereof. For example, identifying the analyte can comprise sequencing the amplification product or a derivative thereof, thereby obtaining sequencing reads. Identifying the analyte can identify information about the analyte, for example, structure information, functional information, or information regarding a level of expression of the analyte in a cell.

[0072] In some cases, identifying the barcode sequence and identifying the analyte occur simultaneously. In other cases, identifying the barcode sequence and identifying the analyte occur sequentially. For example, identifying the barcode sequence can occur prior to identifying the analyte. Alternatively, identifying the analyte can occur prior to identifying the barcode sequence.

[0073] The method can further comprise (e) associating the spatial position of the probe on the nucleic acid scaffolded structure with the analyte in the sample. In some cases, (e) further comprises associating the sequencing reads with the spatial position of the probe in the nucleic acid scaffolded structure.

[0074] The method can further comprise identifying the analyte as associated with the nucleic acid scaffolded structure, for example, via the additional barcode sequence in the probe that is unique to the nucleic acid scaffolded structure. The method can comprise associating the sequencing reads with the corresponding nucleic acid scaffolded structure in addition to associating the sequencing reads with the spatial position of the probe in the nucleic acid scaffolded structure.

[0075] In some cases, where a nucleic acid scaffolded structure is coupled to an additional nucleic acid scaffolded structure, the method can comprise sharing information between the two nucleic acid scaffolded structures or detecting an interaction between two nucleic acid scaffolded structures. For example, the method can comprise hybridizing a first oligonucleotide of one nucleic acid scaffolded structure to a second oligonucleotide of the additional nucleic acid scaffolded structure and performing a nucleic acid extension reaction to copy information from one oligonucleotide to the other. The nucleic acid extension reaction can generate an extension product comprising a sequence associated with the first oligonucleotide and a sequence associated with the second oligonucleotide. In some cases, the sequence associated with first oligonucleotide is a barcode sequence that identifies the nucleic acid scaffolded structure, while the sequence associated with the second oligonucleotide is a barcode sequence that identifies the additional nucleic acid scaffolded structure. For example, in FIGs. 17A and 17B, binding oligonucleotide 17B2 hybridizes to 17B3, thereby coupling nucleic acid scaffolded structures 1701 and 1702 together. A nucleic acid extension reaction is performed to generate extension products 1730 and 1740. The extension products 1730 and 1740 comprise (i) a sequence associated with the first oligonucleotide or with the nucleic acid scaffolded structure 1701 to which it is coupled (e.g., UMI l or the reverse complement thereof UMI l RC) and (ii) a sequence associated with the second oligonucleotide or with the nucleic acid scaffolded structure 1702 to which it is coupled (e.g., UMI 2 or the reverse complement thereof UMI 2 RC). The extension products can further comprise sequences associated with the position barcodes (e.g., 1700E and 17001) that encode the spatial position of the respective binding oligonucleotide on its associated nucleic acid scaffolded. The position barcodes can also provide directional information regarding the interaction between the nucleic acid scaffolded structures. As another example, the method can comprise ligating the first oligonucleotide of one nucleic acid scaffolded structure to the second oligonucleotide of the additional nucleic acid scaffolded structure to generate a ligation product comprising the sequence associated with the first oligonucleotide and the sequence associated with the second oligonucleotide. The method can further comprise sequencing the extension or ligation product. The identifying information (e.g., sequences associated with the UMI barcodes of the respective scaffolded structures) in the resulting sequencing reads can identify the nucleic acid scaffolded structure as being directly coupled to the additional nucleic acid scaffolded structure. The identifying information (e.g., sequences associated with the position barcodes of the coupled binding oligonucleotides) in the resulting sequencing reads can further identify relative positioning of the nucleic acid scaffolded structures and provide directional information regarding the specific interaction between the structures. The detected information can aid in mapping out the positional network of different nucleic acid scaffolded structures.

[0076] The method can further comprise constructing an image or a spatial map using the information identified about the analyte in (d), and the spatial position of the probe on the nucleic acid scaffolded structure identified in (c). The spatial map can be a 2D or 3D spatial map. In some cases, the probes provide x and y dimension coordinates. In some cases, the probes provide x, y, and z dimension coordinates. For example, a 3D nucleic acid scaffolded structure can comprise probes providing x, y, and z dimension coordinates for generating a 3D spatial map of a cell. In some cases, spatial maps are generated from different cells or tissue sections derived from different parts of a tissue to construct a larger spatial map of the tissue. In one example, multiple 2D spatial maps of different tissue layers are stacked to construct a 3D spatial map.

[0077] In some cases, constructing the spatial map further comprises using barcode sequence identifying the nucleic acid scaffolded structure. Constructing the spatial map can further comprise using the sequencing reads that identify the coupling of the nucleic acid scaffolded structure to an additional nucleic acid scaffolded structure. The sequencing reads can identify the nucleic acid scaffolded structure as being adjacent to the additional nucleic acid scaffolded structure. In some cases, constructing the spatial map further comprises using information about the location of the nucleic acid scaffolded structure in the sample. In some cases, a single probe provides information for a single pixel in the image or spatial map. The image or spatial map can comprise multiple pixels, each originating from a single probe. The resolution of the image or spatial map can be linked to the proximity of one probe providing analyte information from another probe providing analyte information on the nucleic acid scaffolded structure. For example, the resolution of the image or spatial map can be at least 0.5 nm, 1 nm, 1.5 nm, 2 nm, 2.5 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, 22 nm, 24 nm, 26 nm, 28 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, 100 nm, 120 nm, 140 nm, 160 nm, 180 nm, 200 nm, 250 nm, or 300 nm. The resolution of the image or spatial map can be from 0.5 to 50 nm, from 1 to 50 nm, from 1.5 to 50 nm, from 2 to 50 nm, from 2.5 to 50 nm, from 3 to 50 nm, from 4 to 50 nm, from 5 to 50 nm, from 6 to 50 nm, from 7 to 50 nm, from 8 to 50 nm, from 9 to 50 nm, from 10 to 50 nm, from 11 to 50 nm, from 12 to 50 nm, from 13 to 50 nm, from 14 to 50 nm, from 15 to 50 nm, from 16 to 50 nm, from 17 to 50 nm, from 18 to 50 nm, from 19 to 50 nm, from 20 to 50 nm, from 22 to 50 nm, from 24 to 50 nm, from 26 to 50 nm, from 28 to 50 nm, or from 30 to 50 nm, 0.5 to 300 nm, from 1 to 300 nm, from 1.5 to 300 nm, from 2 to 300 nm, from 2.5 to 300 nm, from 3 to 300 nm, from 4 to 300 nm, from 5 to 300 nm, from 6 to 300 nm, from 7 to 300 nm, from 8 to 300 nm, from 9 to 300 nm, from 10 to 300 nm, from 11 to 300 nm, from 12 to 300 nm, from 13 to 300 nm, from 14 to 300 nm, from 15 to 300 nm, from 16 to 300 nm, from 17 to 300 nm, from 18 to 300 nm, from 19 to 300 nm, from 20 to 300 nm, from 22 to 300 nm, from 24 to 300 nm, from 26 to 300 nm, from 28 to 300 nm, from 30 to 300 nm, from 35 to 300 nm, from 40 to 300 nm, from 45 to 300 nm, from 50 to 300 nm, from 55 to 300 nm, or from 60 nm to 300.

[0078] In some cases, a plurality of nucleic acid scaffolded structures are provided, wherein each nucleic acid scaffolded structure is coupled to a probe. Each probe can comprise a barcode sequence corresponding to a spatial position of the probe on the nucleic acid scaffolded structure to which the probe is coupled. Each probe can further comprise a barcode sequence corresponding to the nucleic acid scaffolded structure to which the probe is coupled. In some cases, each probe comprises a sensing moiety configured to bind to an analyte. The method can further comprise contacting the sample with the plurality of nucleic acid scaffolded structures and binding probes coupled to the nucleic acid scaffolded structure to analytes in the sample. The method can further comprise identifying a location of each nucleic acid scaffolded structure of the plurality of nucleic acid scaffolded structures, for example, by imaging using microscopy. The method can further comprise identifying the barcode sequences in the probes and using the probes coupled to the nucleic acid scaffolded structures to identify analytes in the sample, as described herein.

[0079] FIG. 1 and FIG. 2 show a flowchart and a schematic, respectively, for a method described herein. A cell 0260 comprising an analyte 0140 can be provided. The analyte 0140 can comprise nucleic acid, for example an RNA transcript. In some cases, the analyte comprises a target sequence 0142 (e.g., a polyA sequence). A nucleic acid scaffolded structure (e.g., a DNA origami) 0210 can also be provided. The nucleic acid scaffolded structure 0210 can comprise a probe 0220 comprising a barcode sequence 0221 corresponding to a spatial position of the probe in the nucleic acid scaffolded structure 0210 (FIG. 2). The probe 0220 can further comprise a target-hybridizing sequence 0222 configured to hybridize to the target sequence 0242 in the analyte 0240.

[0080] The method can further comprise contacting the cell 0260 with the nucleic acid scaffolded structure 0210, wherein the probe 0220 in the nucleic acid scaffolded structure couples to the analyte 0240 in the cell. In some cases, the target-hybridizing sequence 0222 of the probe 0220 hybridizes to the target sequence 0242 in the analyte 0240.

[0081] The method can further comprise detecting the barcode sequence 0221 or reverse complement thereof, thereby identifying the spatial position of the probe in the nucleic acid scaffolded structure. In some cases, the barcode sequence 0221 is detected in situ, for example, via imaging with probes or in situ sequencing. In some cases, the barcode sequence is detected directly in the probe. In other cases, the barcode sequence or reverse complement thereof is detected in a derivative of the probe. For example, the barcode sequence or reverse complement thereof can be detected in a nucleic acid extension product. As another example, the barcode sequence or reverse complement thereof can be further amplified in a nucleic acid amplification reaction and detected in an amplification product of the probe.

[0082] The method can further comprise identifying the analyte 0240 in the cell using the probe in the nucleic acid scaffolded structure.

[0083] In some cases, detection of the barcode sequence 0221 and identification of the analyte 0240 occur simultaneously. In other cases, detection of the barcode sequence 0221 and identification of the analyte 0240 occur sequentially. For example, the barcode sequence 0221 can be detected prior to identifying the analyte 0240. Alternatively, the barcode sequence 0221 can be detected after identifying the analyte 0240. In some cases, the barcode sequence 0221 is detected in situ and the analyte 0240 is identified by sequencing the probe or a derivative thereof in a separate flowcell.

[0084] The method can further comprise associating the spatial position of the probe 0220 in the nucleic acid scaffolded structure with the analyte 0240 in the cell.

[0085] FIG. 6A schematically shows a method for identifying an analyte using a probe, as described herein. An analyte 0640 can be provided. In some cases, the analyte is provided in a cell. The analyte 0040 can comprise nucleic acid, for example an RNA transcript. In some cases, the analyte comprises a first target sequence 0641 and a target sequence 0642 (e.g., a polyA sequence). A nucleic acid scaffolded structure (e.g., a DNA origami) 0610 can be coupled to the target nucleic acid 0640. The nucleic acid scaffolded structure 0610 can comprise a probe 0620 comprising a target-hybridizing sequence 0622 hybridized to the second target sequence 0642 in the target nucleic acid 0640. The probe 0620 can further comprise a barcode sequence 0621 corresponding to a spatial position of the probe in the nucleic acid scaffolded structure 0610.

[0086] The method can comprise performing a nucleic acid extension reaction using the probe 0620 and using the target nucleic acid 0640 as a template to generate a nucleic acid extension product 0650, comprising the barcode sequence, the target-hybridizing sequence 0622, and the cDNA sequence 0651 of the first target sequence 0641. The method can further comprise sequencing the nucleic acid extension product 0650 or a derivative thereof.

[0087] In another aspect, the present disclosure provides a method for coupling nucleic acid scaffolded structures to a surface (e.g., a solid surface). The method can comprise (a) providing a surface, a first nucleic acid scaffolded structure coupled to a first probe, and a second nucleic acid scaffolded structure coupled to a second probe. The first probe can comprise a first sensing moiety configured to bind to a first analyte in a sample. In some cases, the first probe comprises a first barcode sequence corresponding to a first spatial position of the first probe on the first nucleic acid scaffolded structure. The second probe can comprise a second sensing moiety configured to bind to a second analyte in a sample. In some cases, the second probe comprises a second barcode sequence corresponding to a second spatial position of the second probe on the second nucleic acid scaffolded structure.

[0088] The method can further comprise (b) coupling the first nucleic acid scaffolded structure to the surface, and coupling the second nucleic acid scaffolded structure to the surface.

[0089] In some cases, the method further comprises orienting the first nucleic acid scaffolded structure and the second nucleic acid scaffolded structure on the surface based on a pattern on the surface. The orienting can occur prior, after, or during (b). In some cases, the method further comprises, prior to (a), generating the pattern using electron beam lithography or atomic force lithography.

[0090] In some cases, the first nucleic acid scaffolded structure comprises a handle segment, and (b) comprises hybridizing the handle segment to a binding sequence on the surface. In some cases, the method comprises directly coupling the first nucleic acid scaffolded structure to the second nucleic acid structure.

[0091] FIG. 7 and FIG. 8 show a flowchart and a schematic, respectively, for a method of coupling a plurality of nucleic acid scaffolded structures to a surface, as described herein. A surface (e.g., a bead) 0890 and two nucleic acid scaffolded structures 810 and 830 can be provided.

[0092] In some cases, a first capture oligonucleotide 0860 and a second capture oligonucleotide 0880 are attached to the surface 0890.

[0093] The first nucleic acid scaffolded structure 0810 can comprise a first binding oligonucleotide 0850 and a first probe 0820 configured to couple to a first analyte in a cell. For example, the first probe 0820 can comprise a target-hybridizing sequence 0822 configured to hybridize to a target sequence in the first analyte. The first probe can further comprise a first barcode sequence 0821 corresponding to a first spatial position of the first probe in the first nucleic acid scaffolded structure 0810.

[0094] The second nucleic acid scaffolded structure 0830 can comprise a second binding oligonucleotide 0870 and a second probe 0840 configured to couple to a second analyte in a cell. For example, the second probe 0840 can comprise a target-hybridizing sequence 0842 configured to hybridize to a target sequence in the second analyte. The second probe can further comprise a second barcode sequence 0841 corresponding to a second spatial position of the second probe in the second nucleic acid scaffolded structure 0830.

[0095] The method can further comprise coupling the first binding oligonucleotide 0850 of the first nucleic acid scaffolded structure 0810 to the first capture oligonucleotide 0860 attached to the surface. The method can further comprise coupling the second binding oligonucleotide 0870 of the second nucleic acid scaffolded structure 0830 to the second capture oligonucleotide 0880 attached to the surface.

[0096] In some cases, following coupling of the first and second nucleic acid scaffolded structures to the surface, the first probe 0820 of the first nucleic acid scaffolded structure or a derivative thereof is used to identify a first analyte in a cell, as described elsewhere herein. In some cases, the second probe 0840 of the second nucleic acid scaffolded structure or a derivative thereof is used to identify a second analyte in the cell as described elsewhere herein. The method can further comprise detecting the first barcode sequence 0821 and/or the second barcode sequence 0841.

[0097] In another aspect, the present disclosure provides a method for copying information from probes from a nucleic acid scaffolded structure. The method can comprise (a) providing a surface (e.g., a gel) coupled to a first nucleic acid primer and a second nucleic acid primer; and a nucleic acid scaffolded structure. The nucleic acid scaffolded structure can be coupled to a first probe and a second probe. The first probe can comprise a first sensing sequence, wherein the first sensing sequence or a reverse complement thereof is configured to bind to a first analyte in a sample. In some cases, the first probe comprises a first barcode sequence corresponding to a first spatial position of the first probe on the nucleic acid scaffolded structure. The second probe can comprise a second sensing sequence, wherein the second sensing sequence or a reverse complement thereof is configured to bind to a second analyte in a sample. In some cases, the second probe comprises a second barcode sequence corresponding to a second spatial position of the second probe on the nucleic acid scaffolded structure. [0098] The method can further comprise (b) contacting the first nucleic acid primer coupled to the surface and the second nucleic acid primer coupled to the surface with the nucleic acid scaffolded structure.

[0099] The method can further comprise (c) generating a first nucleic acid extension product using the first nucleic acid primer coupled to the surface and the first probe coupled to the nucleic acid scaffolded structure, wherein the first nucleic acid extension product is coupled to the surface. The first nucleic acid extension product can comprise (i) the first sensing sequence or reverse complement thereof, and (ii) the first barcode sequence or reverse complement thereof.

[0100] The method can further comprise (d) generating a second nucleic acid extension product using the second nucleic acid primer coupled to the surface and the second probe coupled to the nucleic acid scaffolded structure, wherein the second nucleic acid extension product is coupled to the surface. The second nucleic acid extension product can comprise (i) the second sensing sequence or reverse complement thereof, and (ii) the second barcode sequence or reverse complement thereof.

[0101] In some cases, an additional nucleic acid scaffolded structure comprising a third probe is provided. The third probe can comprise a third sensing sequence, wherein the third sensing sequence or a reverse complement thereof is configured to bind to a third analyte in a sample. The third probe can further comprise a third barcode sequence corresponding to a third spatial position of the third probe on the additional nucleic acid scaffolded structure. The method can further comprise contacting a third nucleic acid primer coupled to the surface with the additional nucleic acid scaffolded structure. The method can further comprise generating a third nucleic acid extension product using the third nucleic acid primer coupled to the surface and the third probe in the nucleic acid scaffolded structure. The third nucleic acid extension product can comprise the third sensing sequence or reverse complement thereof and the third barcode sequence or reverse complement thereof.

[0102] The method can further comprise, after generating the first nucleic acid extension product coupled to the surface and generating the second nucleic acid extension product coupled to the surface, (e) contacting the surface with a sample comprising the first analyte and the second analyte. In some cases, the first sensing sequence or reverse complement thereof in the first nucleic acid extension product couples to the first analyte in the sample; and wherein the second sensing sequence or reverse complement thereof in the second nucleic acid extension product couples to the second analyte in the sample. [0103] The method can further comprise, after (e), detecting the first barcode sequence or reverse complement thereof in the biological sample and detecting the second barcode sequence or reverse complement thereof in the sample.

[0104] The method can further comprise, after (e), identifying the first analyte in the sample using the first nucleic acid extension product coupled to the surface and identifying the second analyte in the sample using the second nucleic acid extension product coupled to the surface.

[0105] The method can further comprise associating the first barcode sequence with the first analyte and associating the second barcode sequence with the second analyte.

[0106] Examples of replicating microarrays of nucleic acid templates are provided in Xiaonan Fu, Li Sun, Runze Dong, Jane Y. Chen, Runglawan Silakit, Logan F. Condon, Yiing Lin, Shin Lin, Richard D. Palmiter, and Liangcai Gu. Cell, 24, Nov 23, 2022 and International Patent Publication No. WO2024015766A1, which are incorporated by reference herein in their entireties.

[0107] FIG. 14 and FIG. 15A-15C show a flowchart and a schematic, respectively, for a method of copying information from probes from a nucleic acid scaffolded structure, as described herein. A surface 1560 (e.g., a gel) and a nucleic acid scaffolded structure 1510 (e.g., a DNA origami) can be provided. In some cases, the surface 1560 is coupled to a first nucleic acid primer 1540 and a second nucleic acid primer 1550.

[0108] The nucleic acid scaffolded structure 1510 can comprise a first probe 1520 and a second probe 1530. The first probe 1520 can comprise a first binding sequence 1522, wherein a reverse complement of the first binding sequence is configured to couple to a first analyte in a cell. The first probe 1520 can further comprise a first barcode sequence 1521 corresponding to a first spatial position of the first probe in the nucleic acid scaffolded structure. The second probe 1530 can comprise a second binding sequence 1532, wherein a reverse complement of the second binding sequence is configured to couple to a second analyte in a cell. The second probe 1530 can further comprise a second barcode sequence 1531 corresponding to a second spatial position of the second probe in the nucleic acid scaffolded structure.

[0109] The method can comprise contacting the first nucleic acid primer 1540 coupled to the surface and the second nucleic acid primer 1550 coupled to the surface 1560 with the nucleic acid scaffolded structure 1510. The first nucleic acid primer 1540 can hybridize to a portion of the first probe 1520, as shown in FIG. 15A. The second nucleic acid primer 1550 can hybridize to a portion of the second probe 1530. [0110] The method can further comprise generating a first nucleic acid extension product 1570 using the first nucleic acid primer 1540 coupled to the surface and the first probe 1520 in the nucleic acid scaffolded structure, wherein the first nucleic acid extension product is coupled to the surface 1560, as shown in FIG. 15B. The first nucleic acid extension product 1570 can comprise the reverse complement 1522c of the first binding sequence 1522 and the reverse complement 1521c of the first barcode sequence 1521. The sequence 1521c can be associated with a spatial position of the first nucleic acid extension product 1570 in the surface 1560.

[oni] The method can further comprise generating a second nucleic acid extension product 1580 using the second nucleic acid primer 1550 coupled to the surface and the second probe 1530 in the nucleic acid scaffolded structure, wherein the second nucleic acid extension product is coupled to the surface 1560. The second nucleic acid extension product 1580 can comprise the reverse complement 1532c of the second binding sequence 1532 and the reverse complement 1531c of the second barcode sequence 1531. The sequence 1531c can be associated with a spatial position of the second nucleic acid extension product 1580 in the surface 1560.

[0112] The method can further comprise amplifying or processing the first nucleic acid extension product and/or the second nucleic acid extension product to yield a derivative of the first nucleic acid extension product and/or a derivative of the second nucleic acid extension product. In some cases, a derivative of the first nucleic acid extension product or a derivative of the second nucleic acid extension product is coupled to the surface.

[0113] The method can further comprise contacting the surface coupled to the first nucleic acid extension product or a derivative thereof and the second nucleic acid extension product or a derivative thereof with a cell.

[0114] In some cases, the first nucleic acid extension product 1570 or derivative thereof coupled to the surface is then used to identify a first analyte in the cell. For example, the sequence 1522c in the first nucleic acid extension product can hybridize to a first target sequence 1591 in the first analyte 1590 in the cell, as shown in FIG. 15C. In some cases, hybridization of the first nucleic acid extension product or derivative thereof to the first analyte identifies the first analyte. In some cases, a third nucleic acid extension product is generated using the first nucleic acid extension product 1570 and the first analyte 1590, and the third nucleic acid extension product or a derivative thereof is sequenced to identify the first analyte. [0115] In some cases, the second nucleic acid extension product 1580 or derivative thereof coupled to the surface is then used to identify a second analyte in the cell. For example, the sequence 1532c in the second nucleic acid extension product can hybridize to a second target sequence 1593 in the second analyte 1592 in the cell, as shown in FIG. 15C. In some cases, hybridization of the second nucleic acid extension product or derivative thereof to the second analyte identifies the second analyte. In some cases, a fourth nucleic acid extension product is generated using the second nucleic acid extension product 1580 and the second analyte 1592, and the fourth nucleic acid extension product or a derivative thereof is sequenced to identify the second analyte.

[0116] The method can further comprise detecting the first barcode sequence or reverse complement thereof and the second barcode sequence or reverse complement thereof. For example, the method can comprise detecting sequences 1521c and 1531c in situ. Detecting sequences 1521c can identify the first spatial position of the first probe 1520 in the nucleic acid scaffolded structure and be associated with a spatial position of the first nucleic acid extension product 1570 in the surface 1560. Detecting sequences 1531c can identify the second spatial position of the second probe 1530 in the nucleic acid scaffolded structure and be associated with a spatial position of the second nucleic acid extension product 1580 in the surface 1560.

[0117] In some cases, the barcode sequences or reverse complements thereof are detected prior to identifying the analytes. In other cases, the barcode sequences or reverse complements thereof are detected after identifying the analytes. In further cases, detection of the barcode sequences or reverse complements thereof and identification of the analytes occur simultaneously. Using the first barcode sequence or reverse complement thereof, the method can further comprise associating the spatial position of the first nucleic acid extension product or derivative thereof in the surface with the first analyte in the cell. Using the second barcode sequence or reverse complement thereof, the method can further comprise associating the spatial position of the second nucleic acid extension product or derivative thereof in the surface with the second analyte in the cell.

[0118] FIG. 16 schematically shows a method for copying information from probes from a plurality of nucleic acid scaffolded structures onto a surface, as described herein.

Methods of barcoding a nucleic acid scaffolded structure

[0119] Described herein are also methods of adding barcodes to a nucleic acid scaffolded structure and methods of generating barcoded nucleic acid scaffolded structures described elsewhere herein. In one aspect, described herein is a method of barcoding a nucleic acid scaffolded structure, the method comprising: (a) providing: (i) a nucleic acid scaffolded structure, wherein the nucleic acid scaffolded structure comprises a first oligonucleotide and a second oligonucleotide; and (ii) a barcoded nucleic acid template comprising a plurality of barcode sequences, wherein the plurality of barcode sequences comprises a first barcode sequence and a second barcode sequence. The method can comprise (b) generating a barcoded nucleic acid scaffolded structure using the first oligonucleotide, the second oligonucleotide, the first barcode sequence, and the second barcode sequence, wherein the barcoded nucleic acid scaffolded structure comprises (i) a first barcoded oligonucleotide comprising a sequence corresponding to the first barcode sequence, and (ii) a second barcoded oligonucleotide comprising a sequence corresponding to the second barcode sequence.

[0120] In some cases, generating the barcoded nucleic acid scaffolded structure comprises performing nucleic acid extension reactions. The method can comprise coupling the nucleic acid scaffolded structure to the barcoded nucleic acid template, wherein a 3’ end of the first oligonucleotide of the nucleic acid scaffolded structure hybridizes to a first portion of the barcoded nucleic acid template, and wherein a 3’ end of the second oligonucleotide of the nucleic acid scaffolded structure hybridizes to a second portion of the barcoded nucleic acid template. The method can further comprise performing (i) a first nucleic acid extension reaction using the first barcode sequence of the barcoded nucleic acid template and the first oligonucleotide of the nucleic acid scaffolded structure, thereby generating the first barcoded oligonucleotide, and (ii) a second nucleic acid extension reaction using the second barcode sequence of the barcoded nucleic acid template and the second oligonucleotide of the nucleic acid scaffolded structure, thereby generating the second barcoded oligonucleotide.

[0121] In some cases, the plurality of barcode sequences are identical. In other cases, the plurality of barcode sequences comprise different sequences. The method can comprise, prior to (a), generating the barcoded nucleic acid template. For example, the method can comprise performing a nucleic acid amplification reaction on a nucleic acid molecule comprising a barcode sequence of the plurality of barcode sequences. The nucleic acid amplification reaction can comprise rolling circle amplification.

[0122] In another aspect, described herein is a method of generating a plurality of different nucleic acid scaffolded structures, comprising: (a) providing a plurality of nucleic acid scaffolded structures, wherein each nucleic acid scaffolded structure of the plurality of nucleic acid scaffolded structures comprises an oligonucleotide; and (b) combinatorially assembling a barcode sequence on the oligonucleotide of each nucleic acid scaffolded structure. The method can comprise assembling a barcode sequence on a nucleic acid scaffolded structure that distinguishes the nucleic acid scaffolded structure from other nucleic acid scaffolded structures of the plurality of nucleic acid scaffolded structures.

[0123] In some cases, the method comprises, prior to or during the combinatorial assembling, partitioning the plurality of nucleic acid scaffolded structures into a plurality of partitions. The combinatorial assembling can comprise appending one or more nucleotides onto the oligonucleotide of each nucleic acid scaffolded structure within a partition of the plurality of partitions, thereby generating an extended oligonucleotide on each nucleic acid scaffolded structure.

[0124] In another aspect, described herein is a method of generating barcoded nucleic acid scaffolded structures, comprising: (a) partitioning a plurality of nucleic acid scaffolded structures into a plurality of partitions, wherein each nucleic acid scaffolded structure of the plurality of nucleic acid scaffolded structures comprises an oligonucleotide; and (b) appending one or more nucleotides onto the oligonucleotide of each nucleic acid scaffolded structure within a partition of the plurality of partitions, thereby generating an extended oligonucleotide on each nucleic acid scaffolded structure.

[0125] In some cases, each partition of the plurality of partitions comprises a pool of discrete nucleotide monomers. The method can comprise appending a discrete nucleotide monomer of the pool of discrete nucleotide monomers onto the oligonucleotide within each partition. The plurality of partitions can comprise different partitions comprising different pools of discrete nucleotide monomers, resulting in different extended oligonucleotides in the different partitions.

[0126] In other cases, each partition of the plurality of partitions comprises a pool of polynucleotides. The method can comprise appending a polynucleotide of the pool of polynucleotides onto the oligonucleotide within each partition. The plurality of partitions can comprise different partitions comprising different pools of polynucleotides, resulting in different extended oligonucleotides in the different partitions.

[0127] In some aspects, the method further comprises, after generating an extended oligonucleotide on each nucleic acid scaffolded structure, pooling the plurality of nucleic acid scaffold structures. The method can further comprise after pooling the plurality of nucleic acid scaffold structures, partitioning the plurality of nucleic acid scaffold structures into an additional plurality of partitions. The method can further comprise, within each additional partition of the additional plurality of partitions, appending one or more nucleotides onto the extended oligonucleotide of each nucleic acid scaffolded structure.

C. Kits

[0128] In another aspect, the present disclosure provides a kit comprising a plurality of distinct nucleic acid scaffolded structures, as described elsewhere herein. In some cases, each distinct nucleic acid scaffolded structure is coupled to: a plurality of probes, wherein each probe of the plurality of probes comprises (I) a sensing moiety configured to bind to the analyte, and (II) a barcode sequence corresponding to a spatial position of the probe on the distinct nucleic acid scaffolded structure. In some cases, each distinct nucleic acid scaffolded structure comprises an additional barcode sequence identifying the distinct nucleic acid scaffolded structure. In some cases, the kit further comprises instructions for using a nucleic acid scaffolded structure of the plurality of distinct nucleic acid scaffolded structures to detect an analyte.

EXAMPLES

Example 1. Barcoding and analysis of analytes using nucleic acid scaffolded structure

[0129] This example provides a method of analyzing analytes using nucleic acid scaffolded structures. In this example, two nucleic acid scaffolded structures, nucleic acid scaffolded structure 0310 and nucleic acid scaffolded structure 0350 are provided (FIG. 3). Each nucleic acid scaffolded structure can have probes protruding from its surface at specific locations. These probes can comprise a sensing moiety configured to bind to an analyte, as well as one or more barcode sequences that can be used to barcode the analyte.

[0130] In FIG. 3, probe 0320 is coupled to nucleic acid scaffolded structure 0310, and probe 0360 is coupled to nucleic acid scaffolded structure 0350. Probe 0320 comprises barcode sequences 0321a and 0321b and sensing moiety 0322. Barcode sequence 0321a is a position barcode that identifies the spatial position of probe 0320 on the Nucleic acid scaffolded structure 0310. Barcode sequence 0321b is a UMI that identifies the nucleic acid scaffolded structure 0310. Probe 0360 comprises barcode sequences 0361a and 0361b and sensing moiety 0362. Barcode sequence 0361a is a position barcode that identifies the spatial position of probe 0360 on the nucleic acid scaffolded structure 0350. Barcode sequence 0361b is a UMI that identifies nucleic acid scaffolded structure 0350.

[0131] In this example, the different Nucleic acid scaffolded structures are provided to different cells to barcode the analytes in those cells. Nucleic acid scaffolded structure 0310 is provided to a cell comprising analyte 0340. The sensing moiety 0322 on probe 0320 of the nucleic acid scaffolded structure 0310 is a nucleic acid segment that hybridizes to a portion 0342 of the analyte 0340. Nucleic acid scaffolded structure 0350 is provided to a cell comprising analyte 0370. The sensing moiety 0362 on probe 0360 of the nucleic acid scaffolded structure 0350 is a nucleic acid segment that hybridizes to a portion 0372 of the analyte 0372.

[0132] In this example, for each probe hybridized to an analyte, a nucleic acid extension reaction is carried out to generate a barcoded nucleic acid molecule comprising information about the analyte as well as sequences corresponding to the barcode sequences of the probe. The barcoded nucleic acid molecules are the sequenced.

[0133] A sequencing read from a barcoded nucleic acid molecule resulting from probe 0320 hybridized to analyte 0340 comprises information about analyte 0340, information about the spatial position of the analyte on the nucleic acid scaffolded structure based on barcode 0321a, and identifying information about the nucleic acid scaffolded structure 0310 to which the analyte is bound based on barcode 0321b. The identifying information about the nucleic acid scaffolded structure 0310 also associates the analyte 0340 with the cell to which the nucleic acid scaffolded structure 0310 was provided.

[0134] A sequencing read from a barcoded nucleic acid molecule resulting from probe 0360 hybridized to analyte 0370 comprises information about analyte 0370, information about the spatial position of the analyte on the nucleic acid scaffolded structure based on barcode 0361a, and identifying information about the nucleic acid scaffolded structure 0350 to which the analyte is bound based on barcode 0361b. The identifying information about the nucleic acid scaffolded structure 0350 also associates the analyte 0370 with the cell to which the nucleic acid scaffolded structure 0350 was provided. The sequencing reads from each analyte coupled to a nucleic acid scaffolded structure are used to construct an image that relays the identifying information about the analyte and the spatial position of the analyte on the nucleic acid scaffolded structure. Example 2. Barcoding and analysis of a tagmented nucleic acid analyte using a nucleic acid scaffolded structure

[0135] This example provides a method of analyzing a tagmented nucleic acid analyte using a nucleic acid scaffolded structure. FIG. 6B provides an example of a nucleic acid scaffolded structure 0670 that is coupled to a probe 0680 and is used for barcoding and analysis of a tagmented nucleic acid analyte 0690. The probe 0680 comprises a bottom staple section 0681 that anchors the probe to the nucleic acid scaffolded structure, a priming site 0682 for next-generation sequencing (NGS) library preparation via PCR, a position barcode 0683 encoding the probe’s location on the nucleic acid scaffolded structure 0670, and a nucleic acid scaffolded structure-specific unique molecular identifier (UMI) 0685. The probe 0680 further comprises a hybridization segment 0684 used to add the UMI 0685 to the probe in a barcoding reaction, such as, for example, described in Example 3. The probe 0680 further comprises a sensing moiety 0686 configured to bind to an adapter sequence 0686RC in analyte 0690.

[0136] Tagmented nucleic acid analyte 0690 is a post-transposase fragmented product that is generated from a tagmentation reaction that appends a first adapter containing a ME sequence and an 0686RC adapter sequence and a second adapter containing a ME sequence and a R1 segment to a double-stranded genomic DNA fragment comprising genomic DNA sequence 0691. When the analyte 0690 is contacted with the nucleic acid scaffolded structure 0670, and the sensing moiety 0686 of the probe 0680 hybridizes to the adapter sequence 0686RC of the analyte. A nucleic acid ligation reaction and nucleic acid extension reaction are performed to generate a barcoded nucleic acid molecule 0692 that comprises the genomic DNA sequence 0691, the position barcode 0683, the nucleic acid scaffolded structure-specific UMI 0685, and RIRC. The barcoded nucleic acid molecule 0692 can be used to generate an NGS library via PCR.

[0137] The resulting sequencing read associated barcoded nucleic acid molecule 0692 provides information that associates the genomic DNA sequence with the nucleic acid scaffolded structure 0670 and with the spatial position of the probe 0680 on the nucleic acid scaffolded structure. In some cases, the nucleic acid scaffolded structure 0670 is specifically partitioned with a specific sample comprising the tagmented analyte, and identifying the nucleic acid scaffolded structure 0670 further associates the genomic DNA sequence with the sample from which it is derived. Example 3. Barcoding a nucleic acid scaffolded structure using barcode template

[0138] This example provides a method of barcoding a nucleic acid scaffolded structure using a barcode template. FIG. 11A shows a nucleic acid barcode template 1110 comprising multiple copies of a hybridization sequence 1111, multiple copies of a UMI 1112, and multiple copies of a reverse complement of a sensing sequence 1113. Nucleic acid barcode template 1110 is used to barcode a plurality of oligonucleotides on the nucleic acid scaffolded structure 1120. Each oligonucleotide on the nucleic acid scaffolded structure 1120 comprises, each comprising a unique position barcode (e.g., 1 A, IB, and 1C) that identifies the spatial position of the corresponding oligonucleotide on the nucleic acid scaffolded structure 1120. Each oligonucleotide further comprises a binding sequence 1101 that hybridizes to the hybridization sequence 1111 of the nucleic acid barcode template 1110. Nucleic acid extension reactions using a DNA polymerase are performed to extend the oligonucleotides on the nucleic acid scaffolded structure 1120 to generated extended oligonucleotides comprising the reverse complement of the UMI sequence 1112 and the sensing sequence 1113. The extension reactions can be halted using either a restriction enzyme or oligo hybridization, resulting in individual probes with identical UMIs. The sensing sequence 1113 on the probe is configured to hybridize to a sequence of a nucleic acid analyte, and the nucleic acid scaffolded structure coupled to the extended oligonucleotides is used to barcode and analyze the analyte, as described elsewhere herein.

[0139] FIG. 11B shows a schematic of generating the nucleic acid barcode template 1110. A circular DNA comprising the hybridization sequence 1111, the UMI 1112, the reverse complement of a sensing sequence 1113 is provided. Rolling circle amplification is performed to generate the nucleic acid barcode template 1110. Other circular DNA molecules comprising different UMIs are used to generate different nucleic acid barcode templates that are used to barcode other nucleic acid scaffolded structures, such that each nucleic acid scaffolded structure comprises a unique UMI identifying the nucleic acid scaffolded structure.

Example 4. Combinatorially assembling a barcode sequence on a nucleic acid scaffolded structure using a split-pool method with pools of discrete nucleotide monomers

[0140] This example provides a method of combinatorially assembling a barcode sequence on a nucleic acid scaffolded structures using a split-pool method using pools of discrete nucleotide monomers. FIG. 12A shows nucleic acid scaffolded structures 1201 and 1202. Nucleic acid scaffolded structure 1201 comprises a plurality of oligonucleotides 1211, each comprising a unique position barcode (1 A, IB, 1C). Nucleic acid scaffolded structure 1202 comprises a plurality of oligonucleotides 1212, each comprising a unique position barcode (2A, 2B, 2C). In process 1220, the nucleic acid scaffolded structures are split into different partitions, each comprising a pool of discrete nucleotide monomers (A, T, C, or G) and a DNA terminal transferase. The DNA terminal transferase appends a single nucleotide to the 3’ end of each oligonucleotide on the nucleic acid scaffolded structure in each reaction cycle. After the first reaction cycle, extended oligonucleotides of nucleic acid scaffolded structure 1201 each have an appended adenine, while extended oligonucleotides of nucleic acid scaffolded structure 1202 each have an appended cytosine. In process 1230, the nucleic acid scaffolded structures are pooled, followed by re-splitting (process 1240) into different partitions for another round of appending an additional nucleotide to the extended oligonucleotides. After process 1240, extended oligonucleotides of nucleic acid scaffolded structure 1201 have acquired a thymine, while extended oligonucleotides of nucleic acid scaffolded structure 1202 have acquired an adenine. Through multiple split-and-pool cycles, the same random UMI sequence (resulting from the plurality of appended nucleotides) is appended to all the oligonucleotides on the same nucleic acid scaffolded structure, but the vast majority of different nanostructures have different random UMIs, as the probability of two nanostructures consistently being pooled into the same partition each cycle over many cycles remains low.

Example 5. Combinatorially assembling a barcode sequence on a nucleic acid scaffolded structure using a split-pool method with pools of polynucleotides

[0141] This example provides a method of combinatorially assembling a barcode sequence on a nucleic acid scaffolded structures using a split-pool method using pools of discrete nucleotide monomers. FIG. 12B shows nucleic acid scaffolded structures 1201 and 1202. Nucleic acid scaffolded structure 1201 comprises a plurality of oligonucleotides 1211, each comprising a unique position barcode (1A, IB, 1C). Nucleic acid scaffolded structure 1202 comprises a plurality of oligonucleotides 1212, each comprising a unique position barcode (2A, 2B, 2C). In process 1260, the nucleic acid scaffolded structures are split into different partitions, each comprising a pool of polynucleotides. A ligase appends a polynucleotide to the 3’ end of each oligonucleotide on the nucleic acid scaffolded structure in each reaction cycle. After the first reaction cycle, extended oligonucleotides of nucleic acid scaffolded structure 1201 each have an appended polynucleotide 1261, while extended oligonucleotides of nucleic acid scaffolded structure 1202 each have an appended 1262. In process 1270, the nucleic acid scaffolded structures are pooled, followed by re-splitting (process 1280) into different partitions for another round of appending an additional polynucleotide to the extended oligonucleotides. After process 1280, extended oligonucleotides of nucleic acid scaffolded structure 1201 have acquired a polynucleotide 1281, while extended oligonucleotides of nucleic acid scaffolded structure 1202 have acquired a polynucleotide 1282. Through multiple split-and-pool cycles, the same random UMI sequence (resulting from the plurality of appended polynucleotides) is appended to all the oligonucleotides on the same nucleic acid scaffolded structure, but the vast majority of different nanostructures have different random UMIs, as the probability of two nanostructures consistently being pooled into the same partition each cycle over many cycles remains low.

Example 6. Aligning and coupling nucleic acid scaffolded structures to a surface

[0142] This example provides a method of aligning and coupling nucleic acid scaffolded structures to a surface for use for analyte detection and analysis. In this example, electron beam lithography is used to generate patterns for DNA origami docking and orientation. FIG. 13A shows an example of nucleic acid scaffolded structures on a surface prior to alignment, while FIG. 13B shows an example of nucleic acid scaffolded structures on a surface after alignment. Each of the white circles in FIGs. 13A and 13B represents a probe coupled to a nucleic acid scaffolded structure that has a position barcode identifying the spatial position of the probe in the nucleic acid scaffolded structure, and a UMI identifying the nucleic acid scaffolded structure that the probe is coupled to. After alignment of the nucleic acid scaffolded structures on the surface using the patterns generated by electron beam lithography and coupling to the surface, the nucleic acid scaffolded structures are contacted with analytes, which bind to the probes on the nucleic acid scaffolded structures. After binding to an analyte, each probe provides spatial information of the bound analyte along the surface based on (1) the known spatial position of the probe within its nucleic acid scaffolded structure and (2) the known location of the unique nucleic acid scaffolded structure on the surface. Analyte-specific information is further collected by detecting the analyte. The analyte-specific information and spatial information of multiple analytes in a cell or tissue are used to construct a continuous, large-scale image of the cell or tissue. Use of nanoscale nucleic acid scaffolded structures allows image construction at a resolution 1000 times finer than conventional methods.

Example 7. Gel stamping using a nucleic acid scaffolded structure enables large- scale production of sensing arrays for analyte detection and analysis

[0143] This example provides a method of gel stamping using a nucleic acid scaffolded structure, which enables large-scale production of sensing arrays for analyte detection and analysis.

[0144] FIG. 14 shows an example flow chart that provide a nucleic acid scaffolded structure coupled to two probes. Information from the two probes are copied over “stamped” to a surface (e.g., a gel) coupled to two nucleic acid primers by generating nucleic acid extension products that are coupled to the surface and that comprise sequence information from the two probes. FIG. 15A shows an example of a gel surface 1560 coupled to nucleic acid primers and a nucleic acid scaffolded structure 1510 coupled to probes 1530 and 1520. Using nucleic acid extension reactions, the nucleic acid scaffolded structure and the probes are used to produce a sensing array on the gel surface comprising the nucleic acid extension products. The primers 1550 and 1540 bind to the probes 1530 and 1520 and are used to produce nucleic acid extension products 1580 and 1570 coupled to the gel surface 1560 (FIG. 15B). The nucleic acid extension products 1580 and 1570 comprise sequence information originating from the probes. Sequences 1531c and 1521c are position barcode sequences and 1532c and 1522c are sensing sequences configured to bind to analytes. The nucleic acid extension products can also have additional barcodes that identify the nucleic acid scaffolded structure from which they were derived. FIG. 15C shows an example of analyzing analytes 1592 and 1590 using the sensing array comprising the nucleic acid extension products coupled to the surface. Sensing sequences 1532c and 1522c bind to analyte sequences 1593 and 1591. The analytes 1592 and 1590, as well as the barcode sequences 1531c and 1521c are identified, associating the spatial position identified by barcode sequence 1531c with analyte 1592 and the spatial position identified by barcode sequence 1521c with analyte 1590. Example 8. Information sharing between nucleic acid scaffolded structures enables mapping out spatial network of different nucleic acid scaffolded structures

[0145] This example provides a method of information sharing between nucleic acid scaffolded structures to map out a spatial network of different nucleic acid scaffolded structures for analyte detection and analysis. FIGs. 17A and 17B show an example of two nucleic acid scaffolded structures 1701 and 1702 coupled together via binding oligonucleotides 17B2 and 17B3. Nucleic acid scaffolded structures 1701 and 1702 further comprise oligonucleotides 17P1-17P6 that act as probes configured to bind to an analyte and binding oligonucleotides 17B1-17B4 configured to bind to another binding oligonucleotide for information sharing between nucleic acid scaffolded structures. Each of the oligonucleotides 17P1-17P6 and 17B1-17B4 comprises a UMI identifying the nucleic acid scaffolded structure to which the probe is coupled. UMI l identifies nucleic acid scaffolded structure 1701 and UMI 2 identifies nucleic acid scaffolded structure 1702. Each of the oligonucleotides 17P1-17P6 and 17B1-17B4 further comprises a position barcode (1700A-1700J) that identifies the spatial position of the oligonucleotide on its respective nucleic acid scaffolded structure.

[0146] FIG. 17B shows binding oligonucleotide 17B2 is coupled to nucleic acid scaffolded structure 1701 via a bottom staple section 1711 that anchors the binding oligonucleotide 17B2 to the nuclei acid scaffolded structure 1701. Binding oligonucleotide 17B3 is coupled to nucleic acid scaffolded structure 1702 via a bottom staple section 1721 that anchors the binding oligonucleotide 17B3 to the nuclei acid scaffolded structure 1702. Binding oligonucleotide 17B2 further comprises binding sequence 17SE that hybridizes to binding sequence 17SI of the binding oligonucleotide 17B3, thereby coupling nucleic acid scaffolded structures 1701 and 1702 together. Binding oligonucleotide 17B2 further comprises a priming sequence 1712 for nextgeneration sequencing (NGS) library preparation via PCR, a position barcode 1700E encoding the binding oligonucleotide’s position on the nucleic acid scaffolded structure 1701, and a nucleic acid scaffolded structure-specific unique molecular identifier UMI l . Binding oligonucleotide 17B2 also comprises a hybridization segment 1714 used to add the UMI l to the binding oligonucleotide in a barcoding reaction, such as, for example, described in Example 3.

[0147] Binding oligonucleotide 17B3 further comprises a priming sequence 1722 for nextgeneration sequencing (NGS) library preparation via PCR, a position barcode 17001 encoding the binding oligonucleotide’s position on the nucleic acid scaffolded structure 1702, and a nucleic acid scaffolded structure-specific unique molecular identifier UMI 2. Binding oligonucleotide 1720 also comprises a hybridization segment 1724 used to add the UMI 2 to the binding oligonucleotide in a barcoding reaction.

[0148] The nucleic acid scaffolded structures are introduced to a cell comprising analytes. The probes P1-P6 bind to analytes via sensing sequences 17SA, 17SB, 17SC, 17SF, 17SG, and 17SH. Nucleic acid extension reactions and/or ligation reactions are performed to produce extension and/or ligation products comprising a sequence corresponding to the bound analyte, a sequence corresponding to the respective UMI, and a sequence corresponding to the position barcode.

[0149] Nucleic acid extension reactions are also performed to generate extension products 1730 and 1740, sharing information between the nucleic acid scaffolded structures 1701 and 1702. The extension products 1730 and 1740 comprise a sequence associated with the nucleic acid scaffolded structure 1701 (e.g., UMI l or the reverse complement thereof UMI l RC) and a sequence associated with the nucleic acid scaffolded structure 1702 (e.g., UMI_2 or the reverse complement thereof UMI_2 RC). The extension products further comprise sequences associated with the position barcodes (e.g., 1700E and 17001) that encode the spatial position of the respective binding oligonucleotide on its respective nucleic acid scaffolded. The position barcodes also provide directional information regarding interaction between the nucleic acid scaffolded structures.

[0150] The nucleic acid ligation and/or extension products comprising the barcoded analyte information and barcoded nucleic acid scaffolded structure interaction information are then sequenced. The sequencing reads are used to associate analyte information with the UMI of a specific nucleic acid scaffolded structure and a spatial position on the nucleic acid scaffolded structure. The sequencing reads comprising information about the coupling of the nucleic acid scaffolded structures are used to construct pairwise nucleic acid scaffolded structure interaction data and to map out the positional network of the different nucleic acid scaffolded structures. Together, the sequencing reads are used to construct a spatial map of analytes in the cell.

[0151] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method comprising:

(a) providing:

(i) a sample comprising an analyte, and

(ii) a nucleic acid scaffolded structure, wherein the nucleic acid scaffolded structure is coupled to a probe comprising (I) a sensing moiety configured to bind to the analyte, and (II) a barcode sequence corresponding to a spatial position of the probe on the nucleic acid scaffolded structure;

(b) contacting the sample with the nucleic acid scaffolded structure, wherein the sensing moiety of the probe on the nucleic acid scaffolded structure binds to the analyte in the sample, thereby coupling the probe and its barcode sequence to the analyte;

(c) identifying the barcode sequence, thereby identifying the spatial position of the probe on the nucleic acid scaffolded structure;

(d) identifying the analyte in the sample coupled to the probe; and

(e) associating the spatial position of the probe on the nucleic acid scaffolded structure with the analyte in the sample.

2. The method of claim 1, wherein the nucleic acid scaffolded structure is further coupled to a plurality of probes.

3. The method claim 2, wherein the plurality of probes comprises probes configured to bind to different analytes in the sample.

4. The method of claim 2 or 3, wherein the plurality of probes comprises at least 100 probes.

5. The method of any one of claims 2-4, wherein the plurality of probes comprises at least 1000 probes.

6. The method of any one of claims 2-5, wherein the probe is located from 1 to 50 nm away from an additional probe of the plurality of probes.

7. The method of any one of claims 2-6, wherein the sample comprises a plurality of analytes, and wherein, in (b), probes of the plurality of probes on the nucleic acid scaffolded structure couple to the plurality of analytes.

8. The method of any one of claims 1-7, wherein (c) comprises detecting the barcode sequence using sequencing.

9. The method of claim 8, wherein (c) comprises detecting the barcode sequence using in situ sequencing.

10. The method of any one of claims 1-9, wherein the probe further comprises (III) an additional barcode sequence.

11. The method of claim 10, wherein the additional barcode sequence identifies the nucleic acid scaffolded structure, and wherein the method further comprises identifying the analyte as associated with the nucleic acid scaffolded structure.

12. The method of any one of claims 1-11, wherein the analyte comprises a protein or a peptide.

13. The method of claim 12, wherein the analyte further comprises a nucleic acid coupled to the protein or the peptide.

14. The method of any one of claims 1-13, wherein the analyte comprises a small molecule.

15. The method of claim 14, wherein the analyte further comprises a nucleic acid coupled to the small molecule.

16. The method of any one of claims 1-11, wherein the analyte comprises a nucleic acid.

17. The method of claim 16, wherein the analyte comprises an RNA transcript.

18. The method of claim 16, wherein the analyte comprises genomic DNA.

19. The method of any one of claims 1-18, wherein the analyte comprises an adapter sequence.

20. The method of claim 19, further comprising, prior to (a), generating the analyte by coupling the adapter sequence to a nucleic acid molecule.

21. The method of claim 20, wherein the coupling the adapter sequence to the nucleic acid molecule comprises a transposition reaction.

22. The method of any one of claims 19-21, wherein the sensing moiety of the probe comprises a sequence that hybridizes to a portion of the adapter sequence.

23. The method of any one of claims 1-22, wherein the sensing moiety of the probe comprises a sensing sequence that hybridizes to a portion of the analyte.

24. The method of claim 23, wherein the sensing sequence comprises a poly-T sequence.

25. The method of any one of claims 1-24, wherein the sensing moiety of the probe comprises a protein or a peptide.

26. The method of any one of claims 1-25, wherein the sensing moiety of the probe comprises an enzyme.

27. The method of claim 26, wherein the enzyme is a DNA processing enzyme.

28. The method of claim 26 or 27, wherein the enzyme comprises a transposase domain.

29. The method of any one of claims 16-28, wherein the nucleic acid of the analyte comprises a target sequence, and wherein the method further comprises generating a barcoded nucleic acid strand comprising (i) the barcode sequence or a complement thereof, and (ii) the target sequence or complement thereof.

30. The method of claim 29, wherein the probe further comprises (III) an additional barcode sequence that identifies the nucleic acid scaffolded structure, and wherein the barcoded nucleic acid strand further comprises (iii) the additional barcode sequence or a complement thereof.

31. The method of claim 29 or 30, further comprising generating the barcoded nucleic acid strand using a nucleic acid ligation reaction.

32. The method of any one of claims 29-31, further comprising generating the barcoded nucleic acid strand using a nucleic acid extension reaction.

33. The method of any one of claims 16-32, wherein the nucleic acid of the analyte comprises a target sequence, and wherein the method further comprises, prior to (d), amplifying the target sequence using the probe in a nucleic acid amplification reaction to yield an amplification product.

34. The method of claim 33, wherein (d) further comprises detecting the amplification product or a derivative thereof.

35. The method of claim 33 or 34, wherein (d) further comprises sequencing the amplification product or a derivative thereof, thereby obtaining sequencing reads.

36. The method of claim 35, wherein (e) further comprises associating the sequencing reads with the spatial position of the probe in the nucleic acid scaffolded structure.

37. The method of any one of claims 1-36, wherein the nucleic acid scaffolded structure comprises a dimension of from 100 to 2000 nm.

38. The method of any one of claims 1-37, wherein the nucleic acid scaffolded structure comprises a crisscross assembly.

39. The method of any one of claims 1-38, wherein, in (a), the nucleic acid scaffolded structure is coupled to a surface.

40. The method of claim 39, further comprising, prior to (a), coupling the nucleic acid scaffolded structure to the surface.

41. The method of claim 40, wherein the coupling comprises orienting the nucleic acid scaffolded structure on the surface based on a pattern on the surface.

42. The method of claim 41, further comprising, prior to (a), generating the pattern using electron beam lithography or atomic force lithography.

43. The method of any one of claims 39-42, wherein the nucleic acid scaffold structure is coupled to a bead.

44. The method of claim 39, wherein the surface is coupled to a plurality of nucleic acid scaffolded structures.

45. The method of any one of claims 1-44, wherein the sample comprises a cell.

46. The method of any one of claims 1-45, further comprising, prior to (a), coupling the probe to the nucleic acid scaffolded structure.

47. The method of claim 46, wherein the coupling the probe to the nucleic acid scaffolded structure comprises hybridizing a portion of the probe to a binding segment of the nucleic acid scaffolded structure.

48. The method of claim 47, wherein the binding segment of the nucleic acid scaffolded structure comprises a distinct sequence corresponding to a distinct spatial position of the binding segment in the nucleic acid scaffolded structure.

49. The method of claim 47 or 48, wherein the nucleic acid scaffolded structure further comprises a plurality of binding segments, wherein each binding segment of the plurality of binding segments comprises a distinct sequence corresponding to a distinct spatial position of the binding segment in the nucleic acid scaffolded structure.

50. The method of any one of claims 1-49, wherein the identifying in (d) identifies information about the analyte, and wherein the method further comprises (f) constructing an image using:

(i) the information about the analyte identified in (d), and

(ii) the spatial position of the probe on the nucleic acid scaffolded structure identified in (c).

51. The method of claim 50, further comprising, after (b), identifying a location of the nucleic acid scaffolded structure in the sample.

52. The method of claim 51, wherein the constructing the image in (f) further comprises using

(iii) the location of the nucleic acid scaffolded structure in the sample.

53. The method of claim 51 or 52, wherein the identifying the location of the nucleic acid scaffolded structure comprises imaging.

54. The method of any one of claims 1-53, wherein (a) further comprises providing a plurality of nucleic acid scaffolded structures, wherein each nucleic acid scaffolded structure is coupled to a probe comprising (I) a sensing moiety configured to bind to an analyte, and (II) a barcode sequence corresponding to a spatial position of the probe on the nucleic acid scaffolded structure to which the probe is coupled; and wherein (b) further comprises contacting the sample with the plurality of nucleic acid scaffolded structures.

55. The method of claim 54, further comprising after (b), identifying a location of each nucleic acid scaffolded structure of the plurality of nucleic acid scaffolded structures in the sample.

56. The method of claim 55, wherein the identifying the location of each nucleic acid scaffolded structure comprises imaging.

57. A composition comprising a nucleic acid scaffolded structure, wherein the nucleic acid scaffolded structure is coupled to a plurality of probes, wherein each probe of the plurality of probes comprises (I) a sensing moiety configured to bind to an analyte, and (II) a first barcode sequence corresponding to a spatial position of the probe

-SO- on the nucleic acid scaffolded structure, and (III) a second barcode sequence identifying the nucleic acid scaffolded structure.

58. The composition of claim 57, wherein the sensing moiety of the probe comprises a nucleic acid sequence.

59. The composition of claim 57 or 58, wherein the sensing moiety of the probe comprises a protein or a peptide.

60. The composition of any one of claims 57-59, further comprising the analyte, wherein the analyte is bound to the sensing moiety of a probe of the plurality of probes.

61. The composition of claim 60, wherein the analyte comprises an adapter sequence that is bound to the sensing moiety of the probe of the plurality of probes.

62. The composition of any one of claims 57-61, wherein the nucleic acid scaffolded structure is coupled to an additional nucleic acid scaffolded structure.

63. The composition of any one of claims 57-62, wherein the nucleic acid scaffolded structure is coupled to a surface.

64. The composition of claim 63, wherein the surface is coupled to a plurality of nucleic acid scaffolded structures.

65. The composition of claim 64, wherein nucleic acid scaffolded structures of the plurality of nucleic acid scaffolded structures are aligned on the surface based on a pattern on the surface.

66. The composition of any one of claims 57-65, wherein a probe of the plurality of probes is located from 1 to 50 nm away from an additional probe of the plurality of probes

67. A kit comprising:

(a) a plurality of distinct nucleic acid scaffolded structures, wherein each distinct nucleic acid scaffolded structure is coupled to: (i) a plurality of probes, wherein each probe of the plurality of probes comprises (I) a sensing moiety configured to bind to the analyte, (II) a barcode sequence corresponding to a spatial position of the probe on the distinct nucleic acid scaffolded structure; and (III) an additional barcode sequence identifying its corresponding distinct nucleic acid scaffolded structure; and (b) instructions for using a nucleic acid scaffolded structure of the plurality of distinct nucleic acid scaffolded structures to detect an analyte.

68. A method comprising:

(a) providing:

(i) a surface;

(ii) a first nucleic acid scaffolded structure, wherein the first nucleic acid scaffolded structure is coupled to a first probe, wherein the first probe comprises (I) a first sensing moiety configured to bind to a first analyte in a sample, and (II) a first barcode sequence corresponding to a first spatial position of the first probe on the first nucleic acid scaffolded structure; and

(iii) a second nucleic acid scaffolded structure, wherein the second nucleic acid scaffolded structure is coupled to a second probe, wherein the second probe comprises (I) a second sensing moiety configured to bind to a second analyte in a sample, and (II) a second barcode sequence corresponding to a second spatial position of the second probe on the second nucleic acid scaffolded structure; and

(b) coupling the first nucleic acid scaffolded structure to the surface and coupling the second nucleic acid scaffolded structure to the surface.

69. The method of claim 68, further comprising orienting the first nucleic acid scaffolded structure and the second nucleic acid scaffolded structure on the surface based on a pattern on the surface.

70. The method of claim 69, further comprising, prior to (a), generating the pattern using electron beam lithography or atomic force lithography.

71. The method of any one of claims 67-70, wherein the first nucleic acid scaffolded structure comprises a handle segment, and wherein (b) comprises hybridizing the handle segment to a binding sequence on the surface.

72. The method of any one of claims 67-71, further comprising directly coupling the first nucleic acid scaffolded structure to the second nucleic acid scaffolded structure.

73. The method of any one of claims 67-72, wherein the surface is a solid surface.

74. A method comprising:

(a) providing:

(i) a surface coupled to a first nucleic acid primer and a second nucleic acid primer; and

(ii) a nucleic acid scaffolded structure, wherein the nucleic acid scaffolded structure is coupled to:

(A) a first probe comprising:

(I) a first sensing sequence, wherein the first sensing sequence or a reverse complement thereof is configured to bind to a first analyte in a sample, and

(II) a first barcode sequence corresponding to a first spatial position of the first probe on the nucleic acid scaffolded structure; and

(B) a second probe comprising:

(I) a second sensing sequence, wherein the second sensing sequence or a reverse complement thereof is configured to bind to a second analyte in a sample, and

(II) a second barcode sequence corresponding to a second spatial position of the second probe on the nucleic acid scaffolded structure;

(b) contacting the first nucleic acid primer coupled to the surface and the second nucleic acid primer coupled to the surface with the nucleic acid scaffolded structure,

(c) generating a first nucleic acid extension product using the first nucleic acid primer coupled to the surface and the first probe coupled to the nucleic acid scaffolded structure; wherein the first nucleic acid extension product is coupled to the surface, and wherein the first nucleic acid extension product comprises (i) the first sensing sequence or reverse complement thereof, and (ii) the first barcode sequence or reverse complement thereof; and

(d) generating a second nucleic acid extension product using the second nucleic acid primer coupled to the surface and the second probe coupled to the nucleic acid scaffolded structure; wherein the second nucleic acid extension product is coupled to the surface, and wherein the second nucleic acid extension product comprises (i) the second sensing sequence or reverse complement thereof, and (ii) the second barcode sequence or reverse complement thereof.

75. The method of claim 74, wherein (a) further comprises providing (iii) an additional nucleic acid scaffolded structure comprising a third probe, and wherein the method further comprises contacting a third nucleic acid primer coupled to the surface with the additional nucleic acid scaffolded structure.

76. The method of claim 75, further comprising generating a third nucleic acid extension product using the third nucleic acid primer coupled to the surface and the third probe in the nucleic acid scaffolded structure.

77. The method of claim 76, wherein: the third probe comprises:

(A) a third sensing sequence, wherein the third sensing sequence or a reverse complement thereof is configured to bind to a third analyte in a sample, and

(B) a third barcode sequence corresponding to a third spatial position of the third probe on the additional nucleic acid scaffolded structure; and the third nucleic acid extension product comprises: the third sensing sequence or reverse complement thereof, and the third barcode sequence or reverse complement thereof.

78. The method of any one of claims 74-77, further comprising, after generating the first nucleic acid extension product coupled to the surface and generating the second nucleic acid extension product coupled to the surface, (e) contacting the surface with a sample comprising the first analyte and the second analyte.

79. The method of claim 78, wherein, in (e), the first sensing sequence or reverse complement thereof in the first nucleic acid extension product couples to the first analyte in the sample; and wherein the second sensing sequence or reverse complement thereof in the second nucleic acid extension product couples to the second analyte in the sample.

80. The method of claim 79, further comprising, after (e), detecting the first barcode sequence or reverse complement thereof in the sample and detecting the second barcode sequence or reverse complement thereof in the sample.

81. The method of claim 80, further comprising, after (e), identifying the first analyte in the sample using the first nucleic acid extension product coupled to the surface and identifying the second analyte in the sample using the second nucleic acid extension product coupled to the surface.

82. The method of claim 81, further comprising associating the first barcode sequence with the first analyte and associating the second barcode sequence with the second analyte.

83. The method of any one of claims 74-82, wherein the surface comprises a gel.

84. A method of barcoding a nucleic acid scaffolded structure, the method comprising:

(a) providing:

(i) a nucleic acid scaffolded structure, wherein the nucleic acid scaffolded structure comprises a first oligonucleotide and a second oligonucleotide;

(ii) a barcoded nucleic acid template comprising a plurality of barcode sequences, wherein the plurality of barcode sequences comprises a first barcode sequence and a second barcode sequence; and

(b) generating a barcoded nucleic acid scaffolded structure using the first oligonucleotide, the second oligonucleotide, the first barcode sequence, and the second barcode sequence, wherein the barcoded nucleic acid scaffolded structure comprises (i) a first barcoded oligonucleotide comprising a sequence corresponding to the first barcode sequence, and (ii) a second barcoded oligonucleotide comprising a sequence corresponding to the second barcode sequence.

85. The method of claim 84, wherein (b) comprises performing nucleic acid extension reactions.

86. The method of claim 84 or 85, wherein (b) comprises coupling the nucleic acid scaffolded structure to the barcoded nucleic acid template, wherein a 3’ end of the first oligonucleotide of the nucleic acid scaffolded structure hybridizes to a first portion of the barcoded nucleic acid template, and wherein a 3’ end of the second oligonucleotide of the nucleic acid scaffolded structure hybridizes to a second portion of the barcoded nucleic acid template.

87. The method of claim 86, wherein (b) further comprises performing (i) a first nucleic acid extension reaction using the first barcode sequence of the barcoded nucleic acid template and the first oligonucleotide of the nucleic acid scaffolded structure, thereby generating the first barcoded oligonucleotide, and (ii) a second nucleic acid extension reaction using the second barcode sequence of the barcoded nucleic acid template and the second oligonucleotide of the nucleic acid scaffolded structure, thereby generating the second barcoded oligonucleotide.

88. The method of any one of claims 84-87, wherein the plurality of barcode sequences are identical.

89. The method of any one of claims 84-88, further comprising, prior to (a), generating the barcoded nucleic acid template by performing a nucleic acid amplification reaction on a nucleic acid molecule comprising a barcode sequence of the plurality of barcode sequences.

90. The method of claim 89, wherein the nucleic acid amplification reaction comprises rolling circle amplification.

91. A method of generating a plurality of different nucleic acid scaffolded structures, comprising:

(a) providing a plurality of nucleic acid scaffolded structures, wherein each nucleic acid scaffolded structure of the plurality of nucleic acid scaffolded structures comprises an oligonucleotide; and

(b) combinatorially assembling a barcode sequence on the oligonucleotide of each nucleic acid scaffolded structure.

92. The method of claim 91, wherein (b) comprises assembling a barcode sequence on a nucleic acid scaffolded structure that distinguishes the nucleic acid scaffolded structure from other nucleic acid scaffolded structures of the plurality of nucleic acid scaffolded structures.

93. The method of claim 91 or 92, further comprising, prior to or during (b), partitioning the plurality of nucleic acid scaffolded structures into a plurality of partitions, and wherein (b) further comprises appending one or more nucleotides onto the oligonucleotide of each nucleic acid scaffolded structure within a partition of the plurality of partitions, thereby generating an extended oligonucleotide on each nucleic acid scaffolded structure.

94. A method of generating barcoded nucleic acid scaffolded structures, comprising:

(a) partitioning a plurality of nucleic acid scaffolded structures into a plurality of partitions, wherein each nucleic acid scaffolded structure of the plurality of nucleic acid scaffolded structures comprises an oligonucleotide; and

(b) appending one or more nucleotides onto the oligonucleotide of each nucleic acid scaffolded structure within a partition of the plurality of partitions, thereby generating an extended oligonucleotide on each nucleic acid scaffolded structure.

95. The method of any one of claims 93 or 94, wherein each partition of the plurality of partitions comprises a pool of discrete nucleotide monomers, and wherein (b) comprises appending a discrete nucleotide monomer of the pool of discrete nucleotide monomers onto the oligonucleotide within each partition.

96. The method of claim 95, wherein the plurality of partitions comprise different partitions comprising different pools of discrete nucleotide monomers, and wherein (b) generates different extended oligonucleotides in the different partitions.

97. The method of claim 93 or 94, wherein each partition of the plurality of partitions comprises a pool of polynucleotides, and wherein (b) comprises appending a polynucleotide of the pool of polynucleotides onto the oligonucleotide within each partition.

98. The method of claim 97, wherein the plurality of partitions comprise different partitions comprising different pools of polynucleotides, and wherein (b) generates different extended oligonucleotides in the different partitions.

99. The method of any one of claims 93-98, further comprising, after generating an extended oligonucleotide on each nucleic acid scaffolded structure, pooling the plurality of nucleic acid scaffold structures.

100. The method of claim 99, further comprising, after pooling the plurality of nucleic acid scaffold structures, partitioning the plurality of nucleic acid scaffold structures into an additional plurality of partitions.

101. The method of claim 100, further comprising, within each additional partition of the additional plurality of partitions, appending one or more nucleotides onto the extended oligonucleotide of each nucleic acid scaffolded structure.