Compositions and methods for preparing nucleic acid nanostructures using compacted oligonucleotides

Abstract

The present disclosure provides compositions and related methods, for example, for preparing immobilized nucleic acid nanostructures using compacted oligonucleotides. In some embodiments, the rolling circle amplification reaction may be performed with compacted oligonucleotides on a support or in a solution to produce concatemer molecules having multiple copies of polynucleotide units arranged in series. Each polynucleotide unit comprises a sequence of interest and at least one universal adapter sequence that binds one end of a compacted oligonucleotide. The 5'and 3 'regions of the compacted oligonucleotides may hybridize to a concatemer to pull distal portions of the concatemer together, thereby compacting the concatemer to form a nanostructure. The nanostructures have a tighter size and shape compared to concatemers produced in the absence of the compacted oligonucleotides. The compact and stable characteristic of the nucleic acid nanostructure improves sequencing accuracy by increasing signal strength, and the nucleic acid nanostructure maintains its shape and size during multiple sequencing cycles.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 398,178, filed on August 15, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] References to electronic sequence listings

[0004] The contents of the Electronic Sequence Listing (ELEM-005-001WO-SeqList-ST26.xml; size 161,077 bytes; and creation date: August 14, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present disclosure provides compositions and methods for preparing immobilized nucleic acid nanostructures (including nucleic acid nanospheres) using compacted oligonucleotides using the compositions. Background Art

[0006] Polynucleotide sequencing technology has applications in biomedical research and healthcare environments. Improved polynucleotide methods require enhanced surface chemistry, polynucleotide amplification on supports, and base interpretation. Currently, these factors create obstacles in existing sequencing technologies, leading to limitations and poor signal-to-noise ratios in terms of throughput, and ultimately leading to increased costs associated with polynucleotide sequencing. Therefore, there is a need for improved sequencing methods and associated assembly technologies. Summary of the Invention

[0007] In one aspect, the present invention provides a method for producing a high-density nucleic acid nanostructure immobilized on a support, the method comprising:

[0008] a) providing a support having a plurality of first universal surface primers immobilized thereon, wherein the density of the first universal surface primers on the support is 2 About 10 2 -10 15 ;as well as

[0009] b) generating a plurality of immobilized single-stranded nucleic acid concatemer template molecules by:

[0010] 1) hybridizing a plurality of single-stranded circular nucleic acid library molecules to a plurality of immobilized first universal surface primers; and

[0011] 2) performing an on-support rolling circle amplification reaction using: (i) a plurality of strand-displacing polymerases, (ii) a plurality of nucleotides, and (iii) a plurality of compacted oligonucleotides,

[0012] Thereby, the plurality of fixed single-stranded nucleic acid concatemer template molecules are generated,

[0013] o wherein the individual compacted oligonucleotides comprise single-stranded linear oligonucleotides having a first binding region capable of hybridizing to a first portion of a concatemer molecule and a second binding region capable of hybridizing to a second portion of the concatemer molecule,

[0014] o wherein a plurality of immobilized concatemer molecules form a compact nucleic acid nanostructure, and

[0015] o wherein a plurality of concatemers remain immobilized to the support upon formation of the compact nucleic acid nanostructure, thereby producing a density of 2 The nanostructures fixed to the support are about 10 2 -10 15 support.

[0016] In some embodiments, the support is passivated with at least one layer of a hydrophilic polymer coating comprising the plurality of first universal surface primers. In some embodiments, the plurality of fixed first universal surface primers are located at random positions on the support or the hydrophilic polymer coating. In some embodiments, the plurality of fixed first universal surface primers are located at predetermined positions on the support or the hydrophilic polymer coating. In some embodiments, each of the first universal surface primers lacks a cleavable portion that can be converted into an abasic site. In some embodiments, the cleavable portion is uridine, 8-oxo-7,8-dihydroguanine, or deoxyinosine. In some embodiments, the plurality of nucleotides used in the rolling circle amplification reaction include dATP, dCTP, dGTP, and dTTP, and wherein the nucleotides lack a cleavable portion that can be converted into an abasic site. In some embodiments, the plurality of nucleotides used in the rolling circle amplification reaction include dATP, dCTP, dGTP, dTTP, and nucleotides having a cleavable portion that can be converted into an abasic site. In some embodiments, the nucleotide having the scissile moiety comprises uridine, 8-oxo-7,8-dihydroguanine, or deoxyinosine. In some embodiments, the rolling circle amplification reaction of step (b) produces a plurality of single-stranded nucleic acid concatemer template molecules, wherein individual concatemer template molecules comprise at least two nucleotides, each nucleotide having a scissile moiety distributed at a random position along the individual immobilized concatemer template molecules.

[0017] In some embodiments, the plurality of compacted oligonucleotides in step (b) comprise the same sequence. In some embodiments, the sequence is according to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, or 156.

[0018] In some embodiments, the plurality of compacted oligonucleotides in step (b) comprises a mixture of two or more different populations of compacted oligonucleotides, each population having a different sequence, wherein the compacted oligonucleotides in different populations have different sequences. In some embodiments, the mixture comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 different populations of compacted oligonucleotides. In some embodiments, each population of compacted oligonucleotides in the mixture comprises a sequence according to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, or 156.

[0019] In some embodiments, the compact nucleic acid nanostructure comprises one or more rings, or comprises a spherical shape, an elongated shape, a preliminary ring shape, or a toroidal shape. In some embodiments, the spherical shape is a nanosphere. In some embodiments, the elongated shape is a nanorod. In some embodiments, the toroidal shape is a nanotoroid.

[0020] In some embodiments, the nucleic acid nanostructure comprises a compact nucleic acid structure having a full width at half maximum (FWHM) that is less than the FWHM of a concatemer that is not collapsed / folded into the nanostructure.

[0021] In some embodiments, the method further comprises imaging the high-density nucleic acid nanostructures immobilized on the support.

[0022] In some embodiments, the method further comprises:

[0023] a) contacting the immobilized nucleic acid nanostructures with the labeled oligonucleotides under conditions suitable for hybridization of the oligonucleotides labeled with a detectable reporter moiety to the plurality of immobilized nucleic acid nanostructures to produce a plurality of immobilized labeled nanostructures; and

[0024] b) imaging the plurality of immobilized labeled nanostructures.

[0025] In some embodiments, the method further comprises contacting the individual immobilized nanostructures with (i) a plurality of soluble sequencing primers, (ii) a plurality of sequencing polymerases, and (iii) a plurality of nucleotide reagents under conditions suitable for: hybridizing the plurality of soluble sequencing primers to the individual immobilized nanostructures to generate a plurality of nucleic acid duplexes along the individual nanostructures, and

[0026] • Combining at least one nucleic acid duplex with a sequencing polymerase and nucleotide reagents.

[0027] In some embodiments, the multiple nucleotide reagents comprise a plurality of nucleotides, each nucleotide comprising an aromatic base, a pentose, and at least one phosphate group. In some embodiments, at least one of the multiple nucleotides further comprises a detectable reporter gene moiety. In some embodiments, the detectable reporter gene moiety is a fluorophore.

[0028] In some embodiments, the method further comprises:

[0029] a) contacting the plurality of immobilized nucleic acid nanostructures with labeled nucleotides; and

[0030] b) imaging the high-density nucleic acid nanostructure immobilized on the support.

[0031] In some embodiments, the plurality of nucleotide reagents comprises a plurality of nucleotide analogs, each nucleotide analog comprising an aromatic base, a pentose sugar having a 3' chain terminator that inhibits polymerase-catalyzed nucleotide incorporation, and at least one phosphate group. In some embodiments, at least one nucleotide analog in the plurality of nucleotide analogs further comprises a detectable reporter moiety. In some embodiments, the detectable reporter moiety is a fluorophore.

[0032] In some embodiments, the method further comprises

[0033] a) contacting the plurality of immobilized nucleic acid nanostructures with labeled labeled nucleotide analogs; and

[0034] b) imaging the high-density nucleic acid nanostructure immobilized on the support.

[0035] In some embodiments, the plurality of nucleotide agents comprises a plurality of multivalent molecules, wherein an individual multivalent molecule comprises: (1) a core; and (2) a plurality of nucleotide arms, the plurality of nucleotide arms comprising: (i) a core attachment moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms, the spacer is attached to the linker, the linker is attached to the nucleotide unit, and the nucleotide unit comprises an aromatic base, a pentose sugar, and at least one phosphate group.

[0036] In some embodiments, the method further comprises forming a plurality of binding complexes, the method comprising the steps of:

[0037] a) binding a first sequencing primer, a first sequencing polymerase, and a first multivalent molecule to a first portion of the individual immobilized nanostructures, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule is bound to the first sequencing polymerase; and

[0038] b) binding a second sequencing primer, a second sequencing polymerase, and the first multivalent molecule to a second portion of the same single immobilized nanostructure, thereby forming a second binding complex, wherein the second nucleotide unit of the first multivalent molecule binds to the second sequencing polymerase, and wherein the first binding complex and the second binding complex comprising the same multivalent molecule form an avidity complex.

[0039] In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules further comprises at least one detectable reporter moiety. In some embodiments, the at least one detectable reporter moiety comprises at least one fluorophore.

[0040] In some embodiments, the method further comprises:

[0041] a) contacting the plurality of immobilized nucleic acid nanostructures with a labeled multivalent molecule; and

[0042] b) imaging the high-density nucleic acid nanostructure immobilized on the support.

[0043] In some embodiments, the method further comprises contacting the plurality of fixed nanostructures with a cellular biological sample. In some embodiments, the cellular biological sample comprises a single cell, a slice of a single cell, a plurality of cells, a slice of a plurality of cells, a tissue, a slice of a tissue, an organ, a slice of an organ, an organism, or a slice of an organism.

[0044] In some embodiments, the plurality of immobilized nucleic acid nanostructures are in fluid communication with each other to allow a reagent solution to flow onto the support, such that the plurality of immobilized nucleic acid nanostructures on the support react with the reagent solution in a massively parallel manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The features of the present invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description which sets forth illustrative embodiments, in which the principles of the present disclosure are utilized, and in the accompanying drawings:

[0046] Figure 1 Schematic diagrams of several embodiments of linear compacted oligonucleotides are shown, each comprising a first binding region, an intervening junction, and a second binding region. In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation and a second binding region arranged in a 5' to 3' orientation ( Figure 1 (i)). In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 3' to 5' orientation and a second binding region arranged in a 3' to 5' orientation ( Figure 1 (ii)). In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 3' to 5' orientation and a second binding region arranged in a 5' to 3' orientation ( Figure 1 (iii)). In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation and a second binding region arranged in a 3' to 5' orientation ( Figure 1 (iv)).

[0047] Figure 2A Schematic diagrams of several embodiments of linear compacted oligonucleotides are shown, each comprising a first binding region, a first intervening junction, a second binding region, a second intervening junction, and a third binding region. In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a second binding region arranged in a 5' to 3' orientation, and a third binding region arranged in a 5' to 3' orientation ( Figure 2A (i)). In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a second binding region arranged in a 5' to 3' orientation, and a third binding region arranged in a 3' to 5' orientation ( Figure 2A (ii)). In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a second binding region arranged in a 3' to 5' orientation, and a third binding region arranged in a 3' to 5' orientation ( Figure 2A (iii)).

[0048] Figure 2BSchematic diagrams of several embodiments of linear compacted oligonucleotides are shown, each comprising a first binding region, a first intervening junction, a second binding region, a second intervening junction, and a third binding region. In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a second binding region arranged in a 3' to 5' orientation, and a third binding region arranged in a 5' to 3' orientation ( Figure 2B (iv)). In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 3' to 5' orientation, a second binding region arranged in a 5' to 3' orientation, and a third binding region arranged in a 5' to 3' orientation ( Figure 2B (v)). In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 3' to 5' orientation, a second binding region arranged in a 3' to 5' orientation, and a third binding region arranged in a 5' to 3' orientation ( Figure 2B (vi)).

[0049] Figure 2C Schematic diagrams of several embodiments of linear compacted oligonucleotides are shown, each comprising a first binding region, a first intervening junction, a second binding region, a second intervening junction, and a third binding region. In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 3' to 5' orientation, a second binding region arranged in a 5' to 3' orientation, and a third binding region arranged in a 5' to 3' orientation ( Figure 2C (vii)). In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 3' to 5' orientation, a second binding region arranged in a 3' to 5' orientation, and a third binding region arranged in a 5' to 3' orientation ( Figure 2C (viii)). In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 3' to 5' orientation, a second binding region arranged in a 5' to 3' orientation, and a third binding region arranged in a 3' to 5' orientation ( Figure 2C (ix)).

[0050] Figure 3A Schematic diagrams of several embodiments of compacted oligonucleotides are shown, each comprising three binding arms connected together by at least one internal intervening junction, wherein individual binding arms comprise binding regions. In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening junction and a first binding region arranged in a 5' to 3' orientation, wherein the 3' end of the first binding region is directed away from the internal intervening junction; (2) an internal intervening junction and a second binding region arranged in a 5' to 3' orientation, wherein the 3' end of the second binding region is directed away from the internal intervening junction; and (3) an internal intervening junction and a third binding region arranged in a 5' to 3' orientation, wherein the 3' end of the third binding region is directed away from the internal intervening junction. Figure 3A(i)). In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening linker and a first binding region arranged in a 3' to 5' orientation, wherein the 5' end of the first binding region is directed away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 3' to 5' orientation, wherein the 5' end of the second binding region is directed away from the internal intervening linker; and (3) an internal intervening linker and a third binding region arranged in a 3' to 5' orientation, wherein the 5' end of the third binding region is directed away from the internal intervening linker ( Figure 3A (ii)).

[0051] Figure 3B Schematic diagrams of several embodiments of compacted oligonucleotides are shown, each comprising three binding arms connected together by at least one internal intervening junction, wherein individual binding arms comprise binding regions. In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening junction and a first binding region arranged in a 5' to 3' orientation, wherein the 3' end of the first binding region is directed away from the internal intervening junction; (2) an internal intervening junction and a second binding region arranged in a 5' to 3' orientation, wherein the 3' end of the second binding region is directed away from the internal intervening junction; and (3) an internal intervening junction and a third binding region arranged in a 3' to 5' orientation, wherein the 5' end of the third binding region is directed away from the internal intervening junction. Figure 3B (iii)). In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening linker and a first binding region arranged in a 5' to 3' orientation, wherein the 3' end of the first binding region is directed away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 3' to 5' orientation, wherein the 5' end of the second binding region is directed away from the internal intervening linker; and (3) an internal intervening linker and a third binding region arranged in a 3' to 5' orientation, wherein the 5' end of the third binding region is directed away from the internal intervening linker. Figure 3B (iv)).

[0052] Figure 4 Schematic diagrams of embodiments of compacted oligonucleotides comprising three binding arms connected by at least one internal intervening linker, wherein each binding arm comprises a first binding region, an intervening linker, and a second binding region. In some embodiments, the compacted oligonucleotide comprises three binding arms, wherein each binding arm comprises: an internal intervening linker, a first binding region arranged in a 5' to 3' orientation, an intervening linker, and a second binding region arranged in a 5' to 3' orientation, wherein the 3' end of the second binding region is directed away from the internal intervening linker.

[0053] Figure 5Schematic diagrams of several embodiments of compacted oligonucleotides are shown, each comprising four binding arms connected together by at least one internal intervening junction, wherein an individual binding arm comprises a binding region. In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening junction and a first binding region arranged in a 5' to 3' orientation, wherein the 3' end of the first binding region is directed away from the internal intervening junction; (2) an internal intervening junction and a second binding region arranged in a 5' to 3' orientation, wherein the 3' end of the second binding region is directed away from the internal intervening junction; (3) an internal intervening junction and a third binding region arranged in a 5' to 3' orientation, wherein the 3' end of the third binding region is directed away from the internal intervening junction; and (4) an internal intervening junction and a fourth binding region arranged in a 5' to 3' orientation, wherein the 3' end of the fourth binding region is directed away from the internal intervening junction. Figure 5 (i)). In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening linker and a first binding region arranged in a 3' to 5' orientation, wherein the 5' end of the first binding region is directed away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 3' to 5' orientation, wherein the 5' end of the second binding region is directed away from the internal intervening linker; (3) an internal intervening linker and a third binding region arranged in a 3' to 5' orientation, wherein the 5' end of the third binding region is directed away from the internal intervening linker; and (4) an internal intervening linker and a fourth binding region arranged in a 3' to 5' orientation, wherein the 5' end of the fourth binding region is directed away from the internal intervening linker. Figure 5 (ii)).

[0054] Figure 6A Shown is a schematic diagram of an embodiment of a double-sided comb-like compacting oligonucleotide, which comprises a plurality of binding arms connected to a central joint portion, wherein an independent binding arm comprises a binding region. In certain embodiments, the compacting oligonucleotide comprises at least three binding arms. In certain embodiments, the compacting oligonucleotide comprises a plurality of binding arms with identical sequences. In certain embodiments, an independent binding arm comprises a first binding region arranged in a 5' to 3' orientation, wherein the 3' end of the first binding region guides away from the joint portion. In certain embodiments, an independent binding arm is connected to the joint portion by an internal intervening joint.

[0055] Figure 6BShown is a schematic diagram of an embodiment of a double-sided comb-shaped compacted oligonucleotide, which comprises a plurality of binding arms connected to a central joint portion, wherein a separate binding arm comprises a binding region. In certain embodiments, the compacted oligonucleotide comprises at least three binding arms. In certain embodiments, the compacted oligonucleotide comprises a plurality of binding arms with one of two different sequences. In certain embodiments, a separate binding arm comprises a first binding region arranged in a 5' to 3' orientation, wherein the 3' end of the first binding region guides away from the joint portion. In certain embodiments, a separate binding arm comprises a second binding region arranged in a 5' to 3' orientation, wherein the 3' end of the second binding region guides away from the joint portion. In certain embodiments, a separate binding arm is connected to the joint portion by an internal intervening joint.

[0056] Figure 6C Shown is a schematic diagram of an embodiment of a double-sided comb-shaped compacted oligonucleotide, which comprises a plurality of binding arms connected to a central joint portion, wherein a separate binding arm comprises a binding region. In some embodiments, the compacted oligonucleotide comprises at least three binding arms. In some embodiments, the compacted oligonucleotide comprises a plurality of binding arms with one of three different sequences. In some embodiments, a separate binding arm comprises a first binding region arranged in a 5' to 3' orientation, wherein the 3' end of the first binding region guides away from the joint portion. In some embodiments, a separate binding arm comprises a second binding region arranged in a 5' to 3' orientation, wherein the 3' end of the second binding region guides away from the joint portion. In some embodiments, a separate binding arm comprises a third binding region arranged in a 5' to 3' orientation, wherein the 3' end of the third binding region guides away from the joint portion. In some embodiments, a separate binding arm is connected to the joint portion by an internal intervening joint.

[0057] Figure 7A Shown is a schematic diagram of an embodiment of a multi-part compaction oligonucleotide comprising a primary linear compaction oligonucleotide that hybridizes with a secondary linear compaction oligonucleotide. In certain embodiments, the primary linear compaction oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a first intervening joint, a second binding region arranged in a 5' to 3' orientation, a second intervening joint, and a connector region arranged in a 5' to 3' orientation. In certain embodiments, the secondary linear compaction oligonucleotide comprises a fastener region arranged in a 3' to 5' orientation, an intervening joint, and a third binding region arranged in a 3' to 5' orientation. In certain embodiments, the connector region of the primary compaction oligonucleotide can hybridize with the fastener region of the secondary compaction oligonucleotide.

[0058] Figure 7BSchematic diagram of an embodiment of a multi-part compaction oligonucleotide is shown, which comprises a primary linear compaction oligonucleotide that hybridizes with a secondary linear compaction oligonucleotide. In certain embodiments, the primary linear compaction oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a first intervening joint, a second binding region arranged in a 5' to 3' orientation, a second intervening joint, and a connector region arranged in a 5' to 3' orientation. In certain embodiments, the secondary linear compaction oligonucleotide comprises a third binding region arranged in a 3' to 5' orientation, an intervening joint, and a fastener region arranged in a 3' to 5' orientation. In certain embodiments, the connector region of the primary compaction oligonucleotide can hybridize with the fastener region of the secondary compaction oligonucleotide.

[0059] Figure 8A Schematic diagram of an embodiment of a multi-part compaction oligonucleotide is shown, which comprises a primary linear compaction oligonucleotide that hybridizes with a secondary linear compaction oligonucleotide. In certain embodiments, the primary linear compaction oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a first intervening joint, a second binding region arranged in a 5' to 3' orientation, a second intervening joint, and a connector region arranged in a 3' to 5' orientation. In certain embodiments, the secondary linear compaction oligonucleotide comprises a fastener region arranged in a 5' to 3' orientation, an intervening joint, and a third binding region arranged in a 5' to 3' orientation. In certain embodiments, the connector region of the primary compaction oligonucleotide can hybridize with the fastener region of the secondary compaction oligonucleotide.

[0060] Figure 8B Schematic diagram of an embodiment of a multi-part compaction oligonucleotide is shown, which comprises a primary linear compaction oligonucleotide that hybridizes with a secondary linear compaction oligonucleotide. In certain embodiments, the primary linear compaction oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a first intervening joint, a second binding region arranged in a 5' to 3' orientation, a second intervening joint, and a connector region arranged in a 3' to 5' orientation. In certain embodiments, the secondary linear compaction oligonucleotide comprises a third binding region arranged in a 5' to 3' orientation, an intervening joint, and a fastener region arranged in a 5' to 3' orientation. In certain embodiments, the connector region of the primary compaction oligonucleotide can hybridize with the fastener region of the secondary compaction oligonucleotide.

[0061] Figure 9Schematic diagram of an embodiment of a multi-part compaction oligonucleotide is shown, which comprises a primary linear compaction oligonucleotide that hybridizes with a secondary linear compaction oligonucleotide. In certain embodiments, the primary linear compaction oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a first intervening joint, a second binding region arranged in a 5' to 3' orientation, a second intervening joint, and a connector region arranged in a 5' to 3' orientation. In certain embodiments, the secondary linear compaction oligonucleotide comprises a third binding region arranged in a 5' to 3' orientation, an intervening joint, and a fastener region arranged in a 3' to 5' orientation. In certain embodiments, the connector region of the primary compaction oligonucleotide can hybridize with the fastener region of the secondary compaction oligonucleotide.

[0062] Figure 10 Schematic diagram of an embodiment of a multi-part compaction oligonucleotide is shown, which comprises a primary linear compaction oligonucleotide that hybridizes with a secondary linear compaction oligonucleotide. In certain embodiments, the primary linear compaction oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a first intervening joint, a second binding region arranged in a 5' to 3' orientation, a second intervening joint, and a connector region arranged in a 3' to 5' orientation. In certain embodiments, the secondary linear compaction oligonucleotide comprises a fastener region arranged in a 5' to 3' orientation, an intervening joint, and a third binding region arranged in a 3' to 5' orientation. In certain embodiments, the connector region of the primary compaction oligonucleotide can hybridize with the fastener region of the secondary compaction oligonucleotide.

[0063] Figure 11 Schematic diagrams of embodiments of multi-part compaction oligonucleotides are shown, comprising a primary linear compaction oligonucleotide that hybridizes with a secondary linear compaction oligonucleotide. In some embodiments, the primary linear compaction oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a first intervening junction, a second binding region arranged in a 5' to 3' orientation, a second intervening junction, and a connector region arranged in a 5' to 3' orientation. In some embodiments, the secondary linear compaction oligonucleotide comprises a fastener region arranged in a 3' to 5' orientation, a first intervening junction, a third binding region arranged in a 3' to 5' orientation, a second intervening junction, and a fourth binding region arranged in a 3' to 5' orientation. In some embodiments, the connector region of the primary compaction oligonucleotide can hybridize with the fastener region of the secondary compaction oligonucleotide.

[0064] Figure 12ASchematic diagrams of embodiments of multi-part compacted oligonucleotides are shown, comprising a primary compacted oligonucleotide having three binding arms, wherein one of the binding arms hybridizes to a secondary linear compacted oligonucleotide. In some embodiments, the primary compacted oligonucleotide comprises three binding arms connected together by at least one internal intervening junction, wherein an individual binding arm comprises a binding region. In some embodiments, the primary compacted oligonucleotide comprises: (1) an internal intervening junction and a first binding region arranged in a 5' to 3' orientation, wherein the 3' end of the first binding region is directed away from the internal intervening junction; (2) an internal intervening junction and a second binding region arranged in a 5' to 3' orientation, wherein the 3' end of the second binding region is directed away from the internal intervening junction; and (3) an internal intervening junction and a connector region arranged in a 5' to 3' orientation, wherein the 3' end of the connector region is directed away from the internal intervening junction. In some embodiments, the secondary linear compacted oligonucleotide comprises a fastener region arranged in a 5' to 3' orientation, an intervening junction, and a third binding region arranged in a 5' to 3' orientation. In some embodiments, the connector region of the primary compaction oligonucleotide can hybridize to the fastener region of the secondary compaction oligonucleotide.

[0065] Figure 12B Schematic diagrams of embodiments of multi-part compacted oligonucleotides are shown, comprising a primary compacted oligonucleotide having three binding arms, wherein one of the binding arms hybridizes to a secondary linear compacted oligonucleotide. In some embodiments, the primary compacted oligonucleotide comprises three binding arms connected together by at least one internal intervening junction, wherein an individual binding arm comprises a binding region. In some embodiments, the primary compacted oligonucleotide comprises: (1) an internal intervening junction and a first binding region arranged in a 3' to 5' orientation, wherein the 5' end of the first binding region is directed away from the internal intervening junction; (2) an internal intervening junction and a second binding region arranged in a 3' to 5' orientation, wherein the 5' end of the second binding region is directed away from the internal intervening junction; and (3) an internal intervening junction and a connector region arranged in a 3' to 5' orientation, wherein the 5' end of the connector region is directed away from the internal intervening junction. In some embodiments, the secondary linear compacted oligonucleotide comprises a fastener region arranged in a 5' to 3' orientation, an intervening junction, and a third binding region arranged in a 5' to 3' orientation. In some embodiments, the connector region of the primary compaction oligonucleotide can hybridize to the fastener region of the secondary compaction oligonucleotide.

[0066] Figure 13AA schematic diagram of an embodiment of a multi-part compacted oligonucleotide is shown, which includes a first nucleic acid strand (100) hybridized to a second nucleic acid strand (200) and a third nucleic acid strand (300). In some embodiments, the first nucleic acid strand includes a first binding region (110) arranged in a 5' to 3' orientation, an intervening junction, and a second binding region arranged in a 5' to 3' orientation. In some embodiments, the second nucleic acid strand includes a third binding region (210) arranged in a 3' to 5' orientation, an intervening junction, and a fourth binding region (220) arranged in a 3' to 5' orientation. In some embodiments, the third binding region (210) can hybridize with at least a portion of the first binding region (110) of the first nucleic acid strand (100). In some embodiments, the fourth binding region (220) can hybridize with at least a portion of the second binding region (120) of the first nucleic acid strand (100). In some embodiments, the third nucleic acid strand (300) includes a fifth binding region (300) arranged in a 3' to 5' orientation. In some embodiments, the fifth binding region (300) can hybridize to at least a portion of the second binding region (120) of the first nucleic acid strand (100). In some embodiments, the fourth binding region (220) and the fifth binding region (300) do not hybridize to the same portion or overlapping portions of the second binding region (120).

[0067] Figure 13B Shown Figure 13A Schematic diagram of a multipart compacted oligonucleotide hybridized to a portion of a nucleic acid concatemer. Figure 13B In the embodiment of the present invention, a portion of the first binding region (110) of the first nucleic acid strand (100) hybridizes with the first portion of the concatemer, which causes a portion of the third binding region (210) to dissociate from the first binding region (110), as shown by the two arrows. The hybridization of a portion of the first binding region (110) and the first portion of the concatemer forms a toehold duplex region. The second binding region (120) of the first nucleic acid strand (100) can remain hybridized with the fourth binding region (220) and the fifth binding region (300).

[0068] Figure 13C Shown Figure 13B Schematic diagram of a multipart compacted oligonucleotide shown in , which hybridizes to different parts of the same nucleic acid concatemer. Figure 13C In the embodiment of the present invention, a portion of the second binding region (120) of the first nucleic acid strand (100) hybridizes with the second portion of the concatemer, which causes a portion of the fifth binding region (300) to dissociate from the second binding region (120), as indicated by the two arrows. The hybridization of the portion of the second binding region (120) and the second portion of the concatemer forms another toehold duplex region. The second nucleic acid strand (200) completely dissociates from the first binding region (110) of the first nucleic acid strand (100).

[0069] Figure 13D Shown Figure 13C Schematic diagram of a multi-part compacted oligonucleotide shown in , wherein the full length of the first binding region (110) hybridizes with the first portion of the nucleic acid concatemer, thereby forming a first toehold duplex region, and the full length of the second binding region (120) hybridizes with the second portion of the same nucleic acid concatemer, thereby forming a second toehold duplex region. The second nucleic acid strand (200) is completely dissociated from the first binding region (110) of the first nucleic acid strand (100). The third nucleic acid strand (300) is completely dissociated from the second binding region (120) of the first nucleic acid strand (100).

[0070] Figures 14A to 14H Table 1 (page 8) is shown, which contains the nucleotide sequences of the compacted oligonucleotides or portions of the compacted oligonucleotides. These sequences are listed in groups of three: (i) the sequence of the first or second binding region of the compacted oligonucleotide; (ii) the reverse sequence of the first or second binding region of the compacted oligonucleotide; and (iii) the full-length sequence of the compacted oligonucleotide with the middle homopolymer region in bold and underlined.

[0071] Figure 15 Table 2 (1 page) is shown, which contains the nucleotide sequences of various universal adapter sequences. The universal adapter sequence can be part of a concatemer molecule having multiple copies of a polynucleotide unit arranged in series, wherein each polynucleotide unit comprises a sequence of interest and at least one universal adapter sequence. The first binding region of the compacting oligonucleotide can hybridize with at least a portion of any one of the universal adapter sequences listed in Table 2. The second binding region of the compacting oligonucleotide sequence can hybridize with at least a portion of any one of the universal adapter sequences listed in Table 2.

[0072] Figure 16A is a schematic diagram illustrating an exemplary linear library molecule comprising: a second surface primer binding site (e.g., SP2; a surface pinning primer binding site); a second index sequence; a first sequencing primer binding site (e.g., a forward sequencing primer binding site); a sequence of interest (e.g., an insert); a second sequencing primer binding site (e.g., a reverse sequencing primer binding site); a first index sequence; and a first surface primer binding site (e.g., SP1; a capture primer binding site). In some embodiments, Figure 16A The linear library molecule shown in is one polynucleotide unit of a concatemer having two or more tandem copies of a polynucleotide unit, wherein each polynucleotide unit comprises a sequence of interest and at least one universal adapter sequence.

[0073] Figure 16Bis a schematic diagram illustrating an exemplary linear library molecule comprising: a second surface primer binding site (e.g., SP2; a surface pinning primer binding site); a first sequencing primer binding site (e.g., a forward sequencing primer binding site); a second index sequence; a sequence of interest (e.g., an insert); a first index sequence; a second sequencing primer binding site (e.g., a reverse sequencing primer binding site); and a first surface primer binding site (e.g., SP1; a capture primer binding site). In some embodiments, Figure 16B The linear library molecule shown in is one polynucleotide unit of a concatemer having two or more tandem copies of a polynucleotide unit, wherein each polynucleotide unit comprises a sequence of interest and at least one universal adapter sequence.

[0074] Figure 17 Schematic diagram showing an exemplary workflow for circularizing linear library molecules. A linear library molecule (A) is hybridized with a double-stranded splint molecule (B), thereby circularizing the library molecule to form a library-splint complex (C) with two cuts. The library molecule (A) comprises: a second surface primer binding site (e.g., SP2; surface pinning primer binding site); a second index sequence; a first sequencing primer binding site (e.g., a forward sequencing primer binding site); a sequence of interest (e.g., an insert); a second sequencing primer binding site (e.g., a reverse sequencing primer binding site); a first index sequence; and a first surface primer binding site (e.g., SP1; a capture primer binding site). The double-stranded splint molecule comprises a first splint chain (a long splint chain) that hybridizes with a second splint chain (a short first splint chain). The first splint chain comprises a first region that hybridizes with a sequence on one end of the linear library molecule and a second region that hybridizes with a sequence on the other end of the linear library molecule. The internal region of the first splint chain hybridizes with the second splint chain.

[0075] Figure 18 Schematic diagram illustrating an exemplary workflow for generating covalently closed circular library molecules using double-stranded splint molecules. The library-splint complex (A) undergoes a ligation reaction to close the two nicks to form a covalently closed circular library molecule (B) that hybridizes to the first splint strand. The first splint strand can be used as an amplification primer to perform a rolling circle amplification reaction (C). The dotted line represents the nascent extension product.

[0076] Figure 19Schematic diagram illustrating an exemplary workflow for circularizing linear library molecules. A linear library molecule (A) hybridizes with a single-stranded splint molecule (B), thereby circularizing the library molecule to form a library-splint complex (C) having one nick. The library molecule (A) comprises: a second surface primer binding site (e.g., SP2; surface pinning primer binding site); a second index sequence; a first sequencing primer binding site (e.g., forward sequencing primer binding site); a sequence of interest (e.g., insert); a second sequencing primer binding site (e.g., reverse sequencing primer binding site); a first index sequence; and a first surface primer binding site (e.g., SP1; capture primer binding site). The single-stranded splint molecule comprises a first region that hybridizes to a sequence on one end of the linear library molecule and a second region that hybridizes to a sequence on the other end of the linear library molecule.

[0077] Figure 20 Schematic diagram showing an exemplary workflow for generating covalently closed circular library molecules using single-stranded splint molecules. The library-splint complex (A) undergoes a ligation reaction to close a nick to form a covalently closed circular library molecule hybridized to the single strand (B). The single-stranded splint strand can be used as an amplification primer to perform a rolling circle amplification reaction (C). The dotted line represents the nascent extension product.

[0078] Figure 21 Schematic diagram of rolling circle amplification reaction on the following exemplary support is shown: (i) through fixed first surface primer, (ii) nucleic acid circular library molecule, (iii) comprising a nucleotide mixture with a nucleotide that can be cut to produce a base-free site with an easy-to-crack portion, (iv) strand displacement polymerase, and (iv) compacted oligonucleotide. The rolling circle amplification reaction produces a fixed single-stranded nucleic acid concatemer template molecule, which has at least one nucleotide with an easy-to-crack portion that can be cut to produce a base-free site in the fixed concatemer template molecule. The arrangement of the various primer binding sequences in the nucleic acid circular library molecule is for illustrative purposes. It will be appreciated by those skilled in the art that many other arrangements are possible. Figures 21 to 28 Shows the Figure 21 The workflow for pairwise sequencing using immobilized concatemer template molecules is depicted in FIG.

[0079] Figure 22 It is shown that by Figure 21 Schematic diagram of an exemplary immobilized nucleic acid nanostructure produced by a rolling circle amplification reaction on a support depicted in FIG. A first binding region of the compacted oligonucleotide hybridizes to a first portion of the immobilized concatemer molecule, and a second binding region of the compacted oligonucleotide hybridizes to a second portion of the concatemer molecule, which causes the concatemer molecule to collapse or fold into the nucleic acid nanostructure.

[0080] Figure 23 It is shown in Figure 22 Schematic diagram of an exemplary forward sequencing reaction performed on an immobilized nucleic acid nanostructure. A forward sequencing reaction can be performed using multiple soluble forward sequencing primers and produce multiple extended forward sequencing primer strands. An immobilized nanostructure can have two or more extended forward sequencing primer strands hybridized thereto.

[0081] Figure 24 is a schematic diagram illustrating an exemplary method for displacing an extended forward sequencing primer strand by performing a primer extension reaction with a strand-displacing polymerase in the absence of a soluble primer to generate a forward extended strand. The primer extension reaction comprises a strand-displacing polymerase, a plurality of nucleotides, and a plurality of compacted oligonucleotides.

[0082] Figure 25 It is shown that by Figure 24 Schematic diagram of the forward extension strand produced by the primer extension reaction shown. For clarity, the compacted oligonucleotide hybridized to the forward extension strand is not shown.

[0083] Figure 26 is a schematic diagram illustrating an exemplary method for generating abasic sites at nucleotides having a cleavable portion in an immobilized nucleic acid nanostructure, and generating gaps at the abasic sites to generate a plurality of gap-containing nanostructures while retaining the plurality of forward extension strands and the plurality of immobilized first surface primers. For clarity, the compacted oligonucleotides hybridized to the forward extension strands are not shown.

[0084] Figure 27 is shown in the removal of Figure 26 Schematic diagram of an exemplary retained forward-extending chain following the formation of a gap-containing nanostructure.

[0085] Figure 28 It is shown in Figure 27 Schematic diagram of an exemplary reverse sequencing reaction performed on the retained forward extension strand shown. The reverse sequencing reaction can be performed using a variety of soluble reverse sequencing primers, a variety of sequencing polymerases, and a variety of nucleotide reagents. The retained forward extension strand can have two or more extended reverse sequencing primer strands hybridized thereto.

[0086] Figure 29 The first surface primer and the second surface primer are fixed thereon. Figure 21Schematic diagram of an exemplary support for immobilized concatemers generated by the RCA workflow on the support depicted in FIG. A portion of the concatemer is hybridized to the immobilized second surface primer. The immobilized concatemer template molecule has two or more copies of the universal binding sequence for the immobilized second surface primer. The portion of the immobilized concatemer template molecule that comprises the universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer.

[0087] Figure 30 Schematic diagram showing an exemplary rolling circle amplification reaction in solution using: (i) a nucleic acid circular library molecule, (ii) a soluble first amplification primer, (iii) a nucleotide mixture comprising nucleotides having a cleavable portion that can be cleaved to generate an abasic site, and (iv) a strand displacement polymerase. The rolling circle amplification reaction produces a single-stranded nucleic acid concatemer molecule in solution having at least one nucleotide with a cleavable portion that can be cleaved to generate an abasic site in the concatemer molecule. The circular library molecule comprises a sequence of interest and at least one universal adapter sequence comprising, for example, a binding sequence for a first surface primer. Figures 30 to 37 Shows the Figure 30 Workflow for pairwise sequencing of concatemer molecules is shown.

[0088] Figure 31 is a schematic diagram illustrating an exemplary method comprising: Figure 30 The rolling circle amplification reaction depicted in is partitioned onto a support having a first surface primer immobilized thereon. The concatemer molecules can hybridize to the immobilized first surface primer.

[0089] Figure 32 is a schematic diagram illustrating an exemplary method, which depicts a rolling circle amplification reaction (e.g., from Figure 31 ) is continued on a support to produce an immobilized concatemer template molecule, the immobilized concatemer template molecule comprising at least one nucleotide with a cleavable portion that can be cleaved to produce an abasic site in the immobilized concatemer template molecule. The rolling circle amplification reaction comprises a plurality of compacted oligonucleotides. The first binding region of the compacted oligonucleotide hybridizes with the first portion of the immobilized concatemer molecule, and the second binding region of the compacted oligonucleotide hybridizes with the second portion of the concatemer molecule, which causes the concatemer molecule to collapse or fold into a nucleic acid nanostructure.

[0090] Figure 33 It is shown in Figure 32Schematic diagram of an exemplary forward sequencing reaction performed on an immobilized nucleic acid nanostructure. The forward sequencing reaction can be performed using multiple soluble forward sequencing primers. The immobilized nucleic acid nanostructure can have two or more extended forward sequencing primer strands hybridized thereto.

[0091] Figure 34 is a schematic diagram illustrating an exemplary method for displacing an extended forward sequencing primer strand by performing a primer extension reaction with a strand-displacing polymerase in the absence of a soluble primer. The primer extension reaction comprises a strand-displacing polymerase, a plurality of nucleotides, and a plurality of compacted oligonucleotides.

[0092] Figure 35 It is shown that by Figure 34 Schematic diagram of the forward extension strand produced by the primer extension reaction shown. For clarity, the compacted oligonucleotide hybridized to the forward extension strand is not shown.

[0093] Figure 36 is a schematic diagram illustrating an exemplary method for generating abasic sites at nucleotides having a cleavable portion in an immobilized nucleic acid nanostructure, and generating gaps at the abasic sites to generate a plurality of gap-containing nanostructures while retaining the plurality of forward extension strands and the plurality of immobilized first surface primers. For clarity, the compacted oligonucleotides hybridized to the forward extension strands are not shown.

[0094] Figure 37 is a forward extension chain showing retention (e.g., from Figure 36 ) is a schematic diagram of an exemplary reverse sequencing reaction performed on a PCR product. The reverse sequencing reaction can be performed using a variety of soluble reverse sequencing primers, a variety of sequencing polymerases, and a variety of nucleotide reagents. The retained forward extended strand is a concatemer molecule that can contain two or more tandem copies of the sequence of interest and various primer binding sites. Such a concatemer molecule can have two or more extended reverse sequencing primer strands hybridized thereon. For simplicity, Figure 37 An exemplary immobilized retained forward extension strand is shown hybridizing to one reverse sequencing primer and undergoing a reverse sequencing reaction to produce an extended reverse sequencing primer strand. A skilled artisan will appreciate that the immobilized retained forward extension strand can hybridize to two or more extended reverse sequencing primer strands.

[0095] Figure 38 is an illustration of an exemplary support having a first surface primer and a second surface primer immobilized thereon and Figure 30-32Schematic diagram of the fixed concatemer produced by the RCA workflow in solution depicted in . A portion of the concatemer hybridizes with the fixed second surface primer. The fixed concatemer template molecule has two or more copies of the universal binding sequence for the fixed second surface primer. The portion of the fixed concatemer template molecule that contains the universal binding sequence for the fixed second surface primer can hybridize with the fixed second surface primer.

[0096] Figure 39 Schematic diagram of rolling circle amplification reaction on the following exemplary support is shown: (i) through fixed first surface primer, (ii) nucleic acid circular library molecule, (iii) lacking nucleotide mixture with nucleotide that can be cut to produce the easy cleavage part of abasic site, (iv) strand displacement polymerase, and (iv) compacting oligonucleotide.The rolling circle amplification reaction produces through fixed single-stranded nucleic acid concatemer template molecule.The arrangement of various primer binding sequences in nucleic acid circular library molecule is for illustrative purposes.Technical staff will understand that many other arrangements are possible. Figures 39 to 45 Shows the Figure 39 The workflow for pairwise sequencing using immobilized concatemer template molecules is depicted in FIG.

[0097] Figure 40 It is shown that by Figure 39 Schematic diagram of an exemplary immobilized nucleic acid nanostructure produced by a rolling circle amplification reaction on a support depicted in FIG. A first binding region of the compacted oligonucleotide hybridizes to a first portion of the immobilized concatemer molecule, and a second binding region of the compacted oligonucleotide hybridizes to a second portion of the concatemer molecule, which causes the concatemer molecule to collapse or fold into the nucleic acid nanostructure.

[0098] Figure 41 It is shown in Figure 40 Schematic diagram of an exemplary forward sequencing reaction performed on an immobilized nucleic acid nanostructure. A forward sequencing reaction can be performed using multiple soluble forward sequencing primers and produce multiple extended forward sequencing primer strands. An immobilized nanostructure can have two or more extended forward sequencing primer strands hybridized thereto.

[0099] Figure 42 is a schematic diagram illustrating an exemplary method for displacing an extended forward sequencing primer strand by performing a primer extension reaction with a strand-displacing polymerase in the absence of a soluble primer to generate a forward extended strand. The primer extension reaction comprises a strand-displacing polymerase, a plurality of nucleotides, and a plurality of compacted oligonucleotides.

[0100] Figure 43 It shows Figure 42Schematic diagram of a continuation of an exemplary strand displacement method is shown, in which a polymerase-catalyzed strand displacement reaction produces: a forward-extending strand that hybridizes to an immobilized concatemer molecule and a partially displaced forward-extending strand, as well as an isolated forward-extending strand that does not hybridize to an immobilized concatemer molecule.

[0101] Figure 44 is a schematic diagram showing an exemplary hybridization complex comprising: a forward extension strand hybridized to an immobilized concatemer molecule and a partially displaced forward extension strand, and an immobilized isolated forward extension strand hybridized to the partially displaced forward extension strand via a compacted oligonucleotide.

[0102] Figure 45 It is shown in Figure 44 Schematic diagram of an exemplary reverse sequencing reaction performed on the hybrid complex shown. The reverse sequencing reaction can be performed using multiple soluble reverse sequencing primers on a partially displaced forward extension strand and an immobilized separated forward extension strand. The reverse sequencing reaction produces extended reverse sequencing primer strands. For simplicity, Figure 45 One copy of the extended reverse sequencing primer strand on the partially displaced forward extension strand and one copy of the extended reverse sequencing primer strand on the immobilized isolated forward extension strand are shown. A skilled artisan will appreciate that the partially displaced forward extension strand and the immobilized isolated forward extension strand may include two or more extended reverse sequencing primer strands hybridized thereto.

[0103] Figure 46 Schematic diagram showing an exemplary rolling circle amplification reaction in solution using: (i) a nucleic acid circular library molecule, (ii) a soluble first amplification primer, (iii) a nucleotide mixture lacking nucleotides having a cleavable portion that can be cleaved to generate an abasic site, and (iv) a strand-displacing polymerase. The rolling circle amplification reaction produces single-stranded nucleic acid concatemer molecules in solution. The circular library molecule comprises a sequence of interest and at least one universal adapter sequence comprising, for example, a binding sequence for a first surface primer. Figures 46 to 53 Shows the Figure 46 Workflow for pairwise sequencing of concatemer molecules is shown.

[0104] Figure 47 is a schematic diagram illustrating an exemplary method comprising: Figure 46 The rolling circle amplification reaction depicted in is partitioned onto a support having a first surface primer immobilized thereon. The concatemer molecules can hybridize to the immobilized first surface primer.

[0105] Figure 48 is a schematic diagram illustrating an exemplary method, which depicts a rolling circle amplification reaction (e.g., from Figure 47) is continued on a support to produce an immobilized concatemer template molecule. The rolling circle amplification reaction comprises a plurality of compacted oligonucleotides. The first binding region of the compacted oligonucleotide hybridizes with the first portion of the immobilized concatemer molecule, and the second binding region of the compacted oligonucleotide hybridizes with the second portion of the concatemer molecule, which causes the concatemer molecule to collapse or fold into a nucleic acid nanostructure.

[0106] Figure 49 It is shown in Figure 48 Schematic diagram of an exemplary forward sequencing reaction performed on an immobilized nucleic acid nanostructure. The forward sequencing reaction can be performed using multiple soluble forward sequencing primers. The immobilized nucleic acid nanostructure can have two or more extended forward sequencing primer strands hybridized thereto.

[0107] Figure 50 is a schematic diagram illustrating an exemplary method for displacing an extended forward sequencing primer strand by performing a primer extension reaction with a strand-displacing polymerase in the absence of a soluble primer. The primer extension reaction comprises a strand-displacing polymerase, a plurality of nucleotides, and a plurality of compacted oligonucleotides.

[0108] Figure 51 It shows Figure 50 Schematic diagram of a continuation of an exemplary strand displacement method is shown, in which a polymerase-catalyzed strand displacement reaction produces: a forward-extending strand that hybridizes to an immobilized concatemer molecule and a partially displaced forward-extending strand, as well as an isolated forward-extending strand that does not hybridize to an immobilized concatemer molecule.

[0109] Figure 52 is a schematic diagram showing an exemplary hybridization complex comprising: a forward extension strand hybridized to an immobilized concatemer molecule and a partially displaced forward extension strand, and an immobilized isolated forward extension strand hybridized to the partially displaced forward extension strand via a compacted oligonucleotide.

[0110] Figure 53 It is shown in Figure 52 Schematic diagram of an exemplary reverse sequencing reaction performed on the hybrid complex shown. The reverse sequencing reaction can be performed using multiple soluble reverse sequencing primers on a partially displaced forward extension strand and an immobilized separated forward extension strand. The reverse sequencing reaction produces extended reverse sequencing primer strands. For simplicity, Figure 53 One copy of the extended reverse sequencing primer strand on the partially displaced forward extension strand and one copy of the extended reverse sequencing primer strand on the immobilized isolated forward extension strand are shown. A skilled artisan will appreciate that the partially displaced forward extension strand and the immobilized isolated forward extension strand may include two or more extended reverse sequencing primer strands hybridized thereto.

[0111] Figure 54 is a schematic diagram of one embodiment of a low binding support comprising a glass substrate and alternating hydrophilic coatings covalently or non-covalently adhered to the glass, and further comprising chemically reactive functional groups that serve as attachment sites for oligonucleotide primers (e.g., capture oligonucleotides and oligonucleotide circularization). In an alternative embodiment, the support can be made of any material such as glass, plastic, or a polymeric material.

[0112] Figure 55A is a schematic diagram of various exemplary configurations of multivalent molecules having starburst or helter-skelter configurations. The nucleotide unit is designated 'N', biotin is designated 'B', and streptavidin is designated 'SA'.

[0113] Figure 55B Schematic diagram of an exemplary multivalent molecule having a dendrimer configuration. The nucleotide unit is designated 'N'.

[0114] Figure 55C Schematic diagram of examples of multiple multivalent molecules formed by reacting streptavidin with 4-arm or 8-arm PEG-NHS with biotin and dNTPs. The nucleotide unit is designated as 'N', biotin as 'B', and streptavidin as 'SA'.

[0115] Figure 56 is a schematic diagram of an exemplary multivalent molecule comprising a universal core attached to multiple nucleotide arms.

[0116] Figure 57 is a schematic diagram of an exemplary multivalent molecule comprising a dendrimer core attached to multiple nucleotide arms.

[0117] Figure 58 A schematic diagram of an exemplary multivalent molecule comprising a core attached to multiple nucleotide arms, wherein the nucleotide arms comprise biotin, a spacer, a linker, and nucleotide units is shown.

[0118] Figure 59 is a schematic diagram of an exemplary nucleotide arm comprising a core attachment moiety, a spacer, a linker, and a nucleotide unit.

[0119] Figure 60 Shown are the chemical structures of exemplary spacers (top) and various exemplary linkers, including an 11-atom linker, a 16-atom linker, a 23-atom linker, and an N3 linker (bottom).

[0120] Figure 61 The chemical structures of various exemplary linkers are shown, including Linkers 1-9.

[0121] Figure 62A The chemical structures of various exemplary linkers joined / attached to the nucleotide units are shown.

[0122] Figure 62B The chemical structures of various exemplary linkers joined / attached to the nucleotide units are shown.

[0123] Figure 62C The chemical structures of various exemplary linkers joined / attached to the nucleotide units are shown.

[0124] Figure 62D The chemical structures of various exemplary linkers joined / attached to the nucleotide units are shown.

[0125] Figure 63 The chemical structure of an exemplary nucleotide arm is shown. In this example, the nucleotide unit is connected to the linker via a propargylamine attachment at the 5 position of a pyrimidine base or at the 7 position of a purine base. The nucleotide arm shows an exemplary biotinylated nucleotide arm.

[0126] Figure 64 is a schematic diagram of a guanine tetrad (eg, a G-tetrad).

[0127] Figure 65 is a schematic diagram of an exemplary intramolecular G-quadruplex structure.

[0128] Figure 66 Shown is a series of fluorescent images of the nucleic acid nanostructure that is fixed to support.By carrying out rolling circle amplification on the support as negative control (none) with various compaction oligonucleotides (100nM) or without compaction oligonucleotide, produce nanostructure.Through fixing nanostructure and through fluorescently labeled probe hybridization, washing and imaging.The SEQ ID NO of compaction oligonucleotide is shown in the upper left corner of each image.Image illustrates, and gained is subject to the influence of measured compaction oligonucleotide type through the shape and size of fixing nanostructure.

[0129] Figure 67 Shown Figure 66 Magnifications of some of the images. Figure 67 A. Negative control: Immobilized concatemers are less compact and have a "fuzzy" appearance. Figure 67 B. Compacted oligonucleotide (SEQ ID NO: 126): The immobilized nucleic acid nanostructures are more compact compared to the negative control. Figure 67 C. Compacted oligonucleotide (SEQ ID NO: 57): compared with negative control and images shown in the inset ( Figure 67 B) The immobilized nucleic acid nanostructures are more compact and discrete compared to .

[0130] Figure 68Be the box plot that illustrates the quantity of each visual field through fixing nanostructure.Carry out rolling circle amplification on support as negative control by using the titration concentration of various compacting oligonucleotide or without compacting oligonucleotide and produce nanostructure.Test compacting oligonucleotide under 25nM, 100nM, 250nM and 500nM.Tested compacting oligonucleotide comprises SEQ ID NO:57,126 and 156 (see the sequence in Table 1).Data illustrate, compacting oligonucleotide does not suppress rolling circle amplification reaction.The quantity through fixing nanostructure (point) of three kinds of different compacting oligonucleotides tested in negative control and this experiment is similar.

[0131] Figure 69 It shows Figure 68 The data show that the FWHM of the nanostructures produced with the compacted oligonucleotides is smaller than that of the negative control. The data also show that the fixed nanostructures produced with the compacted oligonucleotides comprising SEQ ID NO: 57 have a smaller FWHM than those produced with the compacted oligonucleotides comprising SEQ ID NO: 126 or 156.

[0132] Figure 70 It shows Figure 68 Box plot of signal intensity for the immobilized nanostructures described in . The data show that the signals detected from nanostructures generated with compacted oligonucleotides have a smaller distribution (more discrete) compared to the negative control.

[0133] Figure 71 A series of four-color fluorescence images of a first-strand nanostructure immobilized on a support are shown. The first-strand nanostructure was generated by on-support rolling circle amplification using a compacted oligonucleotide and a nucleotide mixture with titrated concentrations of dUTP or without dUTP as a negative control. Sequencing reagents were flowed onto the immobilized nanostructure to form a fluorescent binding complex on the first-strand nanostructure. Images of the resulting binding complex were obtained. The procedure for obtaining the fluorescence images is described in Example 3.

[0134] Figure 72 A series of four-color fluorescence images of the second chain nanostructure fixed to the support are shown. Figure 71 The first strand nanostructure described in Example 3 was generated. A second strand nanostructure was generated by performing a primer extension reaction on the first strand nanostructure using a nucleotide mixture lacking dUTP and a compacted oligonucleotide. The first strand was removed by enzymatic degradation, while retaining the second strand molecules. Sequencing reagents were flowed onto the immobilized second template strand to form a fluorescent binding complex on the second strand nanostructure. An image of the resulting binding complex was obtained. The procedure for obtaining the fluorescent image is described in Example 3.

[0135] Figure 73 Four-color fluorescence images of fluorescent binding complexes on immobilized first-strand nanostructures (top) and second-strand nanostructures (bottom) are shown, where the second strands were generated from their corresponding first strands on the same flow cell. Figure 73 The white outline boxes in the top and bottom images of represent the same field of view. The white arrows indicate the positions of groups of nanostructures that can be easily identified and compared in the top and bottom images. DETAILED DESCRIPTION

[0136] definition:

[0137] The headings provided herein are not limitations of the individual aspects of the disclosure, which aspects can be understood by reference to the specification as a whole.

[0138] Unless otherwise defined, the technology and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art, unless otherwise defined. Generally speaking, the terms related to the technology of molecular biology described herein, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production and hybridization are well-known and commonly used terms in the art. The technology and procedures described herein are usually performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. For example, referring to Sambrook et al., Molecular Cloning:A Laboratory Manual (3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000). See also Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclature used in conjunction with laboratory procedures described herein and technology is well known and commonly used in the art.

[0139] Unless the context otherwise requires, singular terms shall include pluralities and plural terms shall include the singular. The singular forms "a / an" and "the" and any word used in the singular include plural referents unless expressly and unequivocally limited to one referent.

[0140] It will be understood that use of alternative terms (eg, "or") is taken to mean one or both or any combination of the alternatives.

[0141] As used herein, the term "and / or" should be taken to mean a specific disclosure of each of the specified features or components with or without the other. For example, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include: "A and B"; "A or B"; "A" (A alone); and "B" (B alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" herein is intended to cover each of the following aspects: "A, B, and C"; "A, B, or C"; "A or C"; "A or B"; "B or C"; "A and B"; "B and C"; "A and C"; "A" (A alone); "B" (B alone); and "C" (C alone).

[0142] As used herein and in the appended claims, the terms "comprises," "including," "having," and "containing," and grammatical variations thereof, as used herein, are intended to be non-limiting, such that the inclusion of one or more items in a list does not exclude other items that can be substituted or added to the listed items. It should be understood that whenever aspects are described herein with the language "comprising," other similar aspects described with "consisting of" and / or "consisting essentially of" are also provided.

[0143] As used herein, the terms "about" and "approximately" refer to values ​​or compositions within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" or "approximately" may mean within one or more standard deviations. Alternatively, "about" or "approximately" may mean a range of up to 10% (i.e., ±10%) or greater, depending on the limitations of the measurement system. For example, about 5 mg may include any number between 4.5 mg and 5.5 mg. In addition, specifically with respect to biological systems or processes, the term may mean up to an order of magnitude or up to 5 times the value. When a specific value or composition is provided in the present disclosure, unless otherwise stated, the meaning of "about" or "approximately" should be assumed to be within an acceptable error range for that specific value or composition. In addition, where a range and / or subrange of values ​​is provided, the range and / or subrange may include the endpoints of the range and / or subrange.

[0144] The term "biological sample" refers to a section of any one of a single cell, a plurality of cells, a tissue, an organ, an organism or these biological samples. A biological sample can be extracted from an organism (e.g., a biopsy) or obtained from a cell culture grown in a liquid or in a culture dish. A biological sample comprises a fresh, frozen, freshly frozen or archived sample (e.g., formalin-fixed paraffin-embedded; FFPE). A biological sample can be embedded in wax, resin, epoxy resin or agar. For example, a biological sample can be fixed in any one of the following or any combination of two or more of the following: acetone, ethanol, methanol, formaldehyde, paraformaldehyde-Triton or glutaraldehyde. A biological sample can be sliced ​​or unsliced. A biological sample can be stained, decolorized or unstained.

[0145] Nucleic acids of interest can be extracted from biological samples using any of a variety of techniques known to those skilled in the art. For example, a typical DNA extraction procedure involves: (i) collecting a cell sample or tissue sample from which DNA is to be extracted, (ii) disrupting the cell membrane (i.e., cell lysis) to release the DNA and other cytoplasmic components, (iii) treating the lysed sample with a concentrated salt solution to precipitate proteins, lipids, and RNA, followed by centrifugation to separate the precipitated proteins, lipids, and RNA, and (iv) purifying the DNA from the supernatant to remove detergents, proteins, salts, or other reagents used during cell membrane lysis. A variety of suitable commercial nucleic acid extraction and purification kits are consistent with the disclosure herein. Examples include, but are not limited to, the QIAamp kit (for isolating genomic DNA from human samples) and the DNAeasy kit (for isolating genomic DNA from animal or plant samples) from Qiagen (Germantown, MD), or the DNAeasy kit (for isolating genomic DNA from animal or plant samples) from Promega (Madison, WI). and ReliaPrep TM Series of kits.

[0146] As used herein, the terms "nucleic acid," "polynucleotide," and "oligonucleotide," as well as other related terms, are used interchangeably and refer to polymers of nucleotides and are not limited to any specific length. Nucleic acids include recombinant and chemically synthesized forms. Nucleic acids can be isolated. Nucleic acids include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA produced using nucleotide analogs (e.g., peptide nucleic acids (PNA) and non-naturally occurring nucleotide analogs), and chimeric forms containing DNA and RNA. Nucleic acids can be single-stranded or double-stranded. Nucleic acids include polymers of nucleotides, wherein the nucleotides include natural or non-natural bases and / or sugars. Nucleic acids include naturally occurring internucleoside bonds, such as phosphodiester bonds. Nucleic acids may lack phosphate groups. Nucleic acids include non-natural internucleoside bonds, including phosphorothioate, sulfur-containing phosphate, or peptide nucleic acid (PNA) bonds. In some embodiments, nucleic acids include a mixture of one type of polynucleotide or two or more different types of polynucleotides.

[0147] The terms "universal sequence," "universal adapter sequence," and related terms refer to a sequence in a nucleic acid molecule that is shared between two or more polynucleotide molecules. For example, an adapter having the same universal sequence can be joined to multiple polynucleotides such that the population of co-joined molecules carries the same universal adapter sequence. Examples of universal adapter sequences include amplification primer sequences, sequencing primer sequences, or capture primer sequences (e.g., soluble or support-immobilized capture primers). Table 2 ( Figure 15 ) are listed in .

[0148] As used herein, the terms "operably linked" and "operably connected" or related terms refer to the juxtaposition of components. The juxtaposed components can be covalently linked together. For example, two nucleic acid components can be enzymatically linked together, wherein the bond joining the two components together comprises a phosphodiester bond. A first nucleic acid component and a second nucleic acid component can be linked together, wherein the first nucleic acid component can confer a function to the second nucleic acid component. For example, the bond between the primer binding sequence and the sequence of interest forms a nucleic acid library molecule having a portion that can bind to a primer. In another example, a transgene (e.g., a nucleic acid encoding a polypeptide or a target nucleic acid sequence) can be linked to a vector, wherein the link allows the transgene sequence contained in the vector to be expressed or function. In some embodiments, the transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects transgene expression. In some embodiments, the vector includes at least one host cell regulatory sequence comprising a promoter sequence, an enhancer, a transcription and / or translation initiation sequence, a transcription and / or translation termination sequence, a polypeptide secretion signal sequence, and the like. In some embodiments, the host cell regulatory sequence controls the expression of the level, timing, and / or position of the transgene.

[0149] The terms "linked," "connected," "attached," "appended," and variations thereof include any type of fusion, binding, attachment, or association between any combination of compounds or molecules that is sufficiently stable to withstand use in a particular procedure. The procedure may include, but is not limited to, nucleotide binding; nucleotide incorporation; deblocking (e.g., removal of a chain terminating moiety); washing; removal; flow; detection; imaging and / or identification. Such bonds may include, for example, covalent bonding, ionic bonding, hydrogen bonding, dipole-dipole bonding, hydrophilic bonding, hydrophobic bonding, or affinity bonding, bonds or associations involving van der Waals forces, mechanical bonding, and the like. In some embodiments, such bonds occur within a molecule, such as connecting the ends of a single-stranded or double-stranded linear nucleic acid molecule together to form a circular molecule. In some embodiments, such bonds may occur between a combination of different molecules or between a molecule and a non-molecule, including, but not limited to, a bond between a nucleic acid molecule and a solid surface; a bond between a protein and a detectable reporter gene portion; a bond between a nucleotide and a detectable reporter gene portion; and the like. Some examples of bonds can be found in, e.g., Hermanson, G., “Bioconjugate Techniques”, 2nd ed. (2008); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998); Aslam, M., Dent, A., “Bioconjugation: Protein Coupling Techniques for the Biomedical Sciences”, London: Macmillan (1998).

[0150] The term "adapter" and related terms refer to oligonucleotides that can be operably linked (appended) to a target polynucleotide, wherein the adaptor pair conferring function to a co-linked adaptor-target molecule. The adaptor comprises DNA, RNA, chimeric DNA / RNA or its analogs. The adaptor may include at least one ribonucleoside residue. The adaptor may be single-stranded, double-stranded or have a single-stranded and / or double-stranded portion. The adaptor may be configured into a linear form, a stem-loop form, a hairpin form or a Y-shaped form. The adaptor may be of any length, including 4-100 nucleotides or longer. The adaptor may have a blunt end, an overhang end or a combination thereof. The overhang end may include a 5' overhang end and a 3' overhang end. The 5' end of a single-stranded adaptor or a chain of a double-stranded adaptor may have a 5' phosphate group or lack a 5' phosphate group. The adaptor may include a 5' tail (e.g., a tail adaptor) that is not hybridized with the target polynucleotide, or the adaptor may be tailless. The adapter may comprise a sequence that is complementary to at least a portion of a primer, such as an amplification primer, a sequencing primer, or a capture primer (e.g., a soluble or immobilized capture primer). The adapter may comprise a random sequence or a degenerate sequence. The adapter may comprise at least one inosine residue. The adapter may comprise at least one phosphorothioate, phosphorothiol, and / or phosphoramidite bond. The adapter may comprise a barcode sequence that can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in a multiplex assay. The adapter may comprise a unique identification sequence (e.g., a unique molecular index, UMI; or a unique molecular tag) that can be used to uniquely identify the nucleic acid molecule to which the adapter is appended. In some embodiments, the unique identification sequence can be used to increase error correction and accuracy, reduce the rate of false positive variant calls, and / or increase the sensitivity of variant detection. The adapter may comprise at least one restriction enzyme recognition sequence, including any one or any combination of two or more selected from the group consisting of type I, type II, type III, type IV, type Hs, or type IIB.

[0151] The terms "nucleic acid template," "template polynucleotide," "nucleic acid target," "target polynucleotide," "template strand," and other variations refer to a nucleic acid strand that serves as a base nucleic acid molecule for any of the analytical methods described herein (e.g., primer extension, amplification, and / or sequencing). The template nucleic acid can be single-stranded or double-stranded, or the template nucleic acid can have single-stranded or double-stranded portions. The template nucleic acid can be obtained from naturally occurring sources, recombinant forms, or chemically synthesized to include any type of nucleic acid analog. The template nucleic acid can be linear, circular, or in other forms. The template nucleic acid can include an insertion region having an insertion sequence also referred to as a sequence of interest. The template nucleic acid can also include at least one adapter sequence. The template nucleic acid can be a concatemer having two or tandem copies of a sequence of interest and at least one adapter sequence. The insertion region can be isolated in any form, including chromosomal, genomic, organelle (e.g., mitochondrial, chloroplast, or ribosomal) recombinant molecules, cloned, amplified cDNA, RNA (such as pre-mRNA or mRNA), oligonucleotides, whole genomic DNA obtained from fresh frozen paraffin embedded tissue, needle biopsy, circulating tumor cells, cell-free circulating DNA, or any type of nucleic acid library. The insertion region can be isolated from any source, including from organisms such as prokaryotes, eukaryotes (e.g., humans, plants, and animals), fungi, viruses, cells, tissues, normal or diseased cells or tissues, body fluids including blood, urine, serum, lymph, tumors, saliva, anal and vaginal secretions, amniotic fluid samples, sweat, semen, environmental samples, culture samples, or synthetic nucleic acid molecules prepared using recombinant molecular biology or chemical synthesis methods. Insertion regions can be isolated from any organ, including the head, neck, brain, breast, ovary, cervix, colon, rectum, endometrium, gallbladder, intestine, bladder, prostate, testis, liver, lung, kidney, esophagus, pancreas, thyroid, pituitary gland, thymus, skin, heart, larynx, or other organs. Template nucleic acids can be subjected to nucleic acid analysis (including sequencing and composition analysis).

[0152] As used herein, the term "polymerase" and its variants include enzymes comprising a domain that binds nucleotides (or nucleosides), wherein the polymerase can form a complex with a template nucleic acid and a complementary nucleotide. The polymerase can have one or more activities, including but not limited to: base analog detection activity, DNA polymerization activity, reverse transcriptase activity, DNA binding, strand displacement activity, and nucleotide binding and recognition. The polymerase can be any enzyme that can catalyze the polymerization of nucleotides (including analogs thereof) into nucleic acid chains. Typically, but not necessarily, such nucleotide polymerization can occur in a template-dependent manner. Typically, the polymerase includes one or more active sites at which nucleotide binding and / or nucleotide polymerization catalysis can occur. In some embodiments, the polymerase includes other enzymatic activities, such as 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, the polymerase has strand displacement activity. Polymerases may include, but are not limited to, naturally occurring polymerases and any of their subunits and truncations, mutant polymerases, variant polymerases, recombinant, fused or otherwise engineered polymerases, chemically modified polymerases, synthetic molecules or assemblies, and any analogs, derivatives or fragments thereof (e.g., catalytically active fragments) that retain the ability to catalyze nucleotide polymerization. Polymerases include catalytically inactive polymerases, catalytically active polymerases, reverse transcriptases and other enzymes comprising a nucleotide binding domain. In certain embodiments, polymerases can be isolated from cells or produced using recombinant DNA technology or chemical synthesis methods. In certain embodiments, polymerases can be expressed in prokaryotes, eukaryotes, viruses or phage organisms. In certain embodiments, polymerases can be post-translationally modified proteins or fragments thereof. Polymerases can be derived from prokaryotes, eukaryotes, viruses or phages. Polymerases include DNA-guided DNA polymerases and RNA-guided DNA polymerases.

[0153] The term "strand displacement" refers to the ability of a polymerase to locally separate double-stranded nucleic acid chains and synthesize new chains in a template-based manner. The strand displacement polymerase displaces the complementary strand from the template strand and catalyzes the synthesis of the new strand. Strand displacement polymerases include mesophilic and thermophilic polymerases. Strand displacement polymerases include wild-type enzymes and variants (including exonuclease-minus mutants, mutant versions, chimeric enzymes, and truncated enzymes). Examples of strand displacement polymerases include phi29 DNA polymerase, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment (exo-), Bca DNA polymerase (exo-), Klenow fragment of Escherichia coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase can be a wild-type phi29 DNA polymerase (e.g., from Expedeon TMMagniPhi), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific TM ), or chimeric QualiPhi DNA polymerase (e.g., from 4basebio TM ).

[0154] As used herein, the term "fidelity" refers to the accuracy of DNA polymerization performed by a template-dependent DNA polymerase. The fidelity of a DNA polymerase is typically measured by the error rate (the frequency of incorporation of inaccurate nucleotides (i.e., nucleotides that are not complementary to the template nucleotides). The accuracy or fidelity of DNA polymerization is maintained by the polymerase activity and 3'-5' exonuclease activity of the DNA polymerase.

[0155] As used herein, the term "binding complex" refers to a complex formed by combining a nucleic acid duplex, a polymerase, and free nucleotides or nucleotide units of a multivalent molecule, wherein the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a nucleic acid primer. In the binding complex, the free nucleotides or nucleotide units may or may not be bound to the 3' end of the nucleic acid primer at a position opposite to the complementary nucleotide in the nucleic acid template molecule. A "ternary complex" is an example of a binding complex formed by combining a nucleic acid duplex, a polymerase, and free nucleotides or nucleotide units of a multivalent molecule, wherein the free nucleotides or nucleotide units are bound to the 3' end of the nucleic acid primer (as part of the nucleic acid duplex) at a position opposite to the complementary nucleotide in the nucleic acid template molecule.

[0156] The term "residence time" and related terms refer to the length of time that a binding complex remains stable without any component dissociation, wherein the components of the binding complex include nucleic acid templates and nucleic acid primers, polymerases, nucleotide units or free (e.g., unconjugated) nucleotides of multivalent molecules. Nucleotide units or free nucleotides can be complementary or non-complementary to the nucleotide residues in the template molecule. Nucleotide units or free nucleotides can be combined with the 3' end of the nucleic acid primer at a position relative to the complementary nucleotide residues in the nucleic acid template molecule. The stability of the binding complex and the intensity of the binding interaction are indicated by the retention time. The retention time can be measured by observing the start and / or duration of the binding complex (e.g., by observing the signal of the labeled component from the binding complex). For example, labeled nucleotides or labeled reagents comprising one or more nucleotides can be present in the binding complex, thereby allowing the signal from the label to be detected during the residence time of the binding complex. An exemplary label is a fluorescent marker. Binding complex (e.g., a ternary complex) remains stable before being subjected to the conditions of the interactional dissociation that causes polymerase, template molecule, primer and / or nucleotide units or nucleotides. For example, dissociation conditions include contacting the bound complex with any one of detergent, EDTA, and / or water, or any combination thereof.

[0157] The term "primer" and related terms used herein refer to oligonucleotides that can hybridize with DNA and / or RNA polynucleotide templates to form duplex molecules. Primers include natural nucleotides and / or nucleotide analogs. Primers can be recombinant nucleic acid molecules. Primers can have any length, but generally range from 4 to 50 nucleotides. Typical primers include 5' ends and 3' ends. The 3' end of a primer can include a 3'OH portion that serves as a nucleotide polymerization initiation site in a primer extension reaction catalyzed by a polymerase. Alternatively, the 3' end of a primer can lack a 3'OH portion or can include an end 3' blocking group that inhibits nucleotide polymerization in a polymerase-catalyzed reaction. Any one nucleotide along the length of the primer or more than one nucleotide can be labeled with a detectable reporter gene portion. Primers can be in solution (e.g., soluble primers) or can be fixed to a support (e.g., capture primers).

[0158] When used to refer to nucleic acid molecules, the term "hybridize" or "hybridizing" or "hybridization" or other related terms refer to hydrogen bonding between two different nucleic acids to form a duplex nucleic acid. Hybridization also includes hydrogen bonding between two different regions of a single nucleic acid molecule to form a self-hybridizing molecule with a duplex region. Hybridization can include Watson-Crick or Hoogstein binding to form a duplex double-stranded nucleic acid or a double-stranded region within a nucleic acid molecule. The two different regions of a double-stranded nucleic acid or a single nucleic acid can be fully complementary or partially complementary. The complementary nucleic acid chains do not need to hybridize to each other across their entire length. Complementary base pairing can be standard AT or CG base pairing or can be other forms of base pairing interactions. The duplex nucleic acid can include mismatched base-paired nucleotides.

[0159] When used in reference to nucleic acids, the terms "extend," "extending," "extension," and other variants refer to the incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation comprises the polymerization of one or more nucleotides into the 3' OH end of a nucleic acid chain (e.g., a nucleic acid primer), resulting in an extension of the nucleic acid chain (e.g., an extended primer). Nucleotide incorporation can be performed using natural nucleotides and / or nucleotide analogs. Typically, but not necessarily, nucleotide incorporation occurs in a template-dependent manner. Any suitable method for extending nucleic acid molecules can be used, including primer extension catalyzed by a DNA polymerase or an RNA polymerase.

[0160] In some embodiments, any of the amplification primer sequence, sequencing primer sequence, capture primer sequence (capture oligonucleotide), target capture sequence, circularization anchor sequence, sample barcode sequence, spatial barcode sequence, or anchor region sequence can be about 3 to 50 nucleotides in length, about 5 to 40 nucleotides in length, or about 5 to 25 nucleotides in length.

[0161] The term "nucleotide" and related terms refer to molecules comprising an aromatic base, a five-carbon sugar (e.g., ribose or deoxyribose) and at least one phosphate group. Regular or non-regular nucleotides are consistent with the use of the term. In certain embodiments, phosphates include monophosphates, diphosphates, or triphosphates or corresponding phosphate analogs. The term "nucleoside" refers to a molecule comprising an aromatic base and a sugar. Nucleotides and nucleosides can be unlabeled or labeled with a detectable reporter moiety.

[0162] Nucleotides (and nucleosides) typically contain heterocyclic bases, including substituted or unsubstituted nitrogen-containing parent heteroaromatic rings, which are commonly found in nucleic acids, including naturally occurring, substituted, modified or engineered variants or analogs thereof. The base of a nucleotide (or nucleoside) is capable of forming Watson-Crick and / or Hoostein hydrogen bonds with an appropriate complementary base. Exemplary bases include, but are not limited to, purines and pyrimidines, such as: 2-aminopurine, 2,6-diaminopurine, adenine (A), ethyleneadenine, N 6 -Δ 2 -Isopentenyl adenine (6iA), N 6 -Δ 2 -Isopentenyl-2-methylthioadenine (2ms6iA), N 6 -methyladenine, guanine (G), isoguanine, N 2 -dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG), hypoxanthine and O 6 -methylguanine; 7-deaza-purines, such as 7-deazaadenine (7-deazaadenine-A) and 7-deazaguanine (7-deaza-G); pyrimidines, such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5,6-dihydrothymine, O 4 -methylthymine, uracil (U), 4-thiouracil (4sU) and 5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and 4-methylindole; pyrroles such as nitropyrrole; muscimol; inosine; hydroxymethylcytosine; 5-methylcytosine; base (Y); and methylated, glycosylated and acylated base moieties; etc. Additional exemplary bases can be found in Fasman, 1989, in "Practical Handbook of Biochemistry and Molecular Biology", pages 385-394, CRC Press, Boca Raton, Fla.

[0163] Nucleotides (and nucleosides) typically contain a sugar moiety, such as a carbocyclic moiety (Ferraro and Gotor 2000 Chem. Rev. 100:4319-48), an acyclic moiety (Martinez et al., 1999 Nucleic Acids Research 27:1271-1274; Martinez et al., 1997 Bioorganic & Medicinal Chemistry Letters Vol. 7:3013-3016), and other sugar moieties (Joeng, et al., 1993 J. Med. Chem. 36:2627-2638; Kim, et al., 1993 J. Med. Chem. 36:30-7; Eschenmosser 1999 Science 284:2118-2124; and U.S. Pat. No. 5,558,991). Sugar moieties include: ribosyl; 2'-deoxyribosyl; 3'-deoxyribosyl; 2',3'-dideoxyribosyl; 2',3'-didehydrodideoxyribosyl; 2'-alkoxyribosyl; 2'-azidoribosyl; 2'-aminoribosyl; 2'-fluororibosyl; 2'-thiolribosyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-thiolribosyl; 3'-alkylthioribosyl carbocyclic; acyclic or other modified sugars.

[0164] In certain embodiments, the nucleotide comprises a chain of one, two or three phosphorus atoms, wherein the chain is typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramide bond. In certain embodiments, the nucleotide is an analog with a phosphorus chain, wherein the phosphorus atom is linked together with an O, S, NH, methylene or ethylene group in the middle. In certain embodiments, the phosphorus atom in the chain comprises a substituted side group (including O, S or BH3). In certain embodiments, the chain comprises a phosphate group substituted with an analog, and the analog comprises phosphoramide, phosphorothioate, phosphorodithioate and O-methylphosphoramidite groups.

[0165] The term "reporter moiety," "reporter moieties," or related terms refers to a compound that produces or causes a detectable signal. A reporter moiety is sometimes referred to as a "label." Any suitable reporter moiety can be used, including luminescence, photoluminescence, electroluminescence, bioluminescence, chemiluminescence, fluorescence, phosphorescence, chromophores, radioisotopes, electrochemistry, mass spectrometry, Raman, haptens, affinity tags, atoms, or enzymes. The reporter moiety produces a detectable signal caused by a chemical or physical change (e.g., heat, light, electricity, pH, salt concentration, enzyme activity, or a neighboring event). A neighboring event comprises two reporter moieties that are close to, associated with, or bound to each other. It is well known to those skilled in the art that the reporter moiety is selected so that each reporter moiety absorbs excitation radiation and / or fluoresces at a wavelength that can be distinguished from other reporter moieties, to allow monitoring of the presence of different reporter moieties in the same reaction or in different reactions. Two or more different reporter moieties can be selected that have spectrally distinct emission curves or have minimal overlapping spectral emission curves. The reporter moiety can be linked (eg, operably linked) to a nucleotide, nucleoside, nucleic acid, enzyme (eg, a polymerase or reverse transcriptase), or support (eg, a surface).

[0166] As used herein, a "nucleotide unit" or "nucleotide moiety" refers to a nucleotide (e.g., dATP, dTTP, dGTP, dCTP, or dUTP) or an analog thereof, comprising a base, a sugar, and at least one phosphate group. A nucleotide unit can be attached to a multivalent molecule used in a sequencing reaction as described herein. Generally, all nucleotide units attached to the same multivalent molecule will have the same identity (e.g., all A, all T, all C, or all G), although those skilled in the art will appreciate that there may be multivalent molecules in which nucleotide units of different identities may be advantageous.

[0167] Reporter moieties (or labels) include fluorescent labels or fluorophores. Exemplary fluorescent moieties that can be used as fluorescent labels or fluorophores include, but are not limited to, fluorescein and fluorescein derivatives such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynaphthol fluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamido fluorescein, maleimide fluorescein, SAMSA-fluorescein, thiosemicarbazide fluorescein, hydrazine carbonate methylthioacetamido fluorescein, rhodamine and rhodamine derivatives such as TRITC, TMR, lissamine rhodamine, Texas Red, Rhodamine B, Rhodamine 6G, Rhodamine 10, NHS-rhodamine, TMR-iodoacetamide, lissamine rhodamine B sulfonyl chloride, lissamine rhodamine B sulfonyl hydrazide, Texas Red sulfonyl chloride, Texas Red hydrazide, coumarin and coumarin derivatives such as AMCA, AMCA-NHS, AMCA-sulfo-NHS, AMCA-HPDP, DCIA, AMCE-hydrazide, BODIPY and derivatives such as BODIPY FL C3-SE, BODIPY 530 / 550 C3, BODIPY530 / 550 C3-SE, BODIPY 530 / 550 C3 hydrazide, BODIPY 493 / 503 C3 hydrazide, BODIPY FL C3 hydrazide, BODIPY FL IA, BODIPY 530 / 551 IA, Br-BODIPY 493 / 503, Cascade Blue and derivatives such as Cascade Blue acetyl triazoide, Cascade Blue cadaverine, Cascade Blue ethylenediamine, Cascade Blue hydrazide, Lucifer Yellow and derivatives such as Lucifer Yellow iodoacetamide, Lucifer Yellow CH, cyanines and derivatives such as indolium cyanine dyes, benzindolium cyanine dyes, pyridinium cyanine dyes, thiazolium cyanine dyes, quinolinium cyanine dyes, imidazolium cyanine dyes, Cy 3. Cy5, lanthanide chelates and derivatives, such as BCPDA, TBP, TMT, BHHCT, BCOT, europium chelates, terbium chelates, Alexa Fluor dyes, DyLight dyes, Atto dyes, LightCycler Red dyes, CAL Flour dyes, JOE and its derivatives, Oregon Green dye, WellRED dye, IRD dye, phycoerythrin and phycobilin dyes, malachite green, diphenylethylene, DEG dyes, NR dyes, near-infrared dyes, and other dyes known in the art, such as Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th edition;Those described in Lakowicz, Principles of Fluorescence Spectroscopy, 2nd ed., Plenum Press New York (1999) or Hermanson, Bioconjugate Techniques, 2nd ed., or derivatives thereof;Cyanine dyes may exist in sulfonated or non-sulfonated form and consist of two indolenine, benzindolium, pyridinium, thiazolium and / or quinolinium groups separated by a polymethine bridge between the two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3 (which may comprise 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium or 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium oxopyrrolidin-1-yloxy)-6-oxohexyl]-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium-5-sulfonate), Cy5 (which may include 1-(6-((2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl)-3,3-dimethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene}prop-1-en-1-yl)-3,3-dimethyl-3H-indolium-5-sulfonate). )penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium or 1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-2-((1E,3E)-5-((E)-1-(6-((2,5-dioxopyrrolidin-1-yl)oxy)-6-oxohexyl)-3,3-dimethyl-5-sulfonindol-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-indol-1-ium-5-sulfonate) and Cy7 (which may contain 1-(5-carboxypentyl) )-2-[(1E,3E,5E,7Z)-7-(1-ethyl-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium or 1-(5-carboxypentyl)-2-[(1E,3E,5E,7Z)-7-(1-ethyl-5-sulfo-1,3-dihydro-2H-indol-2-ylidene)hepta-1,3,5-trien-1-yl]-3H-indolium-5-sulfonate), where "Cy" stands for 'cyanine' and the first digit identifies the number of carbon atoms between the two indolenine groups. Cy2 is an oxazole derivative rather than an indolenine, and the benzo-derived Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule. ;

[0168] In some embodiments, the reporter moiety can be a FRET pair, so that multiple classifications can be performed in a single excitation and imaging step. As used herein, FRET can include excitation exchange (Forster) transfer or electron exchange (Dexter) transfer.

[0169] As used herein, the term "support" refers to a substrate designed for the deposition of biological molecules or biological samples for measurement and / or analysis. Examples of biological molecules to be deposited on a support include nucleic acids (e.g., DNA, RNA), polypeptides, carbohydrates, lipids, single cells or multiple cells. Examples of biological samples include, but are not limited to, saliva, sputum, mucus, blood, plasma, serum, urine, feces, sweat, tears, and fluids from tissues or organs.

[0170] In some embodiments, the support is solid, semi-solid, or a combination thereof. In some embodiments, the support is porous, semi-porous, non-porous, or any combination of porosity. In some embodiments, the support can be substantially planar, concave, convex, or any combination thereof. In some embodiments, the support can be cylindrical, for example, comprising a capillary or the inner surface of a capillary.

[0171] In some embodiments, the surface of the support can be substantially smooth. In some embodiments, the support can have a regular or irregular texture comprising bumps, etchings, holes, a three-dimensional scaffold, or any combination thereof.

[0172] In some embodiments, the support comprises beads having any shape, including spheres, hemispheres, cylinders, barrels, rings, disks, rods, cones, triangles, cubes, polygons, tubes, or wires.

[0173] The support may be made of any material, including but not limited to glass, fused silica, silicon, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET)), or any combination thereof. Various compositions of both glass and plastic substrates are contemplated.

[0174] The support can have multiple (for example, two or more) nucleic acid templates fixed thereon.The multiple fixed nucleic acid templates have the same sequence or have different sequences.In certain embodiments, the independent nucleic acid template molecules in the multiple nucleic acid templates are fixed to different sites on the support.In certain embodiments, two or more independent nucleic acid template molecules in the multiple nucleic acid templates are fixed to sites on the support.

[0175] The term "array" refers to a support comprising a plurality of sites located at predetermined positions on the support to form an array of sites. The sites may be discrete and separated by gaps. In some embodiments, the predetermined sites on the support may be arranged in rows or columns in one dimension, or in rows and columns in two dimensions. In some embodiments, a plurality of predetermined sites are arranged in an organized manner on the support. In some embodiments, the plurality of predetermined sites are arranged in any organized pattern, including straight lines, hexagonal patterns, grid patterns, patterns with reflection symmetry, patterns with rotational symmetry, and the like. The spacing between different pairs of sites may be the same or may be different. In some embodiments, the support comprises at least 10 2 sites, at least 10 3 sites, at least 10 4 sites, at least 10 5 sites, at least 10 6 sites, at least 10 7 sites, at least 10 8 sites, at least 10 9 sites, at least 10 10 sites, at least 10 11 sites, at least 10 12 sites, at least 10 13 sites, at least 10 14 sites, at least 10 15 sites or more sites, wherein the sites are located at predetermined positions on the support. In some embodiments, the plurality of predetermined sites (e.g., 10 2 to 10 15 In some embodiments, the nucleic acid template is immobilized at a plurality of predetermined sites, for example, at 10 or more sites, to form a nucleic acid template array. In some embodiments, the nucleic acid template is immobilized at a plurality of predetermined sites by hybridizing with an immobilized surface capture primer, or the nucleic acid template is covalently attached to a surface capture primer. In some embodiments, the nucleic acid template is immobilized at a plurality of predetermined sites, for example, at 10 or more sites. 2 to 10 15 sites (e.g., at least 10 2 Site, at least 10 3 sites, at least 10 4 sites, at least 10 5 sites, at least 10 6 sites, at least 10 7 sites, at least 10 8 sites, at least 10 9 sites, at least 10 10 sites, at least 10 11sites, at least 10 12 sites, at least 10 13 sites, at least 10 14 sites, at least 10 15 In some embodiments, the fixed nucleic acid template is cloned to generate fixed nucleic acid polymerase colonies at the plurality of predetermined sites. In some embodiments, the separate fixed nucleic acid polymerase colonies comprise single-stranded or double-stranded concatemers.

[0176] In some embodiments, a support comprising a plurality of sites located at random positions on the support is referred to herein as a support having randomly positioned sites thereon. The positions of the randomly positioned sites on the support are not predetermined. The plurality of randomly positioned sites are arranged in a disordered and / or unpredictable manner on the support. In some embodiments, the support comprises at least 10 2 sites, at least 10 3 sites, at least 10 4 sites, at least 10 5 sites, at least 10 6 sites, at least 10 7 sites, at least 10 8 sites, at least 10 9 sites, at least 10 10 sites, at least 10 11 sites, at least 10 12 sites, at least 10 13 sites, at least 10 14 sites, at least 10 15 sites or more, wherein the sites are randomly positioned on the support. In some embodiments, the plurality of randomly positioned sites (e.g., 10 2 to 10 15 In some embodiments, the nucleic acid template is immobilized at a plurality of randomly located sites by hybridization with an immobilized surface capture primer, or the nucleic acid template is covalently attached to the surface capture primer. In some embodiments, the nucleic acid template is immobilized at a plurality of randomly located sites, for example, at 10 2 to 10 15 In certain embodiments, the nucleic acid template is amplified through a clonal manner to produce a fixed nucleic acid polymerase colony at these multiple randomly located sites. In certain embodiments, the independent nucleic acid polymerase colony comprises a single strand or a double-stranded concatemer.

[0177] The term "immobilized" and related terms, when used to refer to immobilized nucleic acids, refers to nucleic acid molecules that are attached to a support, or to a coating on a support, or embedded within a matrix formed by a coating on a support, by covalent bonds or non-covalent interactions, wherein the nucleic acid molecules comprise surface capture primers, nucleic acid template molecules, and extension products of the capture primers. The extension products of the capture primers include nucleic acid concatemers that can form a nucleic acid polymerase community.

[0178] In certain embodiments, one or more nucleic acid templates are fixed on a support, for example, fixed on a site on a support. In certain embodiments, the one or more nucleic acid templates are for being amplified in a clonal manner. In certain embodiments, the one or more nucleic acid templates are amplified (for example, in a solution) in a clonal manner from a support, and are subsequently deposited on a support and are fixed on a support. In certain embodiments, the clonal amplification reaction of the one or more nucleic acid templates is carried out on a support, resulting in fixing on a support. In certain embodiments, nucleic acid amplification reaction is used to amplify the one or more nucleic acid templates (for example, in a solution or on a support) in a clonal manner, the nucleic acid amplification reaction includes any one of the following or any combination thereof: polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), amplification based on nucleic acid sequence (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification (RCA), ring to ring amplification, helicase-dependent amplification, recombinase-dependent amplification and / or single-stranded binding (SSB) protein-dependent amplification.

[0179] The term "surface primer," "surface capture primer" and related terms refer to single-stranded oligonucleotides that are fixed to a support and comprise a sequence that can hybridize with at least a portion of a nucleic acid template molecule. Surface primers can be used for fixing template molecules to a support via hybridization. Surface primers can be fixed to a support in a manner that resists primer removal during flow, washing, suction, and changes in temperature, pH, salt, chemical, and / or enzyme conditions. Typically, but not necessarily, the 5' end of the surface primer can be fixed to a support. Alternatively, an inner portion or 3' end of the surface primer can be fixed to a support.

[0180] In certain embodiments, the surface primer comprises DNA, RNA or its analogue.Surface primer can comprise the combination of DNA and RNA.The sequence of surface primer can be fully complementary or partially complementary to at least a portion of its length and nucleic acid template molecule (for example, linear or circular template molecule).The support can comprise a plurality of fixed surface primers with the same sequence or with two or more different sequences.Surface primer can be any length, for example 4 to 50 nucleotides or 50 to 100 nucleotides or 100 to 150 nucleotides or longer length or any scope therebetween.

[0181] The surface primer may include an end 3' nucleotide with a sugar 3' OH portion that can be extended for nucleotide polymerization (for example, polymerase-catalyzed polymerization). The surface primer may include an end 3' nucleotide with a portion of the extension that blocks polymerase catalysis. The surface primer may include an end 3' nucleotide with a 3' sugar position linked to a chain termination portion that inhibits nucleotide polymerization. Blocking agents may be used to remove (for example, to block) the 3' chain termination portion so that the 3' end is converted into an extendable 3' OH end. The example of the chain termination portion includes an alkyl group, an alkenyl group, an alkynyl group, an allyl group, an aryl group, a benzyl group, an azido group, an amine group, an amide group, a keto group, an isocyanate group, a phosphate group, a thio group, a disulfide group, a carbonate group, a urea group or a silyl group. The azido-type chain termination portion includes an azido, an azido and an azidomethyl group. Examples of deblocking agents include phosphine compounds such as tris(2-carboxyethyl)phosphine (TCEP) and bissulfotriphenylphosphine (BS-TPP) for azide, azido and azidomethyl chain terminating groups. Examples of deblocking agents include tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine, or with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) for alkyl, alkenyl, alkynyl and allyl chain terminating groups. Examples of deblocking agents include Pd / C for aryl and benzyl chain terminating groups. Examples of deblocking agents include phosphine, β-mercaptoethanol or dithiothreitol (DTT) for amine, amide, ketone, isocyanate, phosphate, thio and disulfide chain terminating groups. Examples of deblocking agents include potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine and Zn in acetic acid (AcOH) for carbonate chain terminating groups. Examples of deblocking agents include tetrabutylammonium fluoride, pyridine-HF, ammonium fluoride, and triethylamine trihydrofluoride for the chain-terminating groups urea and silyl.

[0182] In some embodiments, the plurality of fixed surface capture primers on the support are in fluid communication with each other to allow a solution of reagents (e.g., linear or circular nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, reagents, etc.) to flow onto the support so that the plurality of fixed surface capture primers on the support can react with the reagents substantially simultaneously in a massively parallel manner. In some embodiments, the fluid communication of the plurality of fixed surface capture primers can be used to perform nucleic acid amplification reactions (e.g., RCA, MDA, PCR, and bridge amplification) on the plurality of fixed surface capture primers substantially simultaneously.

[0183] In some embodiments, the plurality of immobilized single-stranded nucleic acid concatemer template molecules on the support are in fluid communication with each other to allow a solution of reagents (e.g., soluble primers, enzymes, nucleotides, divalent cations, buffers, reagents, etc.) to flow onto the support, so that the plurality of immobilized concatemer template molecules on the support can react with the reagents substantially simultaneously in a massively parallel manner. In some embodiments, the fluid communication of the plurality of immobilized single-stranded nucleic acid concatemer template molecules can be used to perform nucleotide binding assays and / or nucleotide polymerization reactions (e.g., primer extension or sequencing) on ​​the plurality of immobilized single-stranded nucleic acid concatemer template molecules substantially simultaneously, and optionally for detection and imaging to perform massively parallel sequencing.

[0184] The terms "amplify," "amplifying," "amplification," and other related terms when used in reference to nucleic acids include generating multiple copies of a preliminary polynucleotide template molecule, wherein the copies comprise a sequence that is complementary to the template sequence, or the copies comprise a sequence that is identical to the template sequence. In some embodiments, the copies comprise a sequence that is substantially identical to the template sequence or a sequence that is substantially identical to the template sequence that is complementary to the template sequence.

[0185] The present disclosure provides various pH buffers. The full names of the pH buffers are listed herein. The term "Tris" refers to the pH buffer tris (hydroxymethyl) -aminomethane. The term "Tris-HCl" refers to the pH buffer tris (hydroxymethyl) -aminomethane hydrochloride. The term "Tricine" refers to the pH buffer N- [tris (hydroxymethyl) methyl] glycine. The term "Bicine" refers to the pH buffer N, N-bis (2-hydroxyethyl) glycine. The term "Bis-Tris propane" refers to the pH buffer 1,3 bis [tris (hydroxymethyl) methylamino] propane. The term "HEPES" refers to the pH buffer 4- (2-hydroxyethyl) -1-piperazineethane sulfonic acid. The term "MES" refers to the pH buffer 2- (N-morpholino) ethane sulfonic acid). The term "MOPS" refers to the pH buffer 3- (N-morpholino) propane sulfonic acid. The term "MOPSO" refers to the pH buffer 3-(N-morpholino)-2-hydroxypropanesulfonic acid. The term "BES" refers to the pH buffer N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid. The term "TES" refers to the pH buffer 2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid. The term "CAPS" refers to the pH buffer 3-(cyclohexylamino)-1-propanesulfonic acid. The term "TAPS" refers to the pH buffer N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid. The term "TAPSO" refers to the pH buffer N-[tris(hydroxymethyl)methyl]-3-amino-2-hydroxypropanesulfonic acid. The term "ACES" refers to the pH buffer N-(2-acetamido)-2-aminoethanesulfonic acid. The term "PIPES" refers to the pH buffer piperazine-1,4-bis(2-ethanesulfonic acid).

[0186] introduction:

[0187] The present disclosure provides compacted oligonucleotides and methods of using compacted oligonucleotides to prepare nucleic acid nanostructures with compact size and shape.

[0188] In certain embodiments, the independent compaction oligonucleotide comprises two or more binding regions, and these two or more binding regions are designed to hybridize with at least two districts of nucleic acid molecules.In certain embodiments, the different binding regions of compaction oligonucleotide are designed to hybridize with the distal portion of the same nucleic acid molecule and the distal portion is pulled together, thereby the nucleic acid molecule is compacted to form a compact nanostructure.For example, but not limited to, the different binding regions of compaction oligonucleotide are designed to hybridize with the distal portion of the same nucleic acid concatemer.In certain embodiments, the different binding regions of compaction oligonucleotide hybridize with the portion of the universal adapter sequence at the distal position on the same concatemer, and the distal portion is pulled together, thereby the concatemer is formed into a DNA nanostructure with a more compact shape and size compared to a concatemer not hybridized with the compaction oligonucleotide.

[0189] In some embodiments, the individual compaction oligonucleotides comprise two or more binding regions, wherein each region is designed to hybridize with the regions of two different nucleic acid molecules. In some embodiments, the different binding regions of the compaction oligonucleotides are designed to hybridize with the two different nucleic acid molecules and form a compact nanostructure. For example, but not limited to, the different binding regions of the compaction oligonucleotides are designed to hybridize with the regions of two different nucleic acid library molecules. In some embodiments, the nucleic acid library molecules comprise linear and / or circular library molecules. In some embodiments, the different binding regions of the compaction oligonucleotides hybridize with portions of the universal adapter sequences on the two different library molecules and form a DNA nanostructure having a more compact shape and size than the library molecules that are not hybridized with the compaction oligonucleotides.

[0190] In some embodiments, a compacted oligonucleotide comprises one or more oligonucleotides and can have any shape, including, for example, linear, branched, star, comb, dendrimer, or other shapes.

[0191] In some embodiments, a compacted oligonucleotide may comprise two, three, four, or more binding regions (e.g., Figure 1 to 1 3) In some embodiments, the compacted oligonucleotides can be modified to increase resistance to exonuclease degradation.

[0192] In some embodiments, the inclusion of compacted oligonucleotides during rolling circle amplification can promote the formation of nanostructures with more compact sizes and shapes compared to concatemers generated in the absence of compacted oligonucleotides. The compact and stable nature of the nucleic acid nanostructures can improve sequencing accuracy by increasing signal intensity, and the nucleic acid nanostructures maintain their shape and size during multiple sequencing cycles and prevent unraveling.

[0193] Rolling circle amplification (RCA) can be performed in the presence of a compaction oligonucleotide to generate a single-stranded concatemer molecule having multiple copies of a polynucleotide unit arranged in series, wherein each polynucleotide unit comprises a sequence of interest and at least one universal adapter sequence (e.g., at least one universal primer binding site). In some embodiments, separate compaction oligonucleotides hybridize to two or more regions of the same concatemer molecule.

[0194] Rolling circle amplification (RCA) can be performed with compacted oligonucleotides to generate single-stranded concatemer molecules having multiple copies of polynucleotide units arranged in series, wherein each polynucleotide unit comprises a sequence of interest and at least one universal adapter sequence (e.g., at least one universal primer binding site). In some embodiments, the compacted oligonucleotides can have any shape (e.g., linear, branched, star-shaped, dendrimer, or other shape) and can include two, three, four, or more binding regions (e.g., Figure 1 to 1 3) In some embodiments, the binding region of the compacted oligonucleotide is designed to hybridize to at least one universal adaptor sequence in a linear library molecule.

[0195] In certain embodiments, the binding region of compaction oligonucleotide is designed to hybridize with at least one universal binding sequence in concatemer molecule.In certain embodiments, the binding region of compaction oligonucleotide can be modified to increase resistance to exonuclease degradation.The different binding regions of compaction oligonucleotide can be designed to hybridize with the distal portion of same concatemer molecule and the distal portion is drawn together, thereby concatemer compaction is to form compact nanostructure.Compared with the concatemer produced when there is no compaction oligonucleotide, during RCA, comprising that compaction oligonucleotide can promote the formation of nanostructure with tighter size and shape.Do not wish to be bound by theory, suppose that the compact and stable characteristic of nucleic acid nanostructure is for example improved order-checking accuracy by increasing signal intensity, and nanostructure keeps its shape and size during multiple order-checking cycles.

[0196] As used herein, "nanostructures" or "nucleic acid nanostructures" are compacted concatemer molecules in which each nanostructure carries many copies of a polynucleotide unit along its length. Each polynucleotide unit can be combined with a sequencing primer, a sequencing polymerase, and a detectably labeled nucleotide reagent to form a detectable sequencing complex. Each nanostructure can be combined with many detectable sequencing complexes. Therefore, the compact size of the nanostructure can improve sequencing accuracy by increasing the local concentration of the detectably labeled nucleotide reagent used during the sequencing workflow, which increases the signal intensity emitted from a given nanostructure to give a discrete detectable signal.

[0197] Large-scale parallel sequencing workflows typically employ multiple steps, including reagent flow, microscopic imaging of detectable sequencing complexes, washing template strands, and repeating these steps for hundreds of cycles. The DNA template molecules immobilized on the flow cell can undergo multiple reagent flow cycles, wherein each flow is intended to change the reaction environment of the immobilized template molecules by changes in temperature, pH, salt, and enzymes. The DNA template molecules may disperse or shift positions in later sequencing cycles, which reduces base call accuracy. Compacting oligonucleotides during RCA produces stable compact nanostructures. Nanostructures as described in the present disclosure resist spreading and drifting, and maintain their compact shape and size in multiple sequencing cycles, which improves base call accuracy in later sequencing cycles.

[0198] The compositions and methods described herein can be used to prepare compact nanostructures that are fixed to random locations on a flow cell at high density. Compact nanostructures are tightly packed and fill most of the space on the flow cell. However, compact nanostructures are distinguishable from their neighboring nanostructures, which facilitates the preparation of high-density nanostructures for high-throughput, massively parallel nucleic acid sequencing. For example, even at approximately 6x10 5 / mm 2 At a density of 100 nm, fluorescently labeled nanostructures can also be imaged as distinct nanospheres arranged at random locations on the flow cell. Including compaction of oligonucleotides during RCA can avoid the need to fabricate sequencing flow cells with ordered, patterned nanopore arrays.

[0199] In some embodiments, the compacted oligonucleotide can be a linear oligonucleotide molecule that can be adapted for integration into existing library preparation workflows employing rolling circle amplification. The first and second regions of the linear compacted oligonucleotide can be designed to hybridize to universal binding sequences in concatemer molecules. The linear compacted oligonucleotides described herein may be simpler than, for example, the multi-branched dendrimers described in U.S. Patent No. 8,445,194.

[0200] In some embodiments, the workflows described herein do not require special conditions or additives to produce stable, compact nanostructures. For example, the workflows described herein do not require the addition of proteins (e.g., streptavidin or histones) to produce stable nanostructures. No additives (such as inorganic cations Co(NH3)6 3+ , polyvinylpyrrolidone or cationic liposomes) to produce stable nanostructures (see, for example, DNA condensation in U.S. Patent No. 9,982,293). These additives may interfere with downstream reactions, including DNA sequencing reactions.

[0201] Composition

[0202] The present disclosure provides a plurality of nucleic acid nanostructures, wherein individual nucleic acid nanostructures comprise nucleic acid concatemer molecules hybridized to at least one compacted oligonucleotide. In some embodiments, the nucleic acid nanostructures are in solution and immobilized to a support, or a mixture of nanostructures are in solution and immobilized to a support.

[0203] In some embodiments, the individual compacted oligonucleotides comprise at least a first binding region that can hybridize to a first portion of a concatemer molecule, and the compacted oligonucleotides comprise at least a second binding region (e.g., a second portion of a concatemer molecule) that can hybridize to a second portion of a concatemer molecule (e.g., the same concatemer molecule). Figure 1 to 1 3) In some embodiments, the first and second regions of separate compaction oligonucleotides can hybridize to two portions of the same concatemer to pull the distal portions of the concatemer together, thereby compacting the concatemer to form a nucleic acid nanostructure.

[0204] In certain embodiments, the hybridization of compaction oligonucleotide and independent concatemer molecule causes concatemer molecule to collapse or be folded into nucleic acid nanostructure.In certain embodiments, nucleic acid nanostructure can comprise one or more rings, or can have spherical shape (for example, nanoball), elongated shape (for example, nanorod), preliminary annular shape or annular shape (for example, nano annular shape).The point image of nucleic acid nanostructure can be expressed as Gaussian point (Gaussian spot), and size can be measured as half maximum full width (FWHM).As indicated by less FWHM, less point size is usually relevant to the improved image of point.In certain embodiments, the FWHM of nanostructure point can be about 10 μ m or less.Nucleic acid nanostructure can be a compact nucleic acid structure, and it has less half maximum full width (FWHM) compared with the concatemer that does not collapse / fold into nanostructure.

[0205] In some embodiments, the individual compacted oligonucleotides in the plurality comprise nucleic acids and can have any shape, including linear, branched, star-shaped, or dendrimer shapes (e.g., bottlebrush shapes). In some embodiments, the compacted oligonucleotides can be folded by intramolecular base pairing with duplex portions via Watson-Crick base pairing, Hoostein base pairing, and / or G-quadruplex structure formation. In some embodiments, the compacted oligonucleotides comprise nucleic acids that can fold into any shape having at least one hairpin, at least one stem-loop, and / or at least one star-shaped shape.

[0206] In some embodiments, individual compacted oligonucleotides in the plurality comprise DNA, RNA, or a combination of DNA and RNA. The compacted oligonucleotides can be of any length, including 20-200 nucleotides, 20-150 nucleotides, 30-100 nucleotides, 40-80 nucleotides, or any range therebetween.

[0207] In some embodiments, an individual compacted oligonucleotide comprises a linear nucleic acid (e.g., Figure 1 ). In some embodiments, the first binding region of the compacted oligonucleotide hybridizes to a first portion of the concatemer molecule. In some embodiments, the second binding region of the same compacted oligonucleotide hybridizes to a second portion of the same concatemer molecule. In some embodiments, the first binding region of the compacted oligonucleotide hybridizes to at least a portion of a first universal binding sequence in the concatemer molecule. In some embodiments, the second binding region of the compacted oligonucleotide hybridizes to at least a portion of a second universal binding sequence in the concatemer molecule. In some embodiments, the first binding region and the second binding region of the compacted oligonucleotide comprise the same sequence or different sequences. In some embodiments, the second binding region of the compacted oligonucleotide comprises the reverse sequence of the first binding region of the compacted oligonucleotide. In some embodiments, the orientation of the first binding region of the compacted oligonucleotide is a 5' to 3' orientation or a 3' to 5' orientation. In some embodiments, the orientation of the second binding region of the compacted oligonucleotide is a 5' to 3' orientation or a 3' to 5' orientation ( Figure 1 ).

[0208] In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation and a second binding region arranged in a 5' to 3' orientation ( Figure 1 (i)). In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 3' to 5' orientation and a second binding region arranged in a 3' to 5' orientation ( Figure 1 (ii)). In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 3' to 5' orientation and a second binding region arranged in a 5' to 3' orientation ( Figure 1 (iii)). In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation and a second binding region arranged in a 3' to 5' orientation ( Figure 1 (iv)).

[0209] In certain embodiments, the intervening joint of compacting oligonucleotide is designed to be flexible.In certain embodiments, the intervening joint of compacting oligonucleotide is designed to be rigid.In certain embodiments, the intervening joint of compacting oligonucleotide comprises any one or any combination among Nucleotide, nucleotide analogs and / or non-nucleotide joints.In certain embodiments, the intervening joint of compacting oligonucleotide shows little or no hybridization with any part of concatemer molecule.

[0210] In some embodiments, the compacted oligonucleotide comprises a linear nucleic acid having a first binding region, a second binding region, a third binding region, and optionally two intervening junctions. In some embodiments, the first intervening junction is located between the first binding region and the second binding region. In some embodiments, the second intervening junction is located between the second binding region and the third binding region (e.g., Figures 2A to 2C In some embodiments, the first binding region of the compacted oligonucleotide hybridizes to a first portion of a concatemer molecule. In some embodiments, the second binding region of the compacted oligonucleotide hybridizes to a second portion of the same concatemer molecule. In some embodiments, the third binding region of the compacted oligonucleotide hybridizes to a third portion of the same concatemer molecule.

[0211] In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a second binding region arranged in a 5' to 3' orientation, and a third binding region arranged in a 5' to 3' orientation ( Figure 2A (i)). In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a second binding region arranged in a 5' to 3' orientation, and a third binding region arranged in a 3' to 5' orientation ( Figure 2A (ii)).

[0212] In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a second binding region arranged in a 3' to 5' orientation, and a third binding region arranged in a 3' to 5' orientation. Figure 2A (iii)).

[0213] In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a second binding region arranged in a 3' to 5' orientation, and a third binding region arranged in a 5' to 3' orientation. Figure 2B (iv)). In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 3' to 5' orientation, a second binding region arranged in a 5' to 3' orientation, and a third binding region arranged in a 5' to 3' orientation ( Figure 2B (v)). In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 3' to 5' orientation, a second binding region arranged in a 3' to 5' orientation, and a third binding region arranged in a 5' to 3' orientation ( Figure 2B (vi)).

[0214] In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 3' to 5' orientation, a second binding region arranged in a 5' to 3' orientation, and a third binding region arranged in a 5' to 3' orientation. Figure 2C (vii)).

[0215] In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 3' to 5' orientation, a second binding region arranged in a 3' to 5' orientation, and a third binding region arranged in a 5' to 3' orientation. Figure 2C (viii)). In some embodiments, the compacted oligonucleotide comprises a first binding region arranged in a 3' to 5' orientation, a second binding region arranged in a 5' to 3' orientation, and a third binding region arranged in a 3' to 5' orientation ( Figure 2C (ix)).

[0216] In some embodiments, the first binding region of the compacted oligonucleotide hybridizes to at least a portion of a first universal binding sequence in a concatemer molecule. In some embodiments, the second binding region of the compacted oligonucleotide hybridizes to at least a portion of a second universal binding sequence in the same concatemer molecule. In some embodiments, the third binding region of the compacted oligonucleotide hybridizes to at least a portion of a third universal binding sequence in the same concatemer molecule.

[0217] In some embodiments, the first, second, and third binding regions of the compacted oligonucleotide comprise the same sequence or different sequences. In some embodiments, the second and third binding regions of the compacted oligonucleotide have the same sequence, and the first binding region has a different sequence. In some embodiments, the first and second regions of the compacted oligonucleotide have the same sequence, and the third binding region has a different sequence. In some embodiments, the first and third binding regions of the compacted oligonucleotide have the same sequence, and the second binding region has a different sequence.

[0218] In some embodiments, the third binding region of the compacted oligonucleotide comprises the reverse sequence of the first binding region of the compacted oligonucleotide. In some embodiments, the second binding region of the compacted oligonucleotide comprises the reverse sequence of the first binding region.

[0219] In certain embodiments, the intervening joint of compacting oligonucleotide is designed to be flexible.In certain embodiments, the intervening joint of compacting oligonucleotide is designed to be rigid.In certain embodiments, the intervening joint of compacting oligonucleotide comprises any one or any combination among Nucleotide, nucleotide analogs and / or non-nucleotide joints.In certain embodiments, the intervening joint of compacting oligonucleotide shows little or no hybridization with any part of concatemer molecule.

[0220] In some embodiments, the compacted oligonucleotide comprises a star-shaped nucleic acid having a first binding region and an internal region and a second binding region, and optionally two intervening junctions. In some embodiments, the first intervening junction is located between the first binding region and the internal region. In some embodiments, the second intervening junction is located between the internal binding region and the second binding region (e.g., Figure 3A and 3BIn some embodiments, the first binding region of the compacted oligonucleotide hybridizes to a first portion of a concatemer molecule. In some embodiments, the interior region of the compacted oligonucleotide hybridizes to a second portion of the same concatemer molecule. In some embodiments, the second binding region of the compacted oligonucleotide hybridizes to a third portion of the same concatemer molecule.

[0221] In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening linker and a first binding region arranged in a 5' to 3' orientation, wherein the 3' end of the first binding region is directed away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 5' to 3' orientation, wherein the 3' end of the second binding region is directed away from the internal intervening linker; and (3) an internal intervening linker and a third binding region arranged in a 5' to 3' orientation, wherein the 3' end of the third binding region is directed away from the internal intervening linker. Figure 3A (i)).

[0222] In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening linker and a first binding region arranged in a 3' to 5' orientation, wherein the 5' end of the first binding region is directed away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 3' to 5' orientation, wherein the 5' end of the second binding region is directed away from the internal intervening linker; and (3) an internal intervening linker and a third binding region arranged in a 3' to 5' orientation, wherein the 5' end of the third binding region is directed away from the internal intervening linker. Figure 3A (ii)).

[0223] In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening linker and a first binding region arranged in a 5' to 3' orientation, wherein the 3' end of the first binding region is directed away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 5' to 3' orientation, wherein the 3' end of the second binding region is directed away from the internal intervening linker; and (3) an internal intervening linker and a third binding region arranged in a 3' to 5' orientation, wherein the 5' end of the third binding region is directed away from the internal intervening linker. Figure 3B (iii)).

[0224] In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening linker and a first binding region arranged in a 5' to 3' orientation, wherein the 3' end of the first binding region is directed away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 3' to 5' orientation, wherein the 5' end of the second binding region is directed away from the internal intervening linker; and (3) an internal intervening linker and a third binding region arranged in a 3' to 5' orientation, wherein the 5' end of the third binding region is directed away from the internal intervening linker. Figure 3B (iv)).

[0225] In some embodiments, the first binding region of the compacted oligonucleotide hybridizes to at least a portion of the first universal binding sequence in the concatemer molecule. In some embodiments, the internal region of the compacted oligonucleotide hybridizes to at least a portion of the second universal binding sequence in the concatemer molecule. In some embodiments, the second binding region of the compacted oligonucleotide hybridizes to at least a portion of the third universal binding sequence in the concatemer molecule. In some embodiments, the first binding region, the internal region, and the second binding region of the compacted oligonucleotide comprise the same sequence or different sequences. In some embodiments, the internal region and the second binding region of the compacted oligonucleotide have the same sequence, and the first binding region has a different sequence. In some embodiments, the first binding region and the internal region of the compacted oligonucleotide have the same sequence, and the second binding region has a different sequence. In some embodiments, the first binding region and the second binding region of the compacted oligonucleotide have the same sequence, and the internal region has a different sequence. In some embodiments, the second binding region of the compacted oligonucleotide comprises the reverse sequence of the first binding region of the compacted oligonucleotide. In some embodiments, the internal region of the compacted oligonucleotide comprises a sequence that is reversed to the first binding region.

[0226] In certain embodiments, the intervening linker of the compacting oligonucleotide is designed to be flexible or rigid. In certain embodiments, the intervening linker of the compacting oligonucleotide comprises any one or any combination of nucleotides, nucleotide analogs and / or non-nucleotide linkers. In certain embodiments, the intervening linker of the compacting oligonucleotide exhibits little or no hybridization with any portion of the concatemer molecule.

[0227] In some embodiments, the compacted oligonucleotide comprises three binding arms, wherein each binding arm comprises: an internal intervening linker, a first binding region arranged in a 5' to 3' orientation, an intervening linker, and a second binding region arranged in a 5' to 3' orientation, wherein the 3' end of the second binding region is directed away from the internal intervening linker ( Figure 4 ).

[0228] In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening linker and a first binding region arranged in a 5' to 3' orientation, wherein the 3' end of the first binding region is directed away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 5' to 3' orientation, wherein the 3' end of the second binding region is directed away from the internal intervening linker; (3) an internal intervening linker and a third binding region arranged in a 5' to 3' orientation, wherein the 3' end of the third binding region is directed away from the internal intervening linker; and (4) an internal intervening linker and a fourth binding region arranged in a 5' to 3' orientation, wherein the 3' end of the fourth binding region is directed away from the internal intervening linker. Figure 5 (i)).

[0229] In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening linker and a first binding region arranged in a 3' to 5' orientation, wherein the 5' end of the first binding region is directed away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 3' to 5' orientation, wherein the 5' end of the second binding region is directed away from the internal intervening linker; (3) an internal intervening linker and a third binding region arranged in a 3' to 5' orientation, wherein the 5' end of the third binding region is directed away from the internal intervening linker; and (4) an internal intervening linker and a fourth binding region arranged in a 3' to 5' orientation, wherein the 5' end of the fourth binding region is directed away from the internal intervening linker. Figure 5 (ii)).

[0230] In some embodiments, the compacted oligonucleotide comprises at least three binding arms. In some embodiments, the compacted oligonucleotide comprises multiple binding arms with the same sequence. In some embodiments, a single binding arm comprises a first binding region arranged in a 5' to 3' orientation, wherein the 3' end of the first binding region is directed away from the joint portion. In some embodiments, a single binding arm is connected to the joint portion ( Figure 6A ).

[0231] In some embodiments, the compacted oligonucleotide comprises at least three binding arms. In some embodiments, the compacted oligonucleotide comprises a plurality of binding arms having one of two different sequences. In some embodiments, a separate binding arm comprises a first binding region arranged in a 5' to 3' orientation, wherein the 3' end of the first binding region guides away from the joint portion. In some embodiments, a separate binding arm comprises a second binding region arranged in a 5' to 3' orientation, wherein the 3' end of the second binding region guides away from the joint portion. In some embodiments, a separate binding arm is connected to the joint portion ( Figure 6B ).

[0232] In some embodiments, the compacted oligonucleotide comprises at least three binding arms. In some embodiments, the compacted oligonucleotide comprises a plurality of binding arms having one of three different sequences. In some embodiments, a separate binding arm comprises a first binding region arranged in a 5' to 3' orientation, wherein the 3' end of the first binding region guides away from the joint portion. In some embodiments, a separate binding arm comprises a second binding region arranged in a 5' to 3' orientation, wherein the 3' end of the second binding region guides away from the joint portion. In some embodiments, a separate binding arm comprises a third binding region arranged in a 5' to 3' orientation, wherein the 3' end of the third binding region guides away from the joint portion. In some embodiments, a separate binding arm is connected to the joint portion ( Figure 6C ).

[0233] In some embodiments, the primary linear compaction oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a first intervening linker, a second binding region arranged in a 5' to 3' orientation, a second intervening linker, and a connector region arranged in a 5' to 3' orientation. In some embodiments, the secondary linear compaction oligonucleotide comprises a fastener region arranged in a 3' to 5' orientation, an intervening linker, and a third binding region arranged in a 3' to 5' orientation. In some embodiments, the connector region of the primary compaction oligonucleotide can hybridize to the fastener region of the secondary compaction oligonucleotide ( Figure 7A ).

[0234] In some embodiments, the primary linear compaction oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a first intervening linker, a second binding region arranged in a 5' to 3' orientation, a second intervening linker, and a connector region arranged in a 5' to 3' orientation. In some embodiments, the secondary linear compaction oligonucleotide comprises a third binding region arranged in a 3' to 5' orientation, an intervening linker, and a fastener region arranged in a 3' to 5' orientation. In some embodiments, the connector region of the primary compaction oligonucleotide can hybridize to the fastener region of the secondary compaction oligonucleotide ( Figure 7B ).

[0235] In some embodiments, the primary linear compaction oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a first intervening linker, a second binding region arranged in a 5' to 3' orientation, a second intervening linker, and a connector region arranged in a 3' to 5' orientation. In some embodiments, the secondary linear compaction oligonucleotide comprises a fastener region arranged in a 5' to 3' orientation, an intervening linker, and a third binding region arranged in a 5' to 3' orientation. In some embodiments, the connector region of the primary compaction oligonucleotide can hybridize to the fastener region of the secondary compaction oligonucleotide ( Figure 8A ).

[0236] In some embodiments, the primary linear compaction oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a first intervening linker, a second binding region arranged in a 5' to 3' orientation, a second intervening linker, and a connector region arranged in a 3' to 5' orientation. In some embodiments, the secondary linear compaction oligonucleotide comprises a third binding region arranged in a 5' to 3' orientation, an intervening linker, and a fastener region arranged in a 5' to 3' orientation. In some embodiments, the connector region of the primary compaction oligonucleotide can hybridize to the fastener region of the secondary compaction oligonucleotide ( Figure 8B ).

[0237] In some embodiments, the primary linear compaction oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a first intervening linker, a second binding region arranged in a 5' to 3' orientation, a second intervening linker, and a connector region arranged in a 5' to 3' orientation. In some embodiments, the secondary linear compaction oligonucleotide comprises a third binding region arranged in a 5' to 3' orientation, an intervening linker, and a fastener region arranged in a 3' to 5' orientation. In some embodiments, the connector region of the primary compaction oligonucleotide can hybridize to the fastener region of the secondary compaction oligonucleotide ( Figure 9 ).

[0238] In some embodiments, the primary linear compaction oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a first intervening linker, a second binding region arranged in a 5' to 3' orientation, a second intervening linker, and a connector region arranged in a 3' to 5' orientation. In some embodiments, the secondary linear compaction oligonucleotide comprises a fastener region arranged in a 5' to 3' orientation, an intervening linker, and a third binding region arranged in a 3' to 5' orientation. In some embodiments, the connector region of the primary compaction oligonucleotide can hybridize to the fastener region of the secondary compaction oligonucleotide ( Figure 10 ).

[0239] In some embodiments, the primary linear compaction oligonucleotide comprises a first binding region arranged in a 5' to 3' orientation, a first intervening junction, a second binding region arranged in a 5' to 3' orientation, a second intervening junction, and a connector region arranged in a 5' to 3' orientation. In some embodiments, the secondary linear compaction oligonucleotide comprises a fastener region arranged in a 3' to 5' orientation, a first intervening junction, a third binding region arranged in a 3' to 5' orientation, a second intervening junction, and a fourth binding region arranged in a 3' to 5' orientation. In some embodiments, the connector region of the primary compaction oligonucleotide can hybridize to the fastener region of the secondary compaction oligonucleotide ( Figure 11 ).

[0240] In some embodiments, the primary compaction oligonucleotide comprises three binding arms connected together by at least one internal intervening junction, wherein the individual binding arms comprise binding regions. In some embodiments, the primary compaction oligonucleotide comprises: (1) an internal intervening junction and a first binding region arranged in a 5' to 3' orientation, wherein the 3' end of the first binding region is directed away from the internal intervening junction; (2) an internal intervening junction and a second binding region arranged in a 5' to 3' orientation, wherein the 3' end of the second binding region is directed away from the internal intervening junction; and (3) an internal intervening junction and a connector region arranged in a 5' to 3' orientation, wherein the 3' end of the connector region is directed away from the internal intervening junction. In some embodiments, the secondary linear compaction oligonucleotide comprises a fastener region arranged in a 5' to 3' orientation, an intervening junction, and a third binding region arranged in a 5' to 3' orientation. In some embodiments, the connector region of the primary compaction oligonucleotide can hybridize to the fastener region of the secondary compaction oligonucleotide ( Figure 12A ).

[0241] In some embodiments, the primary compaction oligonucleotide comprises three binding arms connected together by at least one internal intervening junction, wherein the individual binding arms comprise binding regions. In some embodiments, the primary compaction oligonucleotide comprises: (1) an internal intervening junction and a first binding region arranged in a 3' to 5' orientation, wherein the 5' end of the first binding region is directed away from the internal intervening junction; (2) an internal intervening junction and a second binding region arranged in a 3' to 5' orientation, wherein the 5' end of the second binding region is directed away from the internal intervening junction; and (3) an internal intervening junction and a connector region arranged in a 3' to 5' orientation, wherein the 5' end of the connector region is directed away from the internal intervening junction. In some embodiments, the secondary linear compaction oligonucleotide comprises a fastener region arranged in a 5' to 3' orientation, an intervening junction, and a third binding region arranged in a 5' to 3' orientation. In some embodiments, the connector region of the primary compaction oligonucleotide can hybridize to the fastener region of the secondary compaction oligonucleotide ( Figure 12B ).

[0242] In some embodiments, the first nucleic acid strand comprises a first binding region (110) arranged in a 5' to 3' orientation, an intervening junction, and a second binding region arranged in a 5' to 3' orientation. In some embodiments, the second nucleic acid strand comprises a third binding region (210) arranged in a 3' to 5' orientation, an intervening junction, and a fourth binding region (220) arranged in a 3' to 5' orientation. In some embodiments, the third binding region (210) can hybridize with at least a portion of the first binding region (110) of the first nucleic acid strand (100). In some embodiments, the fourth binding region (220) can hybridize with at least a portion of the second binding region (120) of the first nucleic acid strand (100). In some embodiments, the third nucleic acid strand (300) comprises a fifth binding region (300) arranged in a 3' to 5' orientation. In some embodiments, the fifth binding region (300) can hybridize with at least a portion of the second binding region (120) of the first nucleic acid strand (100). In some embodiments, the fourth binding region (220) and the fifth binding region (300) do not hybridize to the same portion or overlapping portion of the second binding region (120). Figure 13A ).

[0243] exist Figure 13B In the embodiment of the present invention, a portion of the first binding region (110) of the first nucleic acid strand (100) hybridizes with the first portion of the concatemer, which causes a portion of the third binding region (210) to dissociate from the first binding region (110), as shown by the two arrows. The hybridization of a portion of the first binding region (110) and the first portion of the concatemer forms a toehold duplex region. The second binding region (120) of the first nucleic acid strand (100) can remain hybridized with the fourth binding region (220) and the fifth binding region (300).

[0244] exist Figure 13C In the embodiment of the present invention, a portion of the second binding region (120) of the first nucleic acid strand (100) hybridizes with the second portion of the concatemer, which causes a portion of the fifth binding region (300) to dissociate from the second binding region (120), as indicated by the two arrows. The hybridization of the portion of the second binding region (120) and the second portion of the concatemer forms another toehold duplex region. The second nucleic acid strand (200) completely dissociates from the first binding region (110) of the first nucleic acid strand (100).

[0245] exist Figure 13D In the embodiment, the second nucleic acid chain (200) is completely dissociated from the first binding region (110) of the first nucleic acid chain (100). The third nucleic acid chain (300) is completely dissociated from the second binding region (120) of the first nucleic acid chain (100).

[0246] In certain embodiments, the intervening joint of any compaction oligonucleotide described herein can be of any length, for example, a length of about 2-20 nucleotides. The intervening joint comprises a homopolymer with continuous identical bases (e.g., AAA, GGG, CCC, TTT, or UUU). The intervening joint comprises a non-homopolymer sequence. In certain embodiments, the intervening joint comprises at least one inosine. In certain embodiments, the intervening joint comprises a homopolymer with continuous identical bases (e.g., inosine).

[0247] In some embodiments, the intervening linker comprises a spacer. In some embodiments, the spacer comprises a non-nucleotide linker. In some embodiments, the spacer comprises an 18-carbon spacer (e.g., comprising a hexaethylene glycol spacer), a plurality of C3 spacer phosphoramidites, or a spacer 9 comprising a trimethylene glycol spacer. In some embodiments, the spacer comprises a polyethylene glycol spacer, including a PEG2, PEG3, or PEG4 spacer.

[0248] In some embodiments, the intervening linker comprises at least one non-nucleotide linker and at least one PEG spacer in any arrangement. For example, the intervening linker comprises 5'-right arm-([PEG-spacer]-[C18-spacer]) n -Left arm-3', wherein "n" is 1-10. In another example, the intervening linker comprises 5'-Right arm-([C18-spacer]-[PEG-spacer]) n -Left Arm - 3', where "n" is 1-10.

[0249] Any binding region of the compacted oligonucleotide may be fully complementary or partially complementary to a portion of the concatemer molecule along its length. In some embodiments, the binding region of the compacted oligonucleotide is designed to hybridize to a universal binding sequence in the concatemer molecule.

[0250] In some embodiments, the first binding region of the compacted oligonucleotide can hybridize to a first portion of a concatemer molecule, wherein the first portion of the concatemer molecule comprises a universal adaptor sequence according to any one of SEQ ID NOs: 157-176, or a complement thereof (see Table 2).

[0251] In some embodiments, the second binding region of the compacted oligonucleotide can hybridize to a second portion of the concatemer molecule, wherein the second portion of the concatemer molecule comprises a universal adaptor sequence according to any one of SEQ ID NOs: 157-176, or a complementary sequence thereof (see Table 2).

[0252] In some embodiments, any binding region (e.g., the first binding region, the second binding region, the third binding region, the fourth binding region, the fifth binding region, the sixth binding region, or other binding region) of the compacted oligonucleotide can be hybridized to a portion of a concatemer molecule, wherein the portion of the concatemer molecule comprises a universal adapter sequence according to any one of SEQ ID NOs: 157-176, or a complementary sequence thereof (see Table 2).

[0253] In some embodiments, the compacted oligonucleotide comprises two or more binding regions, and all binding regions have the same sequence. In some embodiments, the compacted oligonucleotide comprises two binding regions with different sequences. In some embodiments, the compacted oligonucleotide comprises three or more binding regions, and at least two binding regions have different sequences.

[0254] The first binding region of the compacted oligonucleotide may have the same sequence as the second binding region.

[0255] The first binding region of the compacted oligonucleotide may have a different sequence than the second binding region.

[0256] In some embodiments, the first binding region of the compaction oligonucleotide comprises a sequence according to any one of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, or 154 (see Table 1).

[0257] In some embodiments, the second, third, fourth, fifth, or any subsequent binding region of the compacted oligonucleotide comprises a sequence that is the reverse sequence of the first binding region (e.g., the reverse sequence of any one of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, or 155; see Table 1).

[0258] In some embodiments, the first binding region of the compaction oligonucleotide comprises a sequence according to any one of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, or 155; see Table 1).

[0259] In some embodiments, the second, third, fourth, fifth, or any subsequent binding region of the compacted oligonucleotide comprises a sequence that is the reverse sequence of the first binding region, wherein the second, third, fourth, fifth, or any subsequence binding region comprises any one of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, or 154 (see Table 1).

[0260] In some embodiments, the first binding region of the compacted oligonucleotide can have a sequence that is the reverse sequence of the second binding region (e.g., the reverse sequence of any one of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, or 155; see Table 1).

[0261] In some embodiments, the second binding region of the compaction oligonucleotide comprises a sequence according to any one of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, or 154 (see Table 1).

[0262] In some embodiments, the third binding region of the compaction oligonucleotide comprises a sequence according to any one of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, or 154 (see Table 1).

[0263] In some embodiments, the fourth binding region of the compaction oligonucleotide comprises a sequence according to any one of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, or 154 (see Table 1).

[0264] In some embodiments, the fifth binding region of the compaction oligonucleotide comprises a sequence according to any one of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, or 154 (see Table 1).

[0265] In some embodiments, the subsequent binding region of the compaction oligonucleotide comprises a sequence according to any one of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, or 154 (see Table 1).

[0266] In some embodiments, the compacted oligonucleotide comprises the full length sequence according to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, or 156 (see Table 1).

[0267] In some embodiments, the terminal 3' end of any compacted oligonucleotide may comprise at least one additional base comprising one or more 2'-O-methyl RNA bases (e.g., designated as mUmUmU), or lack an additional 2'-O-methyl RNA base at the terminal 3' end.

[0268] In certain embodiments, the compaction oligonucleotide comprises one or more modified bases or bonds at its 5' or 3' end to confer certain functions. In certain embodiments, the compaction oligonucleotide comprises at least one thiophosphate bond at its 5' and / or 3' end to confer exonuclease resistance. In certain embodiments, at least one nucleotide at or near the 3' end comprises a 2' fluoro base that confer exonuclease resistance. In certain embodiments, the 3' end of the compaction oligonucleotide comprises at least one 2'-O-methyl RNA base that blocks the extension catalyzed by polymerase. For example, the 3' end of the compaction oligonucleotide comprises at least one base, and the at least one base comprises a 2'-O-methyl RNA base (for example, designated as mUmUmU). In certain embodiments, the compaction oligonucleotide comprises at its 3' end 3' reverse dT that blocks the extension catalyzed by polymerase. In certain embodiments, the compaction oligonucleotide comprises 3' phosphorylation that blocks the extension catalyzed by polymerase. In certain embodiments, the compaction oligonucleotide comprises at least one locked nucleic acid (LNA) that increases the thermal stability of the duplex formed by hybridizing the compaction oligonucleotide and the concatemer molecule.

[0269] The compacted oligonucleotide may include at least one region (e.g., a hybridization / binding region) having consecutive guanines. For example, the compacted oligonucleotide may include at least one region having 2, 3, 4, 5, 6, or more consecutive guanines. In some embodiments, the compacted oligonucleotide comprises four consecutive guanines that can form a guanine tetrad structure (see Figure 64 The guanine tetrad structure can be stabilized by Hoogsteen hydrogen bonding. The guanine tetrad structure can be stabilized by a central cation including potassium, sodium, lithium, rubidium, or cesium.

[0270] At least one compacted oligonucleotide can form a guanine tetrad ( Figure 64 ) and hybridizes with the universal binding sequence in the concatemer, which may cause the concatemer to fold to form an intramolecular G-quadruplex structure ( Figure 65 The concatemer can self-collapse to form a compact nanostructure. The formation of guanine tetrads and G-quadruplexes within the nanostructure can increase the stability of the nanostructure, maintaining its compact size and shape, which can withstand changes in pH, temperature, and / or repeated flow of reagents.

[0271] In some embodiments, the plurality of compacted oligonucleotides comprise the same sequence. In some embodiments, the plurality of compacted oligonucleotides comprise a sequence according to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, or 156. In some embodiments, the plurality of compacted oligonucleotides comprises a sequence according to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, or 156, wherein the 3' end of the compacted oligonucleotide further comprises three bases comprising 2'-O-methyl RNA bases (e.g., designated as mUmUmU).

[0272] In some embodiments, the plurality of compacted oligonucleotides comprises a mixture of two or more different populations of compacted oligonucleotides having different sequences. In some embodiments, the plurality of compacted oligonucleotides comprises a mixture of 2, 3, 4, 5, 6, 7, 8, 9, or 10 different populations of compacted oligonucleotides, wherein the compacted oligonucleotides in the different populations have different sequences. In some embodiments, in a mixture of different compacted oligonucleotides, any given population of compacted oligonucleotides comprises a sequence according to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, or 156.

[0273] In certain embodiments, independent nucleic acid concatemer comprises single-stranded nucleic acid molecule.Independent concatemer molecule comprises at least two copies of the polynucleotide unit of series arrangement.In certain embodiments, each polynucleotide unit comprises sequence of interest.In certain embodiments, each polynucleotide unit comprises at least one universal adapter sequence.In certain embodiments, each polynucleotide unit comprises sequence of interest and at least one universal adapter sequence.In certain embodiments, independent concatemer molecule comprises 2-100 copies of polynucleotide unit or 100-250 copies of polynucleotide unit or 250-500 copies of polynucleotide unit or 500-750 copies of polynucleotide unit or 750-1000 copies of polynucleotide unit or more than 1000 copies of polynucleotide unit.In certain embodiments, independent concatemer molecule comprises 1000-10,000 copies of polynucleotide unit.In certain embodiments, concatemer molecule can be produced by using circular library molecule as library template molecule, amplification primer (for example, through fixed primer or soluble primer), amplification polymerase (for example, with chain displacement activity) and multiple nucleotides to carry out rolling circle amplification reaction. In some embodiments, the concatemer comprises a plurality of concatenated polynucleotide units, wherein the sequence of each polynucleotide unit of a given concatemer molecule is complementary to the sequence of a circular library molecule used as a template library molecule.

[0274] In some embodiments, concatemers can be generated by performing a rolling circle amplification reaction using a plurality of nucleotides, the plurality of nucleotides comprising any combination of two or more nucleotides including dATP, dGTP, dCTP, dTTP, and / or dUTP. In some embodiments, individual concatemers in the plurality of concatemer molecules comprise at least two uracil bases distributed at random positions along the individual concatemer template molecules, wherein the uracil bases can be distributed at different positions in different concatemer molecules.

[0275] In some embodiments, the 5' end or 3' end of the separate concatemer can be fixed to a support or coating. In some embodiments, the interior region of the separate concatemer can be fixed to a support or coating. In some embodiments, the concatemer remains fixed to a support or coating after collapsing / folding into the nanostructure.

[0276] In some embodiments, the coating comprises at least one hydrophilic polymer layer and a plurality of surface capture primers having sequences that can hybridize with at least a portion of the circular library molecules. The surface capture primers can be fixed to the coating and / or embedded in the coating. The surface capture primers can be covalently linked to monomeric compounds that form the polymer layer. In some embodiments, the density of the surface capture primers can be 1000 ng / cm2 per mm2. 2 About 10 2 -10 15 The coating can include multiple or more types of surface capture primers. One type of surface capture primer can be used to perform an on-support rolling circle amplification workflow. Another type of surface capture primer can be used to perform an in-solution rolling circle amplification workflow.

[0277] In some embodiments, individual concatemer molecules can be generated by hybridizing circular library molecules to immobilized surface capture primers and performing an on-support rolling circle amplification reaction with an amplification polymerase (e.g., having strand displacement activity) and a plurality of nucleotides to generate concatemers covalently attached to a surface primer that is immobilized to or embedded in a coating (e.g., Figures 21 to 23 , 39 to 40). The surface capture primer used to perform rolling circle amplification on the support can hybridize to at least a portion of the circular library molecules.

[0278] In some embodiments, a separate concatemer molecule can be produced by hybridizing the circular library molecule with a soluble amplification primer and performing a rolling circle amplification reaction in solution with an amplification polymerase (e.g., having strand displacement activity) and a plurality of nucleotides to produce a duplex comprising a concatemer hybridized with the circular library molecule. The duplex can be distributed on a support having at least one hydrophilic polymer layer and a plurality of surface capture primers, the plurality of surface capture primers having a sequence that can hybridize with at least a portion of the concatemer molecule. A portion of the concatemer can be hybridized with the surface capture primer to produce a concatemer fixed by hybridization with the surface primer. The rolling circle amplification reaction can continue after being distributed on the coated support (e.g., Figures 30 to 32 , 46 to 48).

[0279] In some embodiments, the support comprises a planar or non-planar support. The support can be solid or semi-solid. In some embodiments, the support can be porous, semi-porous or non-porous. The support can be made of any material (such as glass, plastic or polymer material).

[0280] In certain embodiments, the surface of the support can be coated with one or more compounds, to produce a passivation layer on the support. In certain embodiments, the passivation layer forms a porous or semi-porous layer. In certain embodiments, independent concatemer molecules can be attached to the support or attached to the passivation layer, so that the concatemer molecules are fixed to the support. In certain embodiments, the support comprises a low non-specific binding surface, which makes it possible to improve nucleic acid hybridization and amplification and sequencing performance on the support. Generally speaking, the support can comprise one or more layers of covalent or non-covalently attached low binding chemical modification layers, such as silane layers, polymer films, and one or more covalent or non-covalently attached oligonucleotides that can be used are fixed to multiple nucleic acid template molecules on the support. In certain embodiments, the support can include a functionalized polymer coating at least covalently attached to a part of the support by the chemical group on the support, a primer grafted with the functionalized polymer coating, and a water-soluble protective coating on the primer and the functionalized polymer coating. In some embodiments, the functionalized polymer coating comprises poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide) (PAZAM). In some embodiments, the support comprises a surface coating comprising at least one layer of a hydrophilic polymer coating. The surface coating may further comprise at least one layer of a plurality of oligonucleotides (e.g., surface primers). The hydrophilic polymer coating may comprise polyethylene glycol (PEG). The hydrophilic polymer coating may comprise a branched PEG having at least 4 branches. In some embodiments, the low nonspecific binding coating has a degree of hydrophilicity that can be measured as a water contact angle, wherein the water contact angle does not exceed 45 degrees.

[0281] In some embodiments, a plurality of concatemer molecules are immobilized to a support or to a coating on a support. In some embodiments, the support comprises 2 About 10 2 -10 15 In some embodiments, the plurality of concatemers remain fixed to the support after collapse or folding into the nanostructure. Thus, the support comprises a density of 100 molecules per mm 2 About 10 2 -10 15 The density of the fixed nanostructures.

[0282] In certain embodiments, concatemer is fixed on the different sites on the support.In certain embodiments, a plurality of concatemer molecules are fixed to the predetermined site (for example, position) on the support.A plurality of concatemers can be arranged on the support by organized predetermined pattern.In certain embodiments, a plurality of concatemer molecules are fixed to the random non-predetermined site (for example, position) on the support.In certain embodiments, a plurality of concatemers remain fixed to the support after collapsing or being folded into nanostructure.Therefore, a plurality of nanostructures can be fixed to the support at the predetermined site on the support or at the random site on the support.

[0283] In some embodiments, multiple immobilized concatemer molecules are in fluid communication with each other to allow a reagent solution (e.g., an enzyme comprising a polymerase, multivalent molecules, nucleotides and / or divalent cations, etc.) to flow onto the support, such that the multiple immobilized concatemer molecules on the support can react with the reagent solution in a massively parallel manner.

[0284] In some embodiments, multiple immobilized nanostructures are fluidically connected to each other to allow a reagent solution (e.g., an enzyme comprising a polymerase, a multivalent molecule, nucleotides and / or divalent cations, etc.) to flow onto the support, so that the multiple immobilized nanostructures on the support can react with the reagent solution in a massively parallel manner.

[0285] In some embodiments, the plurality of fixed nucleic acid nanostructures further comprise a plurality of circular nucleic acid library molecules. In some embodiments, the plurality of circular nucleic acid library molecules are soluble and do not hybridize with the fixed nucleic acid nanostructures. In some embodiments, at least one fixed nucleic acid nanostructure hybridizes with a circular nucleic acid library molecule. In some embodiments, the nucleic acid nanostructure comprises a plurality of tandem polynucleotide units, wherein the sequence of each polynucleotide unit of a given nanostructure molecule is complementary to the sequence of a circular library molecule.

[0286] In certain embodiments, a plurality of nucleic acid nanostructures through fixing further comprise a plurality of circular nucleic acid library molecules, a plurality of amplification polymerases (for example, having strand displacement activity) and a plurality of nucleotides. In certain embodiments, a plurality of circular nucleic acid library molecules are soluble and do not hybridize with the nucleic acid nanostructure through fixing. In certain embodiments, at least one nucleic acid nanostructure through fixing hybridizes with the circular nucleic acid library molecules to form a nucleic acid amplification duplex with a template molecule (for example, a circular library molecule) and a 3' primer start site (for example, the 3' end of the nucleic acid nanostructure). In certain embodiments, the nucleic acid amplification duplex is combined with the amplification polymerase to form a composite amplification polymerase. In certain embodiments, in the composite amplification polymerase, the complementary nucleotide can be combined with the 3' primer start site at a position relative to the complementary nucleotide in the template molecule (for example, a circular library molecule). In certain embodiments, a plurality of nucleotides include any combination of two or more nucleotides (including dATP, dGTP, dCTP, dTTP and / or dUTP).

[0287] In some embodiments, the plurality of fixed nucleic acid nanostructures further comprise a plurality of sequencing primers, a plurality of sequencing polymerases, and a plurality of nucleotide reagents. In some embodiments, the plurality of sequencing primers are soluble and do not hybridize with the fixed nucleic acid nanostructure. In some embodiments, at least one fixed nucleic acid nanostructure hybridizes with at least one sequencing primer to form a nucleic acid sequencing duplex having a template molecule (e.g., a nanostructure molecule) and a 3' primer initiation site (e.g., the 3' end of the sequencing primer). In some embodiments, the nucleic acid sequencing duplex is combined with a sequencing polymerase to form a composite sequencing polymerase. In some embodiments, the composite sequencing polymerase is combined with a nucleotide reagent comprising a canonical nucleotide, a nucleotide analog, or a multivalent molecule.

[0288] In certain embodiments, canonical nucleotides can include aromatic bases, five-carbon sugars, and at least one phosphate group. Canonical nucleotides can be unlabeled, or can be labeled with a detectable reporter group portion (e.g., fluorophore). In certain embodiments, complementary canonical nucleotides can be combined with 3' primer initiation sites at a position relative to the complementary nucleotides in a template molecule (e.g., nanostructure molecule). In certain embodiments, a plurality of fixed nucleic acid nanostructures further include catalytic or non-catalytic divalent cations. Exemplary catalytic divalent cations include magnesium ions and / or manganese ions that promote the incorporation of polymerase-catalyzed nucleotides. Exemplary non-catalytic divalent cations include strontium ions, barium ions, and / or calcium ions that inhibit the incorporation of polymerase-catalyzed nucleotides.

[0289] In some embodiments, the nucleotide analogs can include aromatic bases, pentoses of 3' chain termination moieties with inhibitory polymerase-catalyzed nucleotide incorporation, and at least one phosphate group. Nucleotide analogs can be unlabeled, or can be labeled with a detectable reporter gene moiety (e.g., a fluorophore). In some embodiments, complementary nucleotide analogs can be combined with 3' primer initiation sites at positions relative to complementary nucleotides in template molecules (e.g., nanostructure molecules). In some embodiments, a plurality of fixed nucleic acid nanostructures further include catalytic or non-catalytic divalent cations. Exemplary catalytic divalent cations include magnesium ions and / or manganese ions that promote polymerase-catalyzed nucleotide incorporation. Exemplary non-catalytic divalent cations include strontium ions, barium ions, and / or calcium ions that inhibit polymerase-catalyzed nucleotide incorporation.

[0290] In some embodiments, a multivalent molecule can comprise: (1) a core; and (2) a plurality of nucleotide arms comprising (i) a core attachment moiety, (ii) a spacer comprising a PEG moiety, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit (see Figures 56 to 59 ). In some embodiments, the nucleotide unit comprises an aromatic base, a pentose and at least one phosphate group, and the linker is attached to the nucleotide unit through the base. The multivalent molecule can be unlabeled or can be labeled with a detectable reporter gene portion (e.g., a fluorophore). In some embodiments, the complementary nucleotide unit of the multivalent molecule can bind to the 3' primer start site at a position opposite to the complementary nucleotide in the template molecule (e.g., nanostructure molecule). In some embodiments, a plurality of fixed nucleic acid nanostructures further comprise catalytic or non-catalytic divalent cations. Exemplary catalytic divalent cations include magnesium ions and / or manganese ions that promote polymerase-catalyzed nucleotide incorporation. Exemplary non-catalytic divalent cations include strontium ions, barium ions and / or calcium ions that inhibit polymerase-catalyzed nucleotide incorporation.

[0291] In some embodiments, the plurality of immobilized nucleic acid nanostructures further comprises a plurality of binding complexes, the plurality of binding complexes comprising at least a first binding complex and a second binding complex, wherein (i) the first binding complex comprises a first nucleic acid primer, a first polymerase, and a first multivalent molecule, the first nucleic acid primer, the first polymerase, and the first multivalent molecule being bound to a first portion of the nucleic acid nanostructure to form a first binding complex, wherein a first nucleotide unit of the multivalent molecule is bound to the first polymerase, and (ii) the second binding complex comprises a second nucleic acid primer, a second polymerase, and the first multivalent molecule, the second nucleic acid primer, the second polymerase, and the first multivalent molecule being bound to a second portion of the same nucleic acid nanostructure to form a second binding complex, wherein a second nucleotide unit of the multivalent molecule is bound to the second polymerase, wherein the first binding complex and the second binding complex comprising the same multivalent molecule form an affinity complex. In some embodiments, the first multivalent molecule is unlabeled or labeled with a detectable reporter moiety.

[0292] In some embodiments, the first nucleic acid primer comprises a first sequencing primer, and the second nucleic acid primer comprises a second sequencing primer. In some embodiments, the first polymerase comprises a first sequencing polymerase, and the second polymerase comprises a second sequencing polymerase.

[0293] In some embodiments, the first nucleic acid primer comprises a first amplification primer, and the second nucleic acid primer comprises a second amplification primer. In some embodiments, the first polymerase comprises a first amplification polymerase, and the second polymerase comprises a second amplification polymerase.

[0294] In some embodiments, the plurality of immobilized nucleic acid nanostructures further comprises a cellular biological sample positioned on the immobilized nanostructures.

[0295] In certain embodiments, the cell biological sample comprises a single cell, a plurality of cells, tissues, organs, organisms or sections from any of these cell biological samples. The cell biological sample comprises a fresh, frozen, fresh frozen or archived sample (e.g., formalin fixed paraffin embedded; FFPE). The cell biological sample can be embedded in a matrix material. The cell biological sample can be stained, decolorized or unstained. The cell biological sample can be permeabilized to allow the nucleic acid in the cell sample to migrate from the cell to a plurality of fixed nanostructures.

[0296] Support method

[0297] Producing high-density fixed nanostructures

[0298] The present invention provides a method for producing a high-density nucleic acid nanostructure immobilized on a support, the method comprising: (a) providing a support having a plurality of first universal surface primers immobilized thereon, wherein the density of the first universal surface primers on the support is 100 μm per mm. 2 About 10 2 -10 15 and (b) generating a plurality of immobilized single-stranded nucleic acid concatemer template molecules by hybridizing a plurality of single-stranded circular nucleic acid library molecules to a plurality of immobilized first universal surface primers, and performing an on-support rolling circle amplification reaction using: (i) a plurality of strand-displacing polymerases, (ii) a plurality of nucleotides, and (iii) a plurality of compacted oligonucleotides, thereby generating the plurality of immobilized single-stranded nucleic acid concatemer template molecules ( Figure 21 and 39 In some embodiments, the individual compacted oligonucleotides comprise a single-stranded linear oligonucleotide having a 5' region that can hybridize to a first portion of a concatemer molecule and a compacted oligonucleotide having a 3' region that can hybridize to a second portion of a concatemer molecule (e.g., the same concatemer). Figure 23 and 40 ), wherein the plurality of immobilized concatemer molecules collapse or fold into a compact nucleic acid nanostructure upon binding to the compacted oligonucleotide, and wherein the plurality of concatemers remain immobilized to the support while collapsing or folding into the nanostructure, thereby producing a density of 100 nanometers per mm 2 The nanostructures fixed to the support are about 10 2 -10 15 Individual supports.

[0299] In some embodiments, a nucleic acid nanostructure can comprise one or more rings, or can have a spherical shape (e.g., a nanosphere), an elongated shape (e.g., a nanorod), a pre-toroidal shape, or a toroidal shape (e.g., a nanotoroidal shape).

[0300] In the rolling circle amplification reaction on the support, comprising a plurality of compacted oligonucleotides can improve the FWHM (full width at half maximum) of the point image of the nanostructure. The point image can be represented as a Gaussian point, and size can be measured with FWHM. The smaller point size as indicated by less FWHM is generally relevant to the improved image of the point. In certain embodiments, the FWHM of the nanostructure point can be about 10 μm or less.

[0301] The nucleic acid nanostructure can be a compact nucleic acid structure having a smaller full width at half maximum (FWHM) than a concatemer that is not collapsed / folded into a nanostructure.

[0302] In some embodiments, the compacted oligonucleotide comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The compacted oligonucleotide can be any length, including 20-150 nucleotides, or 30-100 nucleotides, or 40-80 nucleotides, or any range therebetween.

[0303] In some embodiments, the compacted oligonucleotide comprises a first binding region and a second binding region, and optionally an intervening joint between the 5' region and the 3' region. For example, the intervening joint can be of any length. The length is about 2-20 nucleotides. The intervening joint can comprise a homopolymer with continuous identical bases (e.g., AAA, GGG, CCC, TTT or UUU). The intervening joint can comprise a non-homopolymer sequence.

[0304] Compacting the first binding region of oligonucleotide can be along its length and the first part complete complementarity or partial complementarity with concatemer molecule.Compacting the second binding region of oligonucleotide can be along its length and the second part complete complementarity or partial complementarity with concatemer molecule.Compacting the first binding region of oligonucleotide can be with the first universal sequence portion hybridization (referring to Table 2) of the concatemer molecule with the sequence of any one of SEQ ID NO:157-176.Compacting the second binding region of oligonucleotide can be with the second universal sequence portion hybridization (referring to Table 2) of the concatemer molecule with the sequence of any one of SEQ ID NO:157-176.Compacting the first binding region and the second binding region of oligonucleotide can hybridize with concatemer, so that the distal portion of concatemer is drawn together, thereby concatemer compacting is to form nanostructure.

[0305] In some embodiments, any binding region (e.g., the first binding region, the second binding region, the third binding region, the fourth binding region, the fifth binding region, the sixth binding region, or other binding region) of the compacted oligonucleotide can be hybridized to a portion of a concatemer molecule, wherein the portion of the concatemer molecule comprises a universal adapter sequence according to any one of SEQ ID NOs: 157-176, or a complementary sequence thereof (see Table 2).

[0306] In some embodiments, in step (b), the first binding region of the compaction oligonucleotide comprises a sequence according to any one of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, or 154 (see Table 1).

[0307] In some embodiments, in step (b), the second, third, fourth, fifth, or any subsequent binding region of the compacted oligonucleotide comprises a sequence that is the reverse sequence of the first binding region (e.g., the reverse sequence of any one of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, or 155; see Table 1).

[0308] In some embodiments, in step (b), the first binding region of the compacted oligonucleotide comprises a sequence according to any one of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, or 155; see Table 1).

[0309] In some embodiments, in step (b), the second binding region, the third binding region, the fourth binding region, the fifth binding region, or any subsequent binding region of the compacted oligonucleotide comprises a sequence that is the reverse sequence of the first binding region, wherein the second binding region, the third binding region, the fourth binding region, the fifth binding region, or any subsequence binding region comprises SEQ ID NO: NO: any one of 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151 or 154 (see Table 1).

[0310] In some embodiments, in step (b), the first binding region of the compacted oligonucleotide can have a sequence that is the reverse sequence of the second binding region (e.g., the reverse sequence of any one of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, or 155; see Table 1).

[0311] In some embodiments, in step (b), the second binding region of the compaction oligonucleotide comprises a sequence according to any one of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, or 154 (see Table 1).

[0312] In some embodiments, in step (b), the third binding region of the compaction oligonucleotide comprises a sequence according to any one of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, or 154 (see Table 1).

[0313] In some embodiments, in step (b), the fourth binding region of the compaction oligonucleotide comprises a sequence according to any one of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, or 154 (see Table 1).

[0314] In some embodiments, in step (b), the fifth binding region of the compaction oligonucleotide comprises a sequence according to any one of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, or 154 (see Table 1).

[0315] In some embodiments, in step (b), the subsequent binding region of the compaction oligonucleotide comprises a sequence according to any one of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, or 154 (see Table 1).

[0316] In some embodiments, in step (b), the compacted oligonucleotide comprises the full length sequence according to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, or 156 (see Table 1).

[0317] In some embodiments, the 3' region of any compacted oligonucleotide may include an additional three bases at the terminal 3' end that comprise 2'-O-methyl RNA bases (eg, designated as mUmUmU), or lack additional 2'-O-methyl RNA bases at the terminal 3' end.

[0318] In certain embodiments, the compaction oligonucleotide comprises one or more modified bases or bonds at its 5' or 3' end to confer certain functions. In certain embodiments, the compaction oligonucleotide comprises at least one thiophosphate bond at its 5' and / or 3' end to confer exonuclease resistance. In certain embodiments, at least one nucleotide at or near the 3' end comprises a 2' fluoro base that confer exonuclease resistance. In certain embodiments, the 3' end of the compaction oligonucleotide comprises at least one 2'-O-methyl RNA base that blocks the extension catalyzed by polymerase. For example, the 3' end of the compaction oligonucleotide can comprise three bases, and the three bases comprise 2'-O-methyl RNA bases (for example, designated as mUmUmU). In certain embodiments, the compaction oligonucleotide comprises 3' reverse dT that blocks the extension catalyzed by polymerase at its 3' end. In certain embodiments, the compaction oligonucleotide comprises 3' phosphorylation that blocks the extension catalyzed by polymerase. In certain embodiments, the internal region of the compaction oligonucleotide comprises at least one locked nucleic acid (LNA), which increases the thermal stability of the duplex formed by hybridizing the compaction oligonucleotide and the concatemer molecule.

[0319] The compacted oligonucleotide may comprise at least one region having consecutive guanines. For example, the compacted oligonucleotide may comprise at least one region having 2, 3, 4, 5, 6 or more consecutive guanines. In some embodiments, the compacted oligonucleotide comprises four consecutive guanines that can form a guanine tetrad structure (see Figure 64 The guanine tetrad structure can be stabilized by Hoogsteen hydrogen bonding. The guanine tetrad structure can be stabilized by a central cation including potassium, sodium, lithium, rubidium, or cesium.

[0320] The rolling circle amplification reaction can be performed in the presence of multiple compacted oligonucleotides having at least four consecutive guanines. The resulting concatemer contains repeated copies of the universal binding sequence for the compacted oligonucleotides. At least one compacted oligonucleotide can form a guanine tetrad ( Figure 64 ) and hybridizes to the universal binding sequence of the compacted oligonucleotide, and the resulting concatemer can fold to form an intramolecular G-quadruplex structure ( Figure 65 The concatemer can self-collapse to form a compact nanostructure. The formation of guanine tetrads and G-quadruplexes within the nanostructure can increase the stability of the nanostructure, maintaining its compact size and shape, which can withstand changes in pH, temperature, and / or repeated flow of reagents.

[0321] In some embodiments, the plurality of compacted oligonucleotides in step (b) comprise the same sequence. In some embodiments, in step (b), the plurality of compacted oligonucleotides comprise a sequence according to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, or 156. In some embodiments, the plurality of compacted oligonucleotides comprises a sequence according to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, or 156, wherein the 3' end of the compacted oligonucleotide further comprises three bases comprising 2'-O-methyl RNA bases (e.g., designated as mUmUmU).

[0322] In some embodiments, in step (b), the plurality of compacted oligonucleotides comprises a mixture of two or more different populations of compacted oligonucleotides having different sequences. In some embodiments, in step (b), the plurality of compacted oligonucleotides comprises a mixture of 2, 3, 4, 5, 6, 7, 8, 9, or 10 different populations of compacted oligonucleotides, wherein the compacted oligonucleotides in the different populations have different sequences. In some embodiments, in step (b), in the mixture of different compacted oligonucleotides, any given population of compacted oligonucleotides comprises a sequence according to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, or 156.

[0323] The support may be passivated with at least one hydrophilic polymer coating comprising a plurality of first universal surface primers ( Figure 54 ).

[0324] In certain embodiments, a plurality of first universal surface primers through fixing are positioned at random positions on support or coating.When the universal surface primer through fixing is positioned at random positions on support or the support through coating, nucleic acid nano structure is also positioned at random positions.The nucleic acid nano structure of random positioning can be closely packed and fills most of space on the support through coating.When the nanostructure through fixing is combined with oligonucleotide or nucleotide reagent of detectable label, signal intensity can be concentrated in less space, and with oligonucleotide or nucleotide reagent of detectable label being combined with the concatemer that does not collapse / fold into nanostructure, this significantly increases signal intensity and color differentiation.Therefore, nanostructure described herein and the method for producing them have improved signal intensity, and do not need to prepare the support (for example, flow cell) with predetermined organized pattern array.

[0325] In certain embodiments, a plurality of fixed first universal surface primers are positioned at the predetermined position on the support or coating.For example, the fixed first universal surface primer is arranged with an organized pattern.When the fixed universal surface primer is positioned at the predetermined position on the support or the coated support, the nucleic acid nanostructure is also positioned at the predetermined position.

[0326] In some embodiments, the first universal surface primer lacks a cleavable portion (e.g., the first universal surface primer lacks a cleavable portion that can be converted into an abasic site). In some embodiments, the first universal surface primer lacks a cleavable portion comprising uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine.

[0327] In some embodiments, the plurality of nucleotides used to perform the rolling circle amplification reaction includes dATP, dCTP, dGTP, and dTTP, and none of the nucleotides has a scissile moiety (eg, a scissile moiety in the nucleotide can be converted to an abasic site).

[0328] In some embodiments, the plurality of nucleotides used to perform the rolling circle amplification reaction include: dATP, dCTP, dGTP, dTTP, and a nucleotide having a cleavable portion. In some embodiments, the nucleotide having a cleavable portion comprises uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine. The cleavable portion of the nucleotide can be converted into an abasic site.

[0329] In some embodiments, a rolling circle amplification reaction performed with nucleotides having a scissile moiety produces a plurality of single-stranded nucleic acid concatemer template molecules, wherein individual concatemer template molecules comprise at least two nucleotides, each nucleotide having a scissile moiety distributed at a random position along the individual immobilized concatemer template molecules. Figures 21 to 22 In some embodiments, nucleotides having scissile moieties are distributed at different positions in different immobilized concatemer template molecules.

[0330] In certain embodiments, the independent concatemer template molecule in a plurality of comprises at least two copies of the polynucleotide unit arranged in series.In certain embodiments, each polynucleotide unit comprises a sequence of interest.In certain embodiments, each polynucleotide unit comprises at least one universal adapter sequence.In certain embodiments, each polynucleotide unit comprises a sequence of interest and at least one universal adapter sequence.In certain embodiments, the independent concatemer molecule comprises 2-100 copies of a polynucleotide unit or 100-250 copies of a polynucleotide unit or 250-500 copies of a polynucleotide unit or 500-750 copies of a polynucleotide unit or 750-1000 copies of a polynucleotide unit or more than 1000 copies of a polynucleotide unit.In certain embodiments, the independent concatemer molecule comprises 1000-2000 copies of a polynucleotide unit or 1000-10,000 copies of a polynucleotide unit.In certain embodiments, the independent concatemer comprises multiple series polynucleotide units, wherein the sequence of each polynucleotide unit of a given concatemer molecule is complementary to the sequence of the circular library molecule used as a template library molecule.

[0331] In some embodiments, the individual concatemer molecules comprise two or more copies of a sequence of interest, and wherein the immobilized concatemer template molecules further comprise any one of the following, or any combination of two or more of the following: (i) two or more copies of a universal binding sequence (or its complement) for a soluble forward sequencing primer, (ii) two or more copies of a universal binding sequence (or its complement) for a soluble reverse sequencing primer, (iii) two or more copies of a universal binding sequence (or its complement) for an immobilized first universal surface primer, (iv) two or more copies of a universal binding sequence (or its complement) for an immobilized second universal surface primer, (v) two or more copies of a universal binding sequence (or its complement) for a first soluble amplification primer, (vi) two or more copies of a universal binding sequence (or its complement) for a second soluble amplification primer, (vii) two or more copies of a universal binding sequence (or its complement) for a soluble compaction oligonucleotide, (viii) two or more copies of a sample barcode sequence, and / or (ix) two or more copies of a unique molecular index sequence.

[0332] In some embodiments, the method for producing a high density of nucleic acid nanostructures immobilized on a support further comprises positioning a cellular biological sample on the immobilized nanostructures.

[0333] In certain embodiments, the cell biological sample comprises a single cell, a plurality of cells, tissues, organs, organisms or sections from any of these cell biological samples. The cell biological sample comprises a fresh, frozen, fresh frozen or archived sample (e.g., formalin fixed paraffin embedded; FFPE). The cell biological sample can be embedded in a matrix material. The cell biological sample can be stained, decolorized or unstained. The cell biological sample can be permeabilized to allow the nucleic acid in the cell sample to migrate from the cell to a plurality of fixed nanostructures.

[0334] Support method

[0335] Producing high-density fixed nanostructures containing cleavable portions

[0336] The present invention provides a method for producing a plurality of immobilized nucleic acid nanostructures, the method comprising the steps of (a): providing a support having a plurality of first universal surface primers immobilized thereon, wherein the support is passivated with at least one hydrophilic polymer layer, the hydrophilic polymer layer comprising a plurality of first universal surface primers, wherein each first universal surface primer comprises a 3'OH extendable end and lacks a nucleotide having a cleavable portion, and wherein the density of the first universal surface primers is 100 nm per mm 2 About 10 2 -1015 . The first universal surface primer may lack a cleavable portion that can be converted into an abasic site in a nucleic acid chain. For example, the first universal surface primer may lack uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) and deoxyinosine. In some embodiments, the support lacks a plurality of second universal surface primers or comprises a plurality of second universal surface primers. In some embodiments, the support comprises a plurality of first surface primers and a second surface primer.

[0337] In certain embodiments, the first universal surface primer fixed comprises a single-stranded oligonucleotide comprising DNA, RNA, or a combination of DNA and RNA. The first universal surface primer may comprise a sequence that is fully complementary or partially complementary to at least a portion of a circular nucleic acid library molecule along its length. The first universal surface primer may comprise an end 3' nucleotide having a sugar 3' OH portion that can be extended for nucleotide polymerization (e.g., polymerase-catalyzed polymerization).

[0338] In certain embodiments, the first universal surface primer through fixing can be fixed to a support or fixed to the coating on the support.The first universal surface primer through fixing can be embedded and attached (coupled) to the coating on the support.In certain embodiments, the 5 ' end of the first universal surface primer through fixing is fixed to a support or fixed to the coating on the support.Alternatively, the inner part or 3 ' end of the first universal surface primer through fixing can be fixed to a support or fixed to the coating on the support.The support can comprise a plurality of the first universal surface primers through fixing with the same sequence.The first universal surface primer through fixing can be any length, for example, 4 to 50 nucleotides or 50 to 100 nucleotides or 100 to 150 nucleotides or longer length or any scope therebetween.

[0339] In certain embodiments, a plurality of first universal surface primers through fixing are positioned at random positions (for example, non-predetermined positions) on the support through application.When the universal surface primer through fixing is positioned at random positions on the support through application, the nucleic acid nano structure through fixing is also positioned at random positions.The nucleic acid nano structure of random positioning can be closely packed and fill most of space on the support through application.

[0340] In certain embodiments, a plurality of fixed first universal surface primers are positioned at the predetermined position on the support through application.For example, the fixed first universal surface primer is arranged with an organized pattern.When the fixed universal surface primer is positioned at the predetermined position on the support or the support through application, the nucleic acid nanostructure is also positioned at the predetermined position.

[0341] In some embodiments, the plurality of fixed first universal surface primers comprise at least one phosphorothioate diester bond at its 5' end, which can make the first universal surface primer resistant to exonuclease degradation. In some embodiments, the plurality of fixed first universal surface primers comprise 2 to 5 or more consecutive phosphorothioate diester bonds at its 5' end. In some embodiments, the plurality of fixed first universal surface primers comprise at least one ribonucleotide and / or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which can make the first universal surface primer resistant to exonuclease degradation.

[0342] In some embodiments, the immobilized first universal surface primer comprises at least one locked nucleic acid (LNA) comprising a methylene bridge between the 2' oxygen and 4' carbon of a pentose ring. The immobilized first universal surface primer comprising at least one LNA can resist nuclease digestion and can exhibit an increased melting temperature when hybridized to a circular nucleic acid library molecule.

[0343] In some embodiments, the support further comprises a plurality of second universal surface primers ( Figure 29 ). The second universal surface primer has a different sequence from the first universal surface primer through fixation. The second universal surface primer through fixation of step (a) includes a single-stranded oligonucleotide, which includes a combination of DNA, RNA or DNA and RNA. The second universal surface primer comprises a sequence that is completely complementary or partially complementary to at least a portion of the fixed single-stranded concatemer template molecule along its length. The second universal surface primer through fixation can be fixed to a support or fixed to the coating on the support. The second universal surface primer through fixation can be embedded and attached (coupled) to the coating on the support. In certain embodiments, the 5' end of the second surface primer is fixed to a support or fixed to the coating on the support. Alternatively, the inner part or 3' end of the second universal surface primer through fixation can be fixed to a support or fixed to the coating on the support. The support can include a plurality of second universal surface primers through fixation with the same sequence. The fixed second universal surface primer can be of any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides or longer. In some embodiments, the 3' end of the fixed second universal surface primer comprises an extendable 3'OH portion. In some embodiments, the 3' end of the fixed second universal surface primer comprises a 3' non-extendable portion. The 3' end of the fixed second universal surface primer may comprise a portion that blocks primer extension, such as, for example, a phosphate group, a dideoxycytidine group, a reverse dT or an amino group; These fixed second universal surface primers are non-extendable in the primer extension reaction. The fixed second universal surface primer may lack nucleotides with a cleavable portion.

[0344] In some embodiments, a plurality of fixed second universal surface primers comprise at least one phosphorothioate diester bond at its 5' end, which can make the second universal surface primer resistant to exonuclease degradation. In some embodiments, a plurality of fixed second universal surface primers comprise 2 to 5 or more consecutive phosphorothioate diester bonds at its 5' end. In some embodiments, a plurality of fixed second universal surface primers comprise at least one ribonucleotide and / or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which can make the second universal surface primer resistant to exonuclease degradation.

[0345] In some embodiments, an individual immobilized single-stranded nucleic acid concatemer template molecule is covalently attached to an immobilized first universal surface primer, and at least a portion of the individual concatemer template molecule is hybridized to an immobilized second universal surface primer ( Figure 29 ). The fixed second universal surface primer can be used to fix a portion of the fixed concatemer template molecule to a support. In some embodiments, the fixed concatemer template molecule has two or more copies of the universal binding sequence for the fixed second universal surface primer. The portion of the fixed concatemer template molecule comprising the universal binding sequence for the fixed second universal surface primer can hybridize with the fixed second universal surface primer. In some embodiments, the second universal surface primer comprises a terminal 3' blocking group that makes it non-extendable. In some embodiments, the second universal surface primer has a terminal 3' extendable end.

[0346] In some embodiments, the support comprises 2 About 10 2 -10 15 In some embodiments, the support comprises a first universal surface primer per mm 2 About 10 2 -10 15 In some embodiments, the support comprises a second universal surface primer per mm 2 About 10 2 -10 15 An immobilized first universal surface primer and an immobilized second universal surface primer.

[0347] The immobilized surface primers (e.g., the first and second universal surface primers) can be fluidically connected to each other to allow various solutions of linear or circular nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, reagents, etc. to flow onto the support, so that the multiple immobilized surface primers (and primer extension products generated from the immobilized surface primers) react with the solutions in a massively parallel manner.

[0348] In some embodiments, the method for generating a plurality of immobilized nucleic acid nanostructures further comprises the step (b): generating a plurality of immobilized single-stranded nucleic acid concatemer template molecules, wherein individual concatemer template molecules are covalently linked to an immobilized first universal surface primer by hybridizing a plurality of single-stranded covalently closed circular nucleic acid library molecules to a plurality of immobilized first universal surface primers; and performing an on-support rolling circle amplification reaction using: (i) a plurality of strand-displacing polymerases, (ii) a plurality of nucleotides comprising dATP, dCTP, dGTP, dTTP, and a nucleotide having a scissile moiety, and (iii) a plurality of compacted oligonucleotides, thereby generating a plurality of immobilized single-stranded nucleic acid concatemer template molecules (e.g., Figures 22 to 23 ). In some embodiments, the individual compacted oligonucleotides comprise a single-stranded linear oligonucleotide having a first binding region that can hybridize to the first portion of the concatemer molecule and a compacted oligonucleotide having a second binding region that can hybridize to the second portion of the concatemer molecule. In some embodiments, the individual immobilized concatemer molecules collapse or fold into a compact nucleic acid nanostructure (e.g., a first strand nanostructure). In some embodiments, the plurality of concatemers remain fixed to the support while collapsing or folding into the nanostructure, thereby producing a density of 1000 nt / mm 2 The nanostructures fixed to the support are about 10 2 -10 15 In some embodiments, the individual immobilized nanostructures comprise a plurality of tandem polynucleotide units, and each polynucleotide unit has a sequence that is complementary to the sequence of a covalently closed circular nucleic acid library molecule.

[0349] Multiple immobilized nucleic acid nanostructures can be fluidically connected to each other to allow various reagents in solution (including soluble primers, enzymes, nucleotides, divalent cations, buffers, etc.) to flow onto the support, allowing the multiple immobilized nanostructures to react with the solution in a massively parallel manner.

[0350] In some embodiments, in step (b), the first binding region of the compacted oligonucleotide can hybridize to the first portion of the concatemer molecule, wherein the first portion of the concatemer molecule comprises a universal adapter sequence according to any one of SEQ ID NOs: 157-176 or a complementary sequence thereof (see Table 2).

[0351] In some embodiments, in step (b), the second binding region of the compacted oligonucleotide can hybridize to the second portion of the concatemer molecule, wherein the second portion of the concatemer molecule comprises a universal adapter sequence according to any one of SEQ ID NOs: 157-176 or its complement) (see Table 2).

[0352] In some embodiments, in step (b), the compacted oligonucleotide comprises a first binding region, a second binding region, a third binding region, a fourth binding region, a fifth binding region, a sixth binding region, or other binding regions.

[0353] In some embodiments, in step (b), any binding region (e.g., the first binding region, the second binding region, the third binding region, the fourth binding region, the fifth binding region, the sixth binding region, or other binding region) of the compacted oligonucleotide can be hybridized with a portion of a concatemer molecule, wherein the portion of the concatemer molecule comprises a universal adapter sequence according to any one of SEQ ID NOs: 157-176 or its complement (see Table 2).

[0354] In some embodiments, in step (b), the compacted oligonucleotide comprises two or more binding regions, and all binding regions have the same sequence. In some embodiments, the compacted oligonucleotide comprises two binding regions with different sequences. In some embodiments, the compacted oligonucleotide comprises three or more binding regions, and at least two binding regions have different sequences.

[0355] In some embodiments, in step (b), the first binding region of the compacted oligonucleotide can have the same sequence as the second binding region.

[0356] In some embodiments, in step (b), the first binding region of the compacted oligonucleotide may have a different sequence than the second binding region.

[0357] In some embodiments, in step (b), the first binding region of the compaction oligonucleotide comprises a sequence according to any one of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, or 154 (see Table 1).

[0358] In some embodiments, in step (b), the second, third, fourth, fifth, or any subsequent binding region of the compacted oligonucleotide comprises a sequence that is the reverse sequence of the first binding region (e.g., the reverse sequence of any one of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, or 155; see Table 1).

[0359] In some embodiments, in step (b), the first binding region of the compacted oligonucleotide comprises a sequence according to any one of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, or 155; see Table 1).

[0360] In some embodiments, in step (b), the second binding region, the third binding region, the fourth binding region, the fifth binding region, or any subsequent binding region of the compacted oligonucleotide comprises a sequence that is the reverse sequence of the first binding region, wherein the second binding region, the third binding region, the fourth binding region, the fifth binding region, or any subsequence binding region comprises SEQ ID NO: NO: any one of 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151 or 154 (see Table 1).

[0361] In some embodiments, in step (b), the first binding region of the compacted oligonucleotide can have a sequence that is the reverse sequence of the second binding region (e.g., the reverse sequence of any one of SEQ ID NO: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 107, 110, 113, 116, 119, 122, 125, 128, 131, 134, 137, 140, 143, 146, 149, 152, or 155; see Table 1).

[0362] In some embodiments, in step (b), the second binding region of the compaction oligonucleotide comprises a sequence according to any one of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, or 154 (see Table 1).

[0363] In some embodiments, in step (b), the third binding region of the compaction oligonucleotide comprises a sequence according to any one of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, or 154 (see Table 1).

[0364] In some embodiments, in step (b), the fourth binding region of the compaction oligonucleotide comprises a sequence according to any one of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, or 154 (see Table 1).

[0365] In some embodiments, in step (b), the fifth binding region of the compaction oligonucleotide comprises a sequence according to any one of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, or 154 (see Table 1).

[0366] In some embodiments, in step (b), the subsequent binding region of the compaction oligonucleotide comprises a sequence according to any one of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 79, 82, 85, 88, 91, 94, 97, 100, 103, 106, 109, 112, 115, 118, 121, 124, 127, 130, 133, 136, 139, 142, 145, 148, 151, or 154 (see Table 1).

[0367] In some embodiments, in step (b), the compacted oligonucleotide comprises the full length sequence according to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, or 156 (see Table 1).

[0368] In some embodiments, in step (b), the terminal 3' end of any compacted oligonucleotide may comprise at least one additional base comprising one or more 2'-O-methyl RNA bases (e.g., designated as mUmUmU), or the terminal 3' end lacks an additional 2'-O-methyl RNA base.

[0369] In certain embodiments, in step (b), the compaction oligonucleotide comprises one or more modified bases or bonds at its 5' or 3' end to confer certain functions. In certain embodiments, the compaction oligonucleotide comprises at least one thiophosphate bond at its 5' and / or 3' end to confer exonuclease resistance. In certain embodiments, at least one nucleotide at or near the 3' end comprises a 2' fluoro base that confers exonuclease resistance. In certain embodiments, the 3' end of the compaction oligonucleotide comprises at least one 2'-O-methyl RNA base that blocks the extension catalyzed by polymerase. For example, the 3' end of the compaction oligonucleotide comprises at least one base, and the at least one base comprises a 2'-O-methyl RNA base (for example, designated as mUmUmU). In certain embodiments, the compaction oligonucleotide comprises at its 3' end 3' reverse dT that blocks the extension catalyzed by polymerase. In certain embodiments, the compaction oligonucleotide comprises 3' phosphorylation that blocks the extension catalyzed by polymerase. In certain embodiments, the compaction oligonucleotide comprises at least one locked nucleic acid (LNA) that increases the thermal stability of the duplex formed by hybridizing the compaction oligonucleotide and the concatemer molecule.

[0370] In some embodiments, in step (b), the compacted oligonucleotide may include at least one region (e.g., a hybridization / binding region) having consecutive guanines. For example, the compacted oligonucleotide may include at least one region having 2, 3, 4, 5, 6, or more consecutive guanines. In some embodiments, the compacted oligonucleotide comprises four consecutive guanines that can form a guanine tetrad structure (see Figure 64 The guanine tetrad structure can be stabilized by Hoogsteen hydrogen bonding. The guanine tetrad structure can be stabilized by a central cation including potassium, sodium, lithium, rubidium, or cesium.

[0371] In some embodiments, in step (b), at least one compacted oligonucleotide can form a guanine tetrad ( Figure 64 ) and hybridizes with the universal binding sequence in the concatemer, which may cause the concatemer to fold to form an intramolecular G-quadruplex structure ( Figure 65 The concatemer can self-collapse to form a compact nanostructure. The formation of guanine tetrads and G-quadruplexes within the nanostructure can increase the stability of the nanostructure, maintaining its compact size and shape, which can withstand changes in pH, temperature, and / or repeated flow of reagents.

[0372] In some embodiments, in step (b), the plurality of compacted oligonucleotides comprises a mixture of two or more different populations of compacted oligonucleotides having different sequences. In some embodiments, in step (b), the plurality of compacted oligonucleotides comprises a mixture of 2, 3, 4, 5, 6, 7, 8, 9, or 10 different populations of compacted oligonucleotides, wherein the compacted oligonucleotides in the different populations have different sequences. In some embodiments, in step (b), in the mixture of different compacted oligonucleotides, any given population of compacted oligonucleotides comprises a sequence according to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, or 156.

[0373] In certain embodiments, in the method for producing multiple fixed nucleic acid nanostructures in step (b), the nucleic acid nanostructure can include one or more rings, or can have a spherical shape (e.g., nanosphere), an elongated shape (e.g., nanorod), a preliminary annular shape or an annular shape (e.g., nanoannular shape). The nucleic acid nanostructure can be a compact nucleic acid structure having a smaller full width at half maximum (FWHM) than a concatemer that is not collapsed / folded into a nanostructure. Including multiple compacted oligonucleotides in the rolling circle amplification reaction on a support can improve the FWHM (full width at half maximum) of the point image of the nanostructure. The point image can be represented as a Gaussian point, and the size can be measured with FWHM. Smaller point sizes as indicated by smaller FWHM are generally associated with improved images of the point. In certain embodiments, the FWHM of the nanostructure point can be about 10 μm or less.

[0374] In some embodiments, in the method for generating a plurality of immobilized nucleic acid nanostructures of step (b), the individual single-stranded covalently closed circular nucleic acid library molecules in the plurality comprise a sequence of interest, and any one or any combination of two or more of the following: (i) a universal binding sequence for a soluble forward sequencing primer (or its complement), (ii) a universal binding sequence for a soluble reverse sequencing primer (or its complement), (iii) a universal binding sequence for an immobilized first universal surface primer (or its complement), (iv) a universal binding sequence for an immobilized second universal surface primer (or its complement), (v) a universal binding sequence for a first soluble amplification primer (or its complement), (vi) a universal binding sequence for a second soluble amplification primer (or its complement), (vii) a universal binding sequence for a soluble compaction oligonucleotide (or its complement), (viii) a sample barcode sequence, and / or (ix) a unique molecular index sequence (e.g., Figure 18 A and 18B; Figure 20 A and 20B).

[0375] In some embodiments, in the method for producing a plurality of immobilized nucleic acid nanostructures of step (b), the rolling circle amplification reaction produces a plurality of immobilized single-stranded nucleic acid concatemer template molecules (e.g., first strand nanostructures), wherein individual concatemer template molecules comprise at least two copies of polynucleotide units arranged in series. In some embodiments, each polynucleotide unit comprises a sequence of interest. In some embodiments, each polynucleotide unit comprises at least one universal adapter sequence. In some embodiments, each polynucleotide unit comprises a sequence of interest and at least one universal adapter sequence (e.g., Figure 21 ). In some embodiments, a separate concatemer molecule comprises 2-100 copies of a polynucleotide unit, or 100-250 copies of a polynucleotide unit, or 250-500 copies of a polynucleotide unit, or 500-750 copies of a polynucleotide unit, or 750-1000 copies of a polynucleotide unit, or more than 1000 copies of a polynucleotide unit. In some embodiments, a separate concatemer molecule comprises 1000-10,000 copies of a polynucleotide unit. In some embodiments, a separate concatemer comprises a plurality of concatenated polynucleotide units, wherein the sequence of each polynucleotide unit of a given concatemer molecule is complementary to the sequence of a circular library molecule used as a template library molecule.

[0376] In some embodiments, the individual concatemer molecules comprise two or more copies of a sequence of interest, wherein the immobilized concatemer template molecules further comprise any one of the following, or any combination of two or more of the following: (i) two or more copies of a universal binding sequence (or its complement) for a soluble forward sequencing primer, (ii) two or more copies of a universal binding sequence (or its complement) for a soluble reverse sequencing primer, (iii) two or more copies of a universal binding sequence (or its complement) for an immobilized first universal surface primer, (iv) two or more copies of a universal binding sequence (or its complement) for an immobilized second universal surface primer, (v) two or more copies of a universal binding sequence (or its complement) for a first soluble amplification primer, (vi) two or more copies of a universal binding sequence (or its complement) for a second soluble amplification primer, (vii) two or more copies of a universal binding sequence (or its complement) for a soluble compaction oligonucleotide, (viii) two or more copies of a sample barcode sequence, and / or (ix) two or more copies of a unique molecular index sequence.

[0377] In some embodiments, in the method for producing a plurality of fixed nucleic acid nanostructures in step (b), the rolling circle amplification reaction produces concatemer molecules comprising universal binding sequences that can hybridize / bind to certain primers. In some embodiments, the universal binding sequence (or its complement) for the forward sequencing primer can hybridize with at least a portion of the forward sequencing primer. In some embodiments, the universal binding sequence (or its complement) for the reverse sequencing primer can hybridize with at least a portion of the reverse sequencing primer. In some embodiments, the universal binding sequence (or its complement) for the fixed first universal surface primer can hybridize with at least a portion of the fixed first universal surface primer. In some embodiments, the universal binding sequence (or its complement) for the fixed second universal surface primer can hybridize with at least a portion of the fixed second universal surface primer. In some embodiments, the universal binding sequence (or its complement) for the first soluble amplification primer can hybridize with at least a portion of the first soluble amplification primer. In some embodiments, the universal binding sequence (or its complement) for the second soluble amplification primer can hybridize with at least a portion of the second soluble amplification primer. In some embodiments, a universal binding sequence for a soluble compaction oligonucleotide (or its complement) can hybridize to at least a portion of the soluble compaction oligonucleotide.

[0378] In some embodiments, in the method for producing a plurality of fixed nucleic acid nanostructures in step (b), a plurality of fixed single-stranded nucleic acid concatemer template molecules (e.g., first strand nanostructures) comprise two or more copies of a universal binding sequence (or its complement) for a fixed second sequence surface primer. In some embodiments, a separate fixed single-stranded nucleic acid concatemer template molecule is attached (e.g., covalently attached) to the fixed first universal surface primer, and at least a portion of the separate concatemer template molecule hybridizes to the fixed second universal surface primer. The fixed second universal surface primer can be used to fix a portion of the fixed concatemer template molecule to a support (see Figure 29 ). In some embodiments, the second universal surface primer comprises a terminal 3' blocking group that renders it non-extendable.

[0379] In some embodiments, in the method for generating a plurality of immobilized nucleic acid nanostructures in step (b), a rolling circle amplification reaction can be performed with a nucleotide mixture containing dATP, dCTP, dGTP, dTTP, and a nucleotide having a cleavable moiety to generate immobilized concatemer template molecules (e.g., first-strand nanostructures) comprising at least one nucleotide having a cleavable moiety (e.g., Figures 21 to 22 ). The cleavable portion in the fixed concatemer template molecule can be converted into an abasic site. In some embodiments, in the nucleotide mixture, the nucleotide having the cleavable portion comprises uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine. In the fixed concatemer template molecule, uridine can be converted into an abasic site using uracil DNA glycosylase (UDG), 8oxoG can be converted into an abasic site using FPG glycosylase, and deoxyinosine can be converted into an abasic site using 3-methyladenine DNA glycosylase II (AlkA) glycosylase.

[0380] In some embodiments, the nucleotide mixture may include an amount of dUTP such that a target percentage of thymidines in the resulting concatemer molecules are replaced with dUTP. For example, when 30% of the dTTP in the concatemer molecules is to be replaced with dUTP (e.g., 30% is the target percentage), the nucleotide mixture may contain 7.5% dUTP (e.g., 30 / 4 = 7.5%), 17.5% dTTP, and 25% each of dATP, dCTP, and dGTP. The target percentage of dTTP to be replaced with dUTP can be about 0.1% to 1%, or about 1% to 5%, or about 5% to 10%, or about 10% to 20%, or about 20% to 30%, or about 30% to 45%, or about 45% to 50%, or more of the dTTP in the immobilized concatemer template molecules replaced with nucleotides having a cleavable moiety.

[0381] In some embodiments, the nucleotide mixture may include an amount of deoxyinosine such that a target percentage of guanosine in the resulting concatemer molecule is replaced with deoxyinosine. For example, if 30% of the dGTP in the concatemer molecule is to be replaced with deoxyinosine (e.g., 30% is the target percentage), the nucleotide mixture may contain 7.5% deoxyinosine (e.g., 30 / 4 = 7.5%), 17.5% dGTP, and 25% each of dATP, dCTP, and dTTP. The target percentage of dGTP to be replaced with deoxyinosine can be about 0.1% to 1%, or about 1% to 5%, or about 5% to 10%, or about 10% to 20%, or about 20% to 30%, or about 30% to 45%, or about 45% to 50%, or more of the dGTP in the immobilized concatemer template molecule replaced with a nucleotide having a cleavable moiety.

[0382] In some embodiments, the nucleotide mixture may include an amount of 8oxoG such that a target percentage of guanosine in the resulting concatemer molecule is replaced with 8oxoG. For example, if 30% of the dGTP in the concatemer molecule is to be replaced with 8oxoG (e.g., 30% is the target percentage), the nucleotide mixture may contain 7.5% 8oxoG (e.g., 30 / 4 = 7.5%), 17.5% dGTP, and 25% each of dATP, dCTP, and dTTP. The target percentage of dGTP to be replaced with 8oxoG can be about 0.1% to 1%, or about 1% to 5%, or about 5% to 10%, or about 10% to 20%, or about 20% to 30%, or about 30% to 45%, or about 45% to 50%, or more of the dGTP in the immobilized concatemer template molecule replaced with nucleotides having a cleavable moiety.

[0383] In some embodiments, in the method for generating a plurality of immobilized nucleic acid nanostructures of step (b), the rolling circle amplification reaction generates a plurality of single-stranded nucleic acid concatemer template molecules (e.g., first-strand nanostructures), wherein individual concatemer template molecules have at least two nucleotides, each nucleotide having a scissile moiety distributed at a random position along the individual immobilized concatemer template molecules. In some embodiments, the nucleotides having the scissile moiety are distributed at different positions in different immobilized concatemer template molecules.

[0384] In some embodiments, in the method for generating a plurality of immobilized nucleic acid nanostructures of step (b), the method may further comprise removing the single-stranded covalently closed circular nucleic acid library molecules from the concatemer template molecules using at least one washing step, the at least one washing step being performed under conditions suitable for retaining the single-stranded nucleic acid concatemer template molecules, wherein the individual concatemer template molecules are operably linked to the immobilized first universal surface primer.

[0385] In some embodiments, the method for generating a plurality of immobilized nucleic acid nanostructures further comprises step (c): sequencing a plurality of immobilized concatemer template molecules (e.g., first strand nanostructures) to thereby generate a plurality of extended forward sequencing primer chains. The sequencing of step (c) may comprise contacting the plurality of immobilized concatemer template molecules with a plurality of soluble forward sequencing primers under conditions suitable for hybridizing at least one forward sequencing primer to at least one of the forward sequencing primer binding sites / sequences of the immobilized concatemer template molecules, and performing a forward sequencing reaction using one or more types of sequencing polymerases, a plurality of nucleotide reagents, and the hybridized first forward sequencing primer to generate a plurality of forward sequencing products ( Figure 23 In some embodiments, the plurality of nucleotide reagents are detectably labeled. In some embodiments, the sequencing of step (c) further comprises detecting and imaging the plurality of forward sequencing products.

[0386] In some embodiments, the sequencing of step (c) further comprises contacting the plurality of immobilized concatemer template molecules with the plurality of compacted oligonucleotides under conditions suitable for hybridizing the 5' end of at least one compacted oligonucleotide to the first portion of the concatemer molecule and hybridizing a second portion of the same compacted oligonucleotide to the second portion of the same concatemer molecule. In some embodiments, the plurality of compacted oligonucleotides of step (c) comprises any of the sequences described above in step (b).

[0387] In some embodiments, in the sequencing of step (c), the soluble forward sequencing primer comprises a 3'OH extendable end. In some embodiments, the soluble forward sequencing primer comprises a 3' blocking portion that can be removed to produce a 3'OH extendable end. In some embodiments, the soluble forward sequencing primer lacks a nucleotide with an easily cleavable portion. The forward sequencing reaction can produce multiple extended forward sequencing primer chains. In some embodiments, the individual fixed concatemer template molecules have multiple copies of forward sequencing primer binding sites, wherein each forward sequencing primer binding site is capable of hybridizing with the first forward sequencing primer. The individual forward sequencing primer binding site in a given fixed concatemer template molecule can hybridize with the forward sequencing primer and can undergo sequencing reactions. The individual fixed concatemer template molecules can undergo two or more sequence reactions, wherein each sequencing reaction is initiated from the first forward sequencing primer that hybridizes with the forward sequencing primer binding site (e.g., see Figure 23 ).

[0388] In some embodiments, in the sequencing of step (c), the nucleotide reagent comprises a plurality of nucleotides, a plurality of nucleotide analogs, or a plurality of multivalent molecules.

[0389] In certain embodiments, in the sequencing of step (c), the individual nucleotides in the plurality of nucleotides include aromatic bases, pentoses, and at least one phosphate group. In certain embodiments, the plurality of nucleotides are unlabeled. In certain embodiments, at least one nucleotide in the plurality of nucleotides can be labeled with a detectable reporter gene moiety (e.g., a fluorophore). In certain embodiments, the sequencing of step (c) further includes contacting the plurality of fixed concatemer template molecules with catalytic or non-catalytic divalent cations. Exemplary catalytic divalent cations include magnesium ions and / or manganese ions that promote polymerase-catalyzed nucleotide incorporation. Exemplary non-catalytic divalent cations include strontium ions, barium ions, and / or calcium ions that inhibit polymerase-catalyzed nucleotide incorporation.

[0390] In some embodiments, in the sequencing of step (c), the individual nucleotide analogs in a plurality of sequences may comprise aromatic bases, pentoses with 3' chain termination moieties that inhibit polymerase-catalyzed nucleotide incorporation, and at least one phosphate group. In some embodiments, a variety of nucleotide analogs are unlabeled. In some embodiments, at least one nucleotide analog in a plurality of sequences may be labeled with a detectable reporter gene moiety (e.g., a fluorophore). In some embodiments, the sequencing of step (c) further comprises contacting a plurality of fixed concatemer template molecules with catalytic or non-catalytic divalent cations. Exemplary catalytic divalent cations may comprise magnesium ions and / or manganese ions that promote polymerase-catalyzed nucleotide incorporation. Exemplary non-catalytic divalent cations may comprise strontium ions, barium ions, and / or calcium ions that inhibit polymerase-catalyzed nucleotide incorporation.

[0391] In some embodiments, in the sequencing of step (c), the individual multivalent molecules in the plurality comprise (1) a core; and (2) a plurality of nucleotide arms, each of the plurality of nucleotide arms comprising (i) a core attachment moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit (e.g., see Figures 55A to 55C , 56 to 59). In some embodiments, the nucleotide unit comprises an aromatic base, a pentose and at least one phosphate group, and the linker is attached to the nucleotide unit through the base. In some embodiments, a plurality of multivalent molecules are unlabeled. In some embodiments, at least one multivalent molecule in the plurality is labeled with a detectable reporter gene portion (e.g., a fluorophore). In some embodiments, the sequencing of step (c) further comprises contacting a plurality of fixed concatemer template molecules with catalytic or non-catalytic divalent cations. Exemplary catalytic divalent cations may include magnesium ions and / or manganese ions that promote polymerase-catalyzed nucleotide incorporation. Exemplary non-catalytic divalent cations may include strontium ions, barium ions and / or calcium ions that inhibit polymerase-catalyzed nucleotide incorporation.

[0392] In some embodiments, during the sequencing of step (c), the method further comprises forming at least one affinity complex by contacting a plurality of immobilized concatemer template molecules (e.g., first strand nanostructures) with a plurality of soluble forward sequencing primers, a plurality of sequencing polymerases, and a plurality of multivalent molecules to form a plurality of binding complexes including at least a first binding complex and a second binding complex, wherein (i) the first binding complex comprises a first forward sequencing primer, a first sequencing polymerase, and a first multivalent molecule, the first forward sequencing primer, the first sequencing polymerase, and the first multivalent molecule being bound to a first portion of the immobilized concatemer template molecules; In one embodiment, the present invention relates to a method for preparing a plurality of concatemer template molecules, wherein the first portion of the multivalent molecule is bound to the first portion of the multivalent molecule (e.g., the first strand of the nanostructure), thereby forming a first binding complex, wherein the first nucleotide unit of the multivalent molecule is bound to the first polymerase, and (ii) a second binding complex comprising a second forward sequencing primer, a second sequencing polymerase, and the first multivalent molecule, wherein the second forward sequencing primer, the second sequencing polymerase, and the first multivalent molecule are bound to a second portion of the same immobilized concatemer template molecule, thereby forming a second binding complex, wherein the second nucleotide unit of the multivalent molecule is bound to the second polymerase, and wherein the first binding complex and the second binding complex comprising the same multivalent molecule form an avidity complex. In some embodiments, the multivalent molecule is unlabeled or labeled with a detectable reporter moiety.

[0393] In some embodiments, in the on-support rolling circle amplification reaction of step (b), the first binding region of the compacted oligonucleotide hybridizes to the concatemer template molecule at a first portion that does not interfere with (e.g., has little or no overlap) the hybridization of the soluble forward sequencing primer to the forward sequencing primer binding site of the plurality of concatemer template molecules of step (c).

[0394] In some embodiments, in the on-support rolling circle amplification reaction of step (b), the second binding region of the compacted oligonucleotide hybridizes to the concatemer template molecule at a second portion that does not interfere with (e.g., has little or no overlap) the hybridization of the soluble forward sequencing primer to the forward sequencing primer binding site of the plurality of concatemer template molecules of step (c).

[0395] In some embodiments, the method for generating a plurality of immobilized nucleic acid nanostructures further comprises step (d): retaining a plurality of immobilized concatemer template molecules (e.g., first strand nanostructures) and replacing a plurality of extended forward sequencing primer strands with a plurality of forward extension strands hybridized to the retained immobilized nucleic acid concatemer template molecules. Figures 24 to 25), multiple extended forward sequencing primer strands can be removed and replaced with multiple forward extension strands. A primer extension reaction can be performed with multiple compacted oligonucleotides to collapse / fold the forward extension strands, generating a second strand nanostructure. Those skilled in the art will recognize that there are a variety of methods for performing primer extension reactions, some of which are described below.

[0396] In some embodiments, step (d) comprises: contacting the at least one extended forward sequencing primer strand with a plurality of strand displacement polymerases and a plurality of nucleotides under conditions suitable for initiating a strand displacement primer extension reaction using the at least one extended forward sequencing primer strand, and in the absence of a soluble amplification primer, thereby generating a forward extension strand covalently linked to the extended forward sequencing primer strand, wherein the forward extension strand hybridizes to the immobilized concatemer template molecule (e.g., the first strand nanostructure). Figures 24 to 25 ). For example, one of the extended forward sequencing primer chains can serve as a primer for a chain displacement polymerase. The chain displacement polymerase can extend the extended forward sequencing primer chain and displace the forward sequencing primer chain extended downstream, while synthesizing the extended chain that replaces the forward sequencing primer chain extended downstream. The newly extended chain is covalently linked to the extended forward sequencing primer chain. The fixed concatemer template molecule is then retained. In certain embodiments, the multiple nucleotides in the primer extension reaction of step (d) lack nucleotides with an easily cleavable portion. The primer extension reaction can include multiple compaction oligonucleotides to produce a forward extension chain that forms a nanostructure (e.g., a second chain nanostructure). Compared to the forward extension chain produced by a primer extension reaction performed in the absence of compaction oligonucleotides, a separate forward extension chain can collapse into a nanostructure with a more compact size and / or shape. In some embodiments, the individual compacted oligonucleotides comprise a single-stranded linear oligonucleotide having a 5' region that can hybridize to a first portion of a forward extension strand and a compacted oligonucleotide having a 3' region that can hybridize to a second portion of a forward extension strand (eg, the same forward extension strand).

[0397] In some embodiments, any embodiment of step (d) can be performed in the presence of a plurality of compacted oligonucleotides. In some embodiments, the plurality of compacted oligonucleotides in any embodiment of step (d) comprises any sequence as described above in step (b).

[0398] Without wishing to be bound by theory, it is believed that including the compacted oligonucleotide in the primer extension reaction of step (d) can improve the FWHM (full width at half maximum) of the dot image of the nanostructure. The dot image can be represented as a Gaussian dot, and the size can be measured in FWHM. A smaller dot size, as indicated by a smaller FWHM, is generally associated with an improved image of the dot. In some embodiments, the FWHM of the nanostructure dot can be about 10 μm or less.

[0399] Examples of strand displacement polymerases include, but are not limited to, phi29 DNA polymerase, Bst DNA polymerase large fragment, Bsu DNA polymerase large fragment (exo-), Bca DNA polymerase (exo-), Klenow fragment of E. coli DNA polymerase, T5 polymerase, M-MuLV reverse transcriptase, HIV viral reverse transcriptase, Deep Vent DNA polymerase, and KOD DNA polymerase. The phi29 DNA polymerase may be a wild-type phi29 DNA polymerase (e.g., from Expedeon TM MagniPhi), or variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific TM ), or chimeric QualiPhi DNA polymerase (e.g., from 4basebio TM ).

[0400] In some embodiments, the primer extension reaction of step (d) comprises: (i) removing a plurality of extended forward sequencing primer strands while retaining an immobilized concatemer template molecule (e.g., a first strand nanostructure); and (ii) contacting the plurality of retained immobilized concatemer molecules with a plurality of soluble forward sequencing primers (e.g., a second plurality of soluble forward sequencing primers), a plurality of nucleotides (e.g., a second plurality of nucleotides), and a plurality of primer extension polymerases under conditions suitable for hybridizing the plurality of soluble forward sequencing primers to the plurality of retained immobilized concatemer template molecules and for performing a polymerase-catalyzed primer extension reaction, thereby generating a plurality of forward extension strands, wherein the soluble sequencing primers hybridize to the forward sequencing primer binding sequences in the retained immobilized concatemer molecules. The plurality of nucleotides in the primer extension reaction of step (d) lack nucleotides having a cleavable portion. The primer extension reaction of step (d) may comprise a plurality of compacted oligonucleotides to generate a forward extension strand that forms a nanostructure (e.g., a second strand nanostructure). Compared to a forward extension strand produced by a primer extension reaction performed in the absence of a compacting oligonucleotide, the individual forward extension strands can collapse into a nanostructure having a more compact size and / or shape. In some embodiments, the individual compacting oligonucleotides comprise a single-stranded linear oligonucleotide having a 5' region that can hybridize to a first portion of the forward extension strand and a compacting oligonucleotide having a 3' region that can hybridize to a second portion of the forward extension strand (e.g., the same forward extension strand).

[0401] Without wishing to be bound by theory, it is believed that including the compacted oligonucleotide in the primer extension reaction of step (d) can improve the FWHM (full width at half maximum) of the dot image of the nanostructure. The dot image can be represented as a Gaussian dot, and the size can be measured in FWHM. A smaller dot size, as indicated by a smaller FWHM, is generally associated with an improved image of the dot. In some embodiments, the FWHM of the nanostructure dot can be about 10 μm or less.

[0402] In some embodiments, the primer extension reaction of step (d) comprises: (i) removing a plurality of extended forward sequencing primer chains while retaining an immobilized concatemer template molecule (e.g., a first strand nanostructure); and (ii) contacting the plurality of retained immobilized concatemer molecules with a plurality of soluble amplification primers, a plurality of nucleotides (e.g., a second plurality of nucleotides), and a plurality of primer extension polymerases under conditions suitable for hybridizing the plurality of soluble amplification primers to the plurality of retained immobilized concatemer template molecules and suitable for performing a polymerase-catalyzed primer extension reaction, thereby generating a plurality of forward extension chains, wherein the soluble amplification primers hybridize to the soluble amplification primer binding sequences in the retained immobilized concatemer molecules. The plurality of nucleotides in the primer extension reaction of step (d) may lack nucleotides having a cleavable portion. The primer extension reaction of step (d) may include a plurality of compacted oligonucleotides to generate a forward extension chain that forms a nanostructure (e.g., a second strand nanostructure). Compared to a forward extension strand produced by a primer extension reaction performed in the absence of a compacting oligonucleotide, the individual forward extension strands can collapse into a nanostructure having a more compact size and / or shape. In some embodiments, the individual compacting oligonucleotides comprise a single-stranded linear oligonucleotide having a 5' region that can hybridize to a first portion of the forward extension strand and a compacting oligonucleotide having a 3' region that can hybridize to a second portion of the forward extension strand (e.g., the same forward extension strand).

[0403] Without wishing to be bound by theory, it is believed that including the compacted oligonucleotide in the primer extension reaction of step (d) can improve the FWHM (full width at half maximum) of the dot image of the nanostructure. The dot image can be represented as a Gaussian dot, and the size can be measured in FWHM. A smaller dot size, as indicated by a smaller FWHM, is generally associated with an improved image of the dot. In some embodiments, the FWHM of the nanostructure dot can be about 10 μm or less.

[0404] In some embodiments, in any of the embodiments of step (d) above, conditions suitable for hybridizing a plurality of soluble forward sequencing primers to a plurality of retained immobilized single-stranded nucleic acid concatemer template molecules or conditions suitable for hybridizing a plurality of soluble amplification primers to a plurality of retained immobilized single-stranded nucleic acid concatemer template molecules comprise hybridizing the retained immobilized concatemer template molecules to the soluble forward sequencing primers in the presence of a primer extension polymerase, a plurality of nucleotides, and a high-efficiency hybridization buffer. In some embodiments, the high-efficiency hybridization buffer comprises: (i) a first polar aprotic solvent having a dielectric constant of no greater than 40 and a polarity index of 4-9; (ii) a second polar aprotic solvent having a dielectric constant of no greater than 115 and present in the hybridization buffer formulation in an amount effective to denature the double-stranded nucleic acid; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4-8; and (iv) a crowding agent in an amount sufficient to enhance or promote molecular crowding. In some embodiments, the high-efficiency hybridization buffer comprises: (i) a first polar aprotic solvent comprising acetonitrile at 25-50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5-10% by volume of the hybridization buffer; (iii) a pH buffer system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0405] In some embodiments, in any of the above step (d), an enzyme or chemical reagent can be used to remove the plurality of extended forward sequencing primer strands from the immobilized concatemer template molecule (e.g., the first strand nanostructure). For example, a 5' to 3' double-stranded DNA exonuclease (including T7 exonuclease (e.g., from New England Biolabs) can be used. TM , catalog number M0263S)) to enzymatically degrade the multiple extended forward sequencing primer chains. In some embodiments, the multiple extended forward sequencing primer chains can be removed at a temperature that is conducive to nucleic acid denaturation.

[0406] In some embodiments, in any of the embodiments of step (d) described above, a denaturing reagent can be used to remove the plurality of extended forward sequencing primer chains, wherein the denaturing reagent comprises any one or any combination of compounds such as formamide, acetonitrile, guanidine hydrochloride and / or a buffer (e.g., Tris-HCl, MES, HEPES, etc.).

[0407] In some embodiments, in any of the embodiments of step (d) described above, the plurality of extended forward sequencing primer strands can be removed using elevated temperature (e.g., heat) with or without a nucleic acid denaturing agent. The plurality of extended forward sequencing primer strands can be subjected to a temperature of about 45° C. to 50° C., or about 50° C. to 60° C., or about 60° C. to 70° C., or about 70° C. to 80° C., or about 80° C. to 90° C., or about 90° C. to 95° C., or higher.

[0408] In some embodiments, in any of the embodiments of step (d) described above, the plurality of extended forward sequencing primer strands can be removed using 100% formamide at a temperature of about 65° C. for about 3 minutes and washing with a reagent comprising about 50 mM NaCl or an equivalent ionic strength and having a pH of about 6.5 to 8.5.

[0409] In some embodiments, in any of the embodiments of step (d) above, the primer extension polymerase comprises a high-fidelity polymerase. In some embodiments, the primer extension polymerase of step (d) comprises a DNA polymerase capable of catalyzing a primer extension reaction using a template molecule containing uracil (e.g., a uracil-tolerant polymerase). Exemplary polymerases include, but are not limited to, Q5U Hot Start High-Fidelity DNA Polymerase (e.g., from New England Biolabs). TM M0515S), Taq DNA polymerase, One Taq DNA polymerase (e.g., a mixture of Taq and Deep Vent DNA polymerase, from New England Biolabs TM M0480S), LongAmp Taq DNA polymerase (e.g., from New England Biolabs TM M0323S), Epimark Hot Start Taq DNA polymerase (e.g., from New England Biolabs TM M0490S), Bst DNA polymerase (e.g., large fragment, from New England Biolabs TM M0275S), Bsu DNA polymerase (e.g., large fragment, from New England Biolabs TM M0330S), Phi29 DNA polymerase (e.g., from New England Biolabs TM M0269S), E. coli DNA polymerase (e.g., from New England Biolabs TMM0209S from New England Biolabs), Therminator DNA polymerase (e.g., catalog number M0261S from New England Biolabs), Vent DNA polymerase, and Deep Vent DNA polymerase.

[0410] In some embodiments, the method for generating a plurality of immobilized nucleic acid nanostructures further comprises step (e): removing the retained immobilized concatemer template molecules (e.g., first-strand nanostructures) by generating a base-free site at a nucleotide having a cleavable portion in the immobilized single-stranded concatemer template molecules and generating a gap at the base-free site to generate a plurality of single-stranded nucleic acid concatemer template molecules containing gaps, while retaining a plurality of forward extension strands (second-strand nanostructures) and retaining a plurality of immobilized surface primers ( Figures 26 to 27 ).

[0411] The abasic site is produced on the concatemer template strand retained, and the concatemer template strand retained contains a nucleotide with an easy-to-cleave portion. In certain embodiments, the easy-to-cleave portion in the concatemer template molecule retained includes uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG) or deoxyinosine. The abasic site can be removed to produce multiple single-stranded nucleic acid template molecules with gaps while retaining the multiple forward extension chains. The abasic site can be produced by contacting the fixed concatemer template molecule with an enzyme that removes the nucleobase at the nucleotide with an easy-to-cleave portion. The uracil in the concatemer template strand retained can be converted into an abasic site using uracil DNA glycosylase (UDG). The 8oxoG in the concatemer template strand retained can be converted into an abasic site using FPG glycosylase. The deoxyinosine in the concatemer template strand retained can be converted into an abasic site using AlkA glycosylase.

[0412] In some embodiments, in the method for generating a plurality of immobilized nucleic acid nanostructures of step (e), the gaps can be generated by contacting the abasic sites in the immobilized concatemer template molecules with an enzyme or a mixture of enzymes having a lyase activity that cleaves the phosphodiester backbone at the 5' and 3' sides of the abasic sites to release the abasic deoxyribose and generate the gaps ( Figure 26 AP lyase, Endo IV endonuclease, FPG glycosylase / AP lyase, Endo VIII glycosylase / AP lyase can be used to remove abasic sites. In some embodiments, a mixture of uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII (such as, but not limited to, USER TM (From New England Biolabs TM Uracil-specific excision reagent enzyme) or thermolabile USERTM (Also from New England Biolabs TM ) to achieve the creation of abasic sites and the removal of abasic sites to create gaps.

[0413] In some embodiments, in the method for generating a plurality of immobilized nucleic acid nanostructures in step (e), the plurality of template molecules containing gaps can be removed using enzymes, chemical compounds, and / or heat. After the gap removal procedure, the plurality of retained forward extension strands are hybridized to the retained immobilized surface primers ( Figure 27 ). For example, the multiple template molecules containing gaps can be enzymatically degraded using a 5' to 3' double-stranded DNA exonuclease, including T7 exonuclease (e.g., from New England Biolabs, catalog number M0263S). When a 5' to 3' double-stranded DNA exonuclease is used to remove the template molecules containing gaps, the multiple soluble amplification primers in step (e) may comprise at least one phosphorothioate diester bond at their 5' ends, which may render the soluble amplification primers resistant to exonuclease-induced degradation. In some embodiments, the multiple soluble amplification primers in step (d) comprise 2 to 5 or more consecutive phosphorothioate diester bonds at their 5' ends. In some embodiments, the multiple soluble amplification primers in step (d) comprise at least one ribonucleotide and / or at least one 2'-O-methyl or 2'-O-methoxyethyl (MOE) nucleotide, which may render the forward sequencing primer resistant to exonuclease-induced degradation.

[0414] In some embodiments, a chemical reagent that facilitates nucleic acid denaturation can be used to remove the plurality of template molecules containing gaps. The denaturing reagent can include any one or any combination of compounds such as formamide, acetonitrile, guanidine hydrochloride, and / or a buffer (e.g., Tris-HCl, MES, HEPES, etc.).

[0415] In some embodiments, elevated temperature (e.g., heat) can be used with or without a nucleic acid denaturing agent to remove the plurality of template molecules containing gaps. The template molecules containing gaps can be subjected to a temperature of about 45°C to 50°C, or about 50°C to 60°C, or about 60°C to 70°C, or about 70°C to 80°C, or about 80°C to 90°C, or about 90°C to 95°C, or higher.

[0416] In some embodiments, the plurality of template molecules containing gaps can be removed using 100% formamide at about 65°C for about 3 minutes and washing with a reagent comprising about 50 mM NaCl or equivalent ionic strength and having a pH of about 6.5 to 8.5.

[0417] In some embodiments, the method for generating a plurality of immobilized nucleic acid nanostructures further comprises step (f): sequencing the plurality of retained forward extension strands (e.g., second strand nanostructures) to thereby generate a plurality of extended reverse sequencing primer strands. In some embodiments, the sequencing of step (f) comprises contacting the plurality of retained forward extension strands with a plurality of soluble reverse sequencing primers under conditions suitable for hybridizing the reverse sequencing primers to the reverse sequencing primer binding sites of the retained forward extension strands, and performing a reverse sequencing reaction using one or more types of sequencing polymerases, a plurality of nucleotide reagents, and the hybridized reverse sequencing primers to generate a plurality of reverse sequencing products ( Figure 28 ). In some embodiments, the plurality of nucleotide reagents are detectably labeled. In some embodiments, the sequencing of step (f) further comprises detecting and imaging the plurality of reverse sequencing products. The universal adapter sequence can be part of a concatemer molecule having multiple copies of a polynucleotide unit arranged in series, wherein each polynucleotide unit comprises a sequence of interest and at least one universal adapter sequence. The first binding region of the compacted oligonucleotide can hybridize with at least a portion of any one of the universal adapter sequences listed in Table 2. The second binding region of the compacted oligonucleotide sequence can hybridize with at least a portion of any one of the universal adapter sequences listed in Table 2.

[0418] In some embodiments, the sequencing of step (f) further comprises contacting the plurality of retained forward extension strands with the plurality of compacted oligonucleotides under conditions suitable for hybridizing the 5' end of at least one compacted oligonucleotide to a first portion of the plurality of retained forward extension strands and hybridizing the second binding region of the same compacted oligonucleotide to a second portion of the same plurality of retained forward extension strands.

[0419] In some embodiments, in step (f), the plurality of compacted oligonucleotides comprises any sequence as described above in step (b).

[0420] In some embodiments, during the sequencing of step (f), the extended reverse sequencing primer strand hybridizes to the retained forward extension strand. The retained forward extension strand hybridizes to the first universal surface primer. The extended reverse sequencing primer strand is not hybridized to or covalently attached to the first universal surface primer. Thus, the extended reverse sequencing primer strand is not fixed to a support (e.g., see Figure 28 ).

[0421] In some embodiments, in the sequencing of step (f), the soluble reverse sequencing primer comprises a 3'OH extendable end. In some embodiments, the soluble reverse sequencing primer comprises a 3' blocking portion that can be removed to produce a 3'OH extendable end. In some embodiments, the soluble reverse sequencing primer lacks a nucleotide with an easily cleavable portion. The reverse sequencing reaction can produce multiple extended reverse sequencing primer chains. In some embodiments, the separate retained forward extension chain has multiple copies of a reverse sequencing primer binding site / sequence, wherein each reverse sequencing primer binding site is capable of hybridizing with a reverse sequencing primer. The separate reverse sequencing primer binding site in a given retained forward extension chain can then hybridize with a reverse sequencing primer and can undergo a sequencing reaction. The separate retained forward extension chain may undergo two or more sequencing reactions, wherein each sequencing reaction is initiated from a reverse sequencing primer that hybridizes with a reverse sequencing primer binding site (e.g., see Figure 28 ).

[0422] In some embodiments, in the sequencing of step (f), the nucleotide reagent comprises a plurality of nucleotides, a plurality of nucleotide analogs, or a plurality of multivalent molecules.

[0423] In certain embodiments, in the sequencing of step (f), the individual nucleotides in the plurality of nucleotides include aromatic bases, five-carbon sugars, and at least one phosphate group. In certain embodiments, the plurality of nucleotides are unlabeled. In certain embodiments, at least one nucleotide in the plurality of nucleotides can be labeled with a detectable reporter gene moiety (e.g., a fluorophore). In certain embodiments, the sequencing of step (f) further includes contacting the plurality of fixed concatemer template molecules with catalytic or non-catalytic divalent cations. Exemplary catalytic divalent cations can include magnesium ions and / or manganese ions that promote the incorporation of polymerase-catalyzed nucleotides. Exemplary non-catalytic divalent cations can include strontium ions, barium ions, and / or calcium ions that inhibit the incorporation of polymerase-catalyzed nucleotides.

[0424] In some embodiments, in the sequencing of step (f), the individual nucleotide analogs in a plurality of sequences may comprise an aromatic base, a pentose with a 3' chain termination portion that inhibits polymerase-catalyzed nucleotide incorporation, and at least one phosphate group. In some embodiments, a variety of nucleotide analogs are unlabeled. In some embodiments, at least one nucleotide analog in a plurality of sequences may be labeled with a detectable reporter gene portion (e.g., a fluorophore). In some embodiments, the sequencing of step (f) further comprises contacting a plurality of fixed concatemer template molecules with catalytic or non-catalytic divalent cations. Exemplary catalytic divalent cations may comprise magnesium ions and / or manganese ions that promote polymerase-catalyzed nucleotide incorporation. Exemplary non-catalytic divalent cations may comprise strontium ions, barium ions, and / or calcium ions that inhibit polymerase-catalyzed nucleotide incorporation.

[0425] In some embodiments, in the sequencing of step (f), an individual multivalent molecule in the plurality comprises (1) a core; and (2) a plurality of nucleotide arms, each of the plurality of nucleotide arms comprising (i) a core attachment moiety, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms, wherein the spacer is attached to the linker, and wherein the linker is attached to the nucleotide unit (e.g., see Figures 56 to 59 ). In some embodiments, the nucleotide unit comprises an aromatic base, a pentose and at least one phosphate group, and the linker is attached to the nucleotide unit via the base. In some embodiments, a plurality of multivalent molecules are unlabeled. In some embodiments, at least one of the plurality of multivalent molecules is labeled with a detectable reporter gene portion (e.g., a fluorophore). In some embodiments, the sequencing of step (f) further comprises contacting a plurality of fixed concatemer template molecules with catalytic or non-catalytic divalent cations. Exemplary catalytic divalent cations may include magnesium ions and / or manganese ions that promote polymerase-catalyzed nucleotide incorporation. Exemplary non-catalytic divalent cations may include strontium ions, barium ions and / or calcium ions that inhibit polymerase-catalyzed nucleotide incorporation.

[0426] In some embodiments, during the sequencing of step (f), the method further comprises forming at least one affinity complex by contacting a plurality of immobilized concatemer template molecules (e.g., second strand nanostructures) with a plurality of soluble reverse sequencing primers, a plurality of sequencing polymerases, and a plurality of multivalent molecules to form a plurality of binding complexes including at least a first binding complex and a second binding complex, wherein (i) the first binding complex comprises a first reverse sequencing primer, a first sequencing polymerase, and a first multivalent molecule, the first forward sequencing primer, the first sequencing polymerase, and the first multivalent molecule bind to a first portion of the immobilized concatemer template molecules; In some embodiments, the multivalent molecule is bound to a second portion of the immobilized concatemer template molecule (e.g., a second strand nanostructure), thereby forming a first binding complex, wherein the first nucleotide unit of the multivalent molecule is bound to the first polymerase, and (ii) a second binding complex comprising a second reverse sequencing primer, a second sequencing polymerase, and the first multivalent molecule, wherein the second forward sequencing primer, the second sequencing polymerase, and the first multivalent molecule are bound to a second portion of the same immobilized concatemer template molecule, thereby forming a second binding complex, wherein the second nucleotide unit of the multivalent molecule is bound to the second polymerase, and wherein the first binding complex and the second binding complex comprising the same multivalent molecule form an avidity complex. In some embodiments, the multivalent molecule is unlabeled or labeled with a detectable reporter moiety.

[0427] In some embodiments, in any primer extension reaction of step (d), the first binding region of the compacted oligonucleotide hybridizes to the forward extension strand at a first portion that does not interfere with (e.g., has little or no overlap) hybridization of the reverse sequencing primer to the reverse sequencing primer binding site of the plurality of forward extension strands of step (f).

[0428] In some embodiments, in any primer extension reaction of step (d), the second binding region of the compacted oligonucleotide hybridizes to the forward extension strand at a second portion that does not interfere with (e.g., has little or no overlap) the hybridization of the reverse sequencing primer to the reverse sequencing primer binding site of the plurality of forward extension strands of step (f).

[0429] In some embodiments, in the method for generating a plurality of immobilized nucleic acid nanostructures, the compacted oligonucleotides of steps (b), (c), (e), and (f) comprise single-stranded oligonucleotides comprising DNA, RNA, or a combination of DNA and RNA. The compacted oligonucleotides can be of any length, including 20-150 nucleotides, or 30-100 nucleotides, or 40-80 nucleotides, or any range therebetween.

[0430] In certain embodiments, the compacted oligonucleotide comprises a first binding region and a 3' region, and optionally an intervening joint between the first binding region and the second binding region. The intervening joint can be any length, for example, a length of about 2-20 nucleotides. The intervening joint can comprise a homopolymer with continuous identical bases (e.g., AAA, GGG, CCC, TTT or UUU). The intervening joint can comprise a non-homopolymer sequence.

[0431] Compacting the first binding region of oligonucleotide can be along its length and the first part complete complementarity or partial complementarity with concatemer molecule.Compacting the second binding region of oligonucleotide can be along its length and the second part complete complementarity or partial complementarity with concatemer molecule.Compacting the first binding region of oligonucleotide can be with the first universal sequence portion hybridization (referring to Table 2) of the concatemer molecule with the sequence of any one of SEQ ID NO:157-176.Compacting the second binding region of oligonucleotide can be with the second universal sequence portion hybridization (referring to Table 2) of the concatemer molecule with the sequence of any one of SEQ ID NO:157-176.Compacting the first binding region and the second binding region of oligonucleotide can hybridize with concatemer, so that the distal portion of concatemer is drawn together, thereby concatemer compacting is to form nanostructure.

[0432] In some embodiments, the method for generating a plurality of fixed nucleic acid nanostructures further comprises: positioning the cellular biological sample on the fixed nanostructure after step (b). For example, the cellular biological sample can be placed on the fixed nucleic acid nanostructure after step (b) and before step (c).

[0433] In certain embodiments, the cell biological sample comprises a single cell, a plurality of cells, tissues, organs, organisms or sections from any of these cell biological samples. In certain embodiments, the cell biological sample comprises a fresh, frozen, fresh frozen or archived sample (e.g., formalin fixed paraffin embedded; FFPE). The cell biological sample can be embedded in a matrix material. The cell biological sample can be stained, decolorized or unstained. The cell biological sample can be permeabilized to allow the nucleic acid in the cell sample to migrate from the cell to a plurality of fixed nanostructures.

[0434] In some embodiments, in step (f), conditions suitable for hybridizing the reverse sequencing primer to the reverse sequencing primer binding sequence of the retained forward extension strand include: contacting the plurality of soluble reverse sequencing primers and the retained forward extension strand with a high-efficiency hybridization buffer. In some embodiments, the high-efficiency hybridization buffer comprises: (i) a first polar aprotic solvent having a dielectric constant of no greater than 40 and a polarity index of 4-9; (ii) a second polar aprotic solvent having a dielectric constant of no greater than 115 and present in the hybridization buffer formulation in an amount effective to denature double-stranded nucleic acids; (iii) a pH buffer system that maintains the pH of the hybridization buffer formulation in the range of about 4-8; and (iv) a crowding agent in an amount sufficient to enhance or promote molecular crowding. In some embodiments, the high-efficiency hybridization buffer comprises: (i) a first polar aprotic solvent comprising acetonitrile at 25-50% by volume of the hybridization buffer; (ii) a second polar aprotic solvent comprising formamide at 5-10% by volume of the hybridization buffer; (iii) a pH buffer system comprising 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) a crowding agent comprising polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0435] In an alternative embodiment, the sequencing of step (f) comprises: using the immobilized surface primer as a sequencing primer, and performing a sequencing reaction to generate a plurality of reverse sequencing strands.

[0436] In some embodiments, at least one washing step may be performed after any one of steps (a) to (f).The washing step may be performed with a washing buffer comprising a pH buffer, a metal chelator, a salt, and a detergent.

[0437] In some embodiments, the pH buffering compound in the wash buffer comprises any one of the following or any combination of two or more of the following: Tris, Tris-HCl, Tricine, Bicine, Bis-Tris propane, HEPES, MES, MOPS, MOPSO, BES, TES, CAPS, TAPS, TAPSO, ACES, PIPES, ethanolamine (also known as 2-aminomethanol; MEA), a citrate compound, a citrate mixture, NaOH, and / or KOH. In some embodiments, the pH buffering agent may be present in the wash buffer at a concentration of about 1 mM to 100 mM, or about 10 mM to 50 mM, or about 10 mM to 25 mM. In some embodiments, the pH of the pH buffering agent present in any of the reagents described herein may be adjusted to a pH of about 4 to 9, or about 5 to 9, or about 5 to 8.

[0438] In some embodiments, the metal chelator in the wash buffer comprises: EDTA (ethylenediaminetetraacetic acid), EGTA (ethylene glycol tetraacetic acid), HEDTA (hydroxyethylethylenediaminetriacetic acid), DPTA (diethylenetriaminepentaacetic acid), NTA (N,N-bis(carboxymethyl)glycine), anhydrous citrate, sodium citrate, calcium citrate, ammonium citrate, diammonium citrate, citric acid, potassium citrate, or magnesium citrate. In some embodiments, the wash buffer comprises the chelator at a concentration of about 0.01 mM to 50 mM, or about 0.1 mM to 20 mM, or about 0.2 mM to 10 mM.

[0439] In some embodiments, the salt in the wash buffer comprises NaCl, KCl, NH2SO4 or potassium glutamate. In some embodiments, the detergent comprises an ionic detergent such as SDS (sodium dodecyl sulfate). The wash buffer may comprise a monovalent salt at a concentration of about 25 mM to 500 mM, or about 50 mM to 250 mM, or about 100 mM to 200 mM.

[0440] In some embodiments, the detergent in the wash buffer comprises a nonionic detergent such as Triton X-100, Tween 20, Tween 80, or Nonidet P-40. In some embodiments, the detergent comprises a zwitterionic detergent such as CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) or N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfate (DetX). In some embodiments, the detergent comprises LDS (lithium dodecyl sulfate), sodium taurodeoxycholate, sodium taurocholate, sodium glycocholate, sodium deoxycholate, or sodium cholate. In some embodiments, the detergent is included in the wash buffer at a concentration of about 0.01% to 0.05%, or about 0.05% to 0.1%, or about 0.1% to 0.15%, or about 0.15% to 0.2%, or about 0.2% to 0.25%.

[0441] In some embodiments, a method for generating a plurality of immobilized nucleic acid nanostructures using an on-support rolling circle amplification reaction comprises: step (a) providing a support having a plurality of first universal surface primers immobilized thereon; step (b) performing on-support RCA; and step (c) forward sequencing. In some embodiments, the method does not include steps (d)-(f). For example, the method does not include: synthesizing a second strand nanostructure (step (d)); generating abasic sites and gaps (step (e)); and reverse sequencing (step (f)).

[0442] In some embodiments, a method for generating a plurality of immobilized nucleic acid nanostructures using an on-support rolling circle amplification reaction comprises: step (a) providing a support having a plurality of first universal surface primers immobilized thereon; and step (b) performing on-support RCA. In some embodiments, the method does not include steps (c)-(f). For example, the method does not include: forward sequencing (step (c)); synthesis of second strand nanostructures (step (d)); generation of abasic sites and gaps (step (e)); and reverse sequencing (step (f)).

[0443] Support method

[0444] Producing high-density fixed nanostructures lacking breakable portions

[0445] The present invention provides ...

Claims

1. A method for producing a high-density nucleic acid nanostructure immobilized on a support, the method comprising: a) providing a support having a plurality of first universal surface primers immobilized thereon, wherein the density of the first universal surface primers on the support is 2 Approx. 10 2 -10 15 ;as well as b) generating a plurality of fixed single-stranded nucleic acid concatemer template molecules by: 1) hybridizing a plurality of single-stranded circular nucleic acid library molecules with a plurality of immobilized first universal surface primers; and 2) performing an on-support rolling circle amplification reaction using: (i) a plurality of strand displacement polymerases, (ii) a plurality of nucleotides, and (iii) a plurality of compacted oligonucleotides, Thus, the plurality of fixed single-stranded nucleic acid concatemer template molecules are generated, o wherein the individual compacted oligonucleotides comprise a single-stranded linear oligonucleotide having a first binding region capable of hybridizing to a first portion of a concatemer molecule and a second binding region capable of hybridizing to a second portion of the concatemer molecule, o wherein a plurality of immobilized concatemer molecules form a compact nucleic acid nanostructure, and o wherein a plurality of concatemers remain immobilized to the support upon formation of the compact nucleic acid nanostructure, thereby producing a density of 2 The nanostructures fixed to the support are about 10 2 -10 15 support.

2. The method of claim 1, wherein the support is passivated with at least one layer of a hydrophilic polymer coating comprising the plurality of first universal surface primers.

3. The method of claim 1 or 2, wherein the plurality of immobilized first universal surface primers are located at random positions on the support or the hydrophilic polymer coating.

4. The method according to claim 1 or 2, wherein the plurality of immobilized first universal surface primers are located at predetermined positions on the support or the hydrophilic polymer coating.

5. The method according to any one of claims 1 to 4, wherein each of the first universal surface primers lacks a cleavable portion that can be converted into an abasic site.

6. The method of claim 5, wherein the scissile moiety is uridine, 8-oxo-7,8-dihydroguanine or deoxyinosine.

7. The method according to any one of claims 1 to 6, wherein the plurality of nucleotides used in the rolling circle amplification reaction comprises dATP, dCTP, dGTP and dTTP, and wherein the nucleotide lacks a cleavable portion that can be converted into an abasic site.

8. The method according to any one of claims 1 to 6, wherein the plurality of nucleotides used in the rolling circle amplification reaction comprises dATP, dCTP, dGTP, dTTP and a nucleotide having a cleavable portion that can be converted into an abasic site.

9. The method of claim 8, wherein the nucleotide having the scissile moiety comprises uridine, 8-oxo-7,8-dihydroguanine, or deoxyinosine.

10. The method of claim 8 or 9, wherein the rolling circle amplification reaction of step (b) produces a plurality of single-stranded nucleic acid concatemer template molecules, wherein individual concatemer template molecules comprise at least two nucleotides, each nucleotide having a cleavable portion distributed at a random position along the individual fixed concatemer template molecules.

11. The method according to any one of claims 1 to 10, wherein the plurality of compacted oligonucleotides in step (b) comprise the same sequence.

12. The method of claim 11, wherein the sequence is according to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153 or 156.

13. The method according to any one of claims 1 to 12, wherein the plurality of compacted oligonucleotides in step (b) comprises a mixture of two or more different populations of compacted oligonucleotides, each population having a different sequence, wherein the compacted oligonucleotides in different populations have different sequences.

14. The method of claim 13, wherein the mixture comprises 2, 3, 4, 5, 6, 7, 8, 9 or 10 different compacted oligonucleotide populations.

15. The method of claim 13 or 14, wherein each population of compacted oligonucleotides in the mixture comprises a sequence according to any one of SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 105, 108, 111, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, 153, or 156.

16. The method according to any one of claims 1 to 15, wherein the compact nucleic acid nanostructure comprises one or more rings, or comprises a spherical shape, an elongated shape, a pre-annular shape, or a toroidal shape.

17. The method of claim 16, wherein the spherical shape is a nanosphere.

18. The method of claim 16, wherein the elongated shapes are nanorods. The method of claim 16 , wherein the toroidal shape is a nanotoroid.

20. The method of any one of claims 1 to 19, wherein the nucleic acid nanostructure comprises a compact nucleic acid structure having a full width at half maximum (FWHM) that is less than the FWHM of a concatemer that is not collapsed / folded into a nanostructure.

21. The method according to any one of claims 1 to 20, further comprising: The high-density nucleic acid nanostructure immobilized on the support is imaged.

22. The method according to any one of claims 1 to 21, further comprising: c) contacting the immobilized nucleic acid nanostructures with the labeled oligonucleotides under conditions suitable for hybridization of the oligonucleotides labeled with a detectable reporter moiety to the plurality of immobilized nucleic acid nanostructures to produce a plurality of immobilized labeled nanostructures; as well as d) imaging the plurality of immobilized labeled nanostructures.

23. The method according to any one of claims 1 to 21, further comprising: The individual immobilized nanostructures are contacted with (i) a plurality of soluble sequencing primers, (ii) a plurality of sequencing polymerases, and (iii) a plurality of nucleotide reagents under conditions suitable for: hybridizing the plurality of soluble sequencing primers to the individual immobilized nanostructures to generate a plurality of nucleic acid duplexes along the individual nanostructures, and - Combining at least one nucleic acid duplex with a sequencing polymerase and nucleotide reagents.

24. The method of claim 23, wherein the plurality of nucleotide reagents comprises a plurality of nucleotides, each nucleotide comprising an aromatic base, a pentose sugar, and at least one phosphate group.

25. The method of claim 24, wherein at least one nucleotide in the plurality of nucleotides further comprises a detectable reporter moiety.

26. The method of claim 25, wherein the detectable reporter moiety is a fluorophore.

27. The method according to claim 25 or 26, further comprising: c) contacting the plurality of immobilized nucleic acid nanostructures with labeled nucleotides; as well as d) imaging the high-density nucleic acid nanostructure immobilized on the support.

28. The method of any one of claims 23 to 27, wherein the plurality of nucleotide reagents comprises a plurality of nucleotide analogs, each nucleotide analog comprising an aromatic base, a pentose sugar having a 3' chain terminating moiety that inhibits polymerase-catalyzed nucleotide incorporation, and at least one phosphate group.

29. The method of claim 28, wherein at least one nucleotide analog in the plurality of nucleotide analogs further comprises a detectable reporter gene moiety.

30. The method of claim 29, wherein the detectable reporter moiety is a fluorophore.

31. The method according to claim 29 or 30, further comprising: c) contacting the plurality of immobilized nucleic acid nanostructures with a labeled labeled nucleotide analog; as well as d) imaging the high-density nucleic acid nanostructure immobilized on the support.

32. A method according to any one of claims 23 to 31, wherein the plurality of nucleotide reagents comprises a plurality of multivalent molecules, wherein individual multivalent molecules comprise: (1) a core; and (2) a plurality of nucleotide arms, wherein the plurality of nucleotide arms comprise: (i) a core attachment portion, (ii) a spacer, (iii) a linker, and (iv) a nucleotide unit, wherein the core is attached to the plurality of nucleotide arms, the spacer is attached to the linker, the linker is attached to the nucleotide unit, and the nucleotide unit comprises an aromatic base, a pentose, and at least one phosphate group.

33. The method of claim 32, further comprising forming a plurality of binding complexes, the method comprising the steps of: c) binding a first sequencing primer, a first sequencing polymerase, and a first multivalent molecule to a first portion of the individual immobilized nanostructures, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule is bound to the first sequencing polymerase; as well as d) binding a second sequencing primer, a second sequencing polymerase, and the first multivalent molecule to a second portion of the same single immobilized nanostructure, thereby forming a second binding complex, wherein the second nucleotide unit of the first multivalent molecule binds to the second sequencing polymerase, wherein the first binding complex and the second binding complex comprising the same multivalent molecule form an affinity complex.

34. The method of claim 32 or 33, wherein at least one multivalent molecule of the plurality of multivalent molecules further comprises at least one detectable reporter gene moiety.

35. The method of claim 29, wherein the at least one detectable reporter moiety comprises at least one fluorophore.

36. The method according to claim 34 or 35, further comprising: c) contacting the plurality of immobilized nucleic acid nanostructures with a labeled multivalent molecule; as well as d) imaging the high-density nucleic acid nanostructure immobilized on the support.

37. The method according to any one of claims 1 to 20, further comprising: The plurality of immobilized nanostructures is contacted with a cellular biological sample.

38. The method of claim 36, wherein the cellular biological sample comprises a single cell, a section of a single cell, a plurality of cells, a section of a plurality of cells, a tissue, a section of a tissue, an organ, a section of an organ, an organism, or a section of an organism.

39. The method of any one of claims 1 to 38, wherein the plurality of fixed nucleic acid nanostructures are fluidically connected to each other to allow a reagent solution to flow onto the support, such that the plurality of fixed nucleic acid nanostructures on the support react with the reagent solution in a massively parallel manner.

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