Methods for preparing nucleic acid nanostructures using compacted oligonucleotides

Abstract

The present disclosure provides compositions and related methods for preparing immobilized nucleic acid nanostructures, for example, using compacting oligonucleotides. In some embodiments, rolling circle amplification reactions can be performed using compacting oligonucleotides on a support or in solution to generate concatemer molecules with multiple copies of tandemly arranged polynucleotide units. Each polynucleotide unit comprises a sequence of interest and at least one universal adapter sequence attached to one end of the compacting oligonucleotide. The 5' and 3' regions of the compacting oligonucleotide can hybridize to the concatemers, pulling the distal portions of the concatemers together and causing compaction of the concatemers to form nanostructures. The nanostructures have a more compact size and shape compared to concatemers generated in the absence of the compacting oligonucleotide. The compact and stable characteristics of the nucleic acid nanostructures improve sequencing accuracy by increasing signal intensity and retain their shape and size over multiple sequencing cycles.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 398,178, filed August 15, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Electronic Sequence Listing Reference The contents of the Electronic Sequence Listing (ELEM-005-001WO-SeqList-ST26.xml, size 161,077 bytes, and created on August 14, 2023) are incorporated herein by reference in their entirety.

[0003] The present disclosure provides compositions and methods using the compositions to prepare immobilized nucleic acid nanostructures, including nucleic acid nanoballs, using compacting oligonucleotides. [Background technology]

[0004] Polynucleotide sequencing technology has applications in biomedical research and medical environments. Improved methods for polynucleotide sequencing require enhanced surface chemistry, on-support polynucleotide amplification, and base calling. Currently, these factors create barriers in existing sequencing technologies, causing limited throughput and poor signal-to-noise ratios, ultimately increasing the costs associated with polynucleotide sequencing. Therefore, there is a need for improved sequencing methods and related assembly techniques. Summary of the Invention

[0005] In one aspect, the present disclosure provides a method for generating high density nucleic acid nanostructures immobilized on a support, 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 greater than or equal to 1 mm 2 Approximately 10 per 2 ~1015 and b) generating a plurality of immobilized single-stranded nucleic acid concatemeric template molecules, the generating comprising: 1) hybridizing a plurality of single-stranded circular nucleic acid library molecules to a plurality of immobilized first universal surface primers; 2) performing an on-support rolling circle amplification reaction with (i) a plurality of strand-displacing polymerases, (ii) a plurality of nucleotides, and (iii) a plurality of compacting oligonucleotides; thereby generating a plurality of immobilized single-stranded nucleic acid concatemeric template molecules; each compacted oligonucleotide comprises a single-stranded linear oligonucleotide having a first binding region capable of hybridizing to a first portion of the concatemeric molecule and a second binding region capable of hybridizing to a second portion of the concatemeric molecule; Multiple immobilized concatemeric molecules form compact nucleic acid nanostructures, a plurality of concatemers remain immobilized on the support as they form the compact nucleic acid nanostructure, thereby forming a nanostructure immobilized on the support; 2 Approximately 10 per 2 ~about 10 15 The present invention provides a method for producing a support having a density of

[0006] In some embodiments, the support is passivated with at least one layer of a hydrophilic polymer coating comprising a plurality of first universal surface primers. In some embodiments, the plurality of immobilized first universal surface primers are located on the support or hydrophilic polymer coating at random positions. In some embodiments, the plurality of immobilized first universal surface primers are located on the support or hydrophilic polymer coating at predetermined positions. In some embodiments, each of the first universal surface primers lacks a cleavable moiety that can be converted to an abasic site. In some embodiments, the cleavable moiety is uridine, 8-oxo-7,8-dihydroguanine, or deoxyinosine. In some embodiments, the plurality of nucleotides for the rolling circle amplification reaction comprises dATP, dCTP, dGTP, and dTTP, and the nucleotide lacks a cleavable moiety that can be converted to an abasic site. In some embodiments, the plurality of nucleotides for the rolling circle amplification reaction comprises dATP, dCTP, dGTP, dTTP, and a nucleotide having a cleavable moiety that can be converted to an abasic site. In some embodiments, the nucleotide having a cleavable moiety comprises uridine, 8-oxo-7,8-dihydroguanine, or deoxyinosine. In some embodiments, the rolling circle amplification reaction of step (b) generates a plurality of single-stranded nucleic acid concatemeric template molecules, each concatemeric template molecule comprising at least two nucleotides each having a cleavable moiety distributed at random positions along each immobilized concatemeric template molecule.

[0007] In some embodiments, the multiple compacting oligonucleotides in step (b) comprise the same sequence, in some embodiments, the sequence is set forth in any one of SEQ ID NOs: 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.

[0008] 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, and the compacted oligonucleotides of the 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 compacted oligonucleotide population in the mixture comprises a sequence set forth in any of SEQ ID NOs: 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.

[0009] In some embodiments, the compact nucleic acid nanostructure comprises one or more loops, or comprises a spherical, elongated, prototoroid, or toroid shape. In some embodiments, the spherical shape is a nanoball. In some embodiments, the elongated shape is a nanorod. In some embodiments, the toroid shape is a nanotoroid.

[0010] In some embodiments, nucleic acid nanostructures comprise compact nucleic acid structures that have a smaller full width at half maximum (FWHM) than concatemers that have not collapsed / folded into nanostructures.

[0011] In some embodiments, the method further comprises imaging the high density nucleic acid nanostructures immobilized on the support.

[0012] In some embodiments, the method comprises: a) contacting a plurality of immobilized nucleic acid nanostructures with oligonucleotides labeled with a detectable reporter moiety under conditions suitable for hybridizing the labeled oligonucleotides to the immobilized nucleic acid nanostructures to produce a plurality of immobilized labeled nanostructures; b) imaging the plurality of immobilized labeled nanostructures.

[0013] In some embodiments, the method comprises combining individual immobilized nanostructures with (i) a plurality of soluble sequencing primers, (ii) a plurality of sequencing polymerases, and (iii) a plurality of nucleotide reagents; hybridizing a plurality of soluble sequencing primers to each of the immobilized nanostructures to generate a plurality of nucleic acid duplexes along each of the nanostructures; - further comprising contacting at least one nucleic acid duplex with a sequencing polymerase and a nucleotide reagent under conditions suitable for binding.

[0014] In some embodiments, the plurality of nucleotide reagents comprises a plurality of nucleotides, each nucleotide comprising an aromatic base, a five-carbon sugar, and at least one phosphate group. In some embodiments, at least one of the nucleotides in the plurality of nucleotides further comprises a detectable reporter moiety. In some embodiments, the detectable reporter moiety is a fluorophore.

[0015] In some embodiments, the method comprises: a) contacting a plurality of immobilized nucleic acid nanostructures with labeled nucleotides; b) imaging the high-density nucleic acid nanostructures immobilized on the support.

[0016] In some embodiments, the plurality of nucleotide reagents comprises a plurality of nucleotide analogs each comprising an aromatic base, a 5-carbon sugar having a 3' chain-terminating moiety that inhibits polymerase-catalyzed nucleotide incorporation, and at least one phosphate group. In some embodiments, at least one of the nucleotide analogs in the plurality of nucleotide analogs further comprises a detectable reporter moiety. In some embodiments, the detectable reporter moiety is a fluorophore.

[0017] In some embodiments, the method comprises: a) contacting a plurality of immobilized nucleic acid nanostructures with labeled nucleotide analogs; b) imaging the high-density nucleic acid nanostructures immobilized on the support.

[0018] In some embodiments, the plurality of nucleotide reagents comprises a plurality of multivalent molecules, each multivalent molecule comprising (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, and the linker is attached to the nucleotide unit, and the nucleotide unit comprises an aromatic base, a five-carbon sugar, and at least one phosphate group.

[0019] In some embodiments, the method further comprises forming a plurality of binding complexes, wherein the forming comprises: a) binding a first sequencing primer, a first sequencing polymerase, and a first multivalent molecule to a first portion of an individual immobilized nanostructure, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule binds to the first sequencing polymerase; b) binding a second sequencing primer, a second sequencing polymerase, and the first multivalent molecule to a second portion of the same individual 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 the first and second binding complexes comprising the same multivalent molecule form an avidity complex.

[0020] In some embodiments, at least one of the multivalent molecules in the plurality of multivalent molecules further comprises at least one detectable reporter moiety, hi some embodiments, the at least one detectable reporter moiety comprises at least one fluorophore.

[0021] In some embodiments, the method comprises: a) contacting a plurality of immobilized nucleic acid nanostructures with a labeled multivalent molecule; b) imaging the high-density nucleic acid nanostructures immobilized on the support.

[0022] In some embodiments, the method further comprises contacting the plurality of immobilized nanostructures with a cellular biological sample, hi some embodiments, 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.

[0023] In some embodiments, the multiple immobilized nucleic acid nanostructures are in fluid communication with each other, allowing a solution of reagents to flow over the support such that the multiple immobilized nucleic acid nanostructures on the support react with the solution of reagents in a massively parallel manner.

[0024] The features of the present disclosure 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 that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [Brief explanation of the drawings]

[0025] [Figure 1] 1(i) and 1(ii) show schematic diagrams of several embodiments of a linear compaction oligonucleotide, each comprising a first binding region, an intervening linker, and a second binding region. In some embodiments, the compaction 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 (FIG. 1(i)). In some embodiments, the compaction 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 (FIG. 1(ii)). In some embodiments, the compaction 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 (FIG. 1(iii)). In some embodiments, the compaction 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 (FIG. 1(iv)). [Figure 2A]2A(i) and 2A(ii) show schematic diagrams of several embodiments of linear compaction oligonucleotides, each comprising a first binding region, a first intervening linker, a second binding region, a second intervening linker, and a third binding region. In some embodiments, the compaction 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 (FIG. 2A(i)). In some embodiments, the compaction 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 (FIG. 2A(iii)). [Figure 2B] 2B(v) Schematic diagrams of several embodiments of a linear compaction oligonucleotide, each comprising a first binding region, a first intervening linker, a second binding region, a second intervening linker, and a third binding region. In some embodiments, the compaction 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 (FIG. 2B(iv)). In some embodiments, the compaction 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 (FIG. 2B(v)). In some embodiments, the compaction 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 (FIG. 2B(vi)). [Figure 2C]Schematic diagrams of some embodiments of linear compacted oligonucleotides each containing a first binding region, a first intervening linker, a second binding region, a second intervening linker, and a third binding region are shown. In some embodiments, the compacted oligonucleotide includes a first binding region arranged in the 3' to 5' direction, a second binding region arranged in the 5' to 3' direction, and a third binding region arranged in the 5' to 3' direction (Figure 2C(vii)). In some embodiments, the compacted oligonucleotide includes a first binding region arranged in the 3' to 5' direction, a second binding region arranged in the 3' to 5' direction, and a third binding region arranged in the 5' to 3' direction (Figure 2C(viii)). In some embodiments, the compacted oligonucleotide includes a first binding region arranged in the 3' to 5' direction, a second binding region arranged in the 5' to 3' direction, and a third binding region arranged in the 3' to 5' direction (Figure 2C(ix)). [Figure 3A]3A(i) shows schematic diagrams of some embodiments of a compacted oligonucleotide, each comprising three linkage arms connected together by at least one internal intervening linker, each comprising a linkage region. In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening linker and a first linkage region arranged in a 5' to 3' orientation, with the 3' end of the first linkage region facing away from the internal intervening linker; (2) an internal intervening linker and a second linkage region arranged in a 5' to 3' orientation, with the 3' end of the second linkage region facing away from the internal intervening linker; and (3) an internal intervening linker and a third linkage region arranged in a 5' to 3' orientation, with the 3' end of the third linkage region facing away from the internal intervening linker (FIG. 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, with the 5' end of the first binding region facing away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 3' to 5' orientation, with the 5' end of the second binding region facing away from the internal intervening linker; and (3) an internal intervening linker and a third binding region arranged in a 3' to 5' orientation, with the 5' end of the third binding region facing away from the internal intervening linker (Figure 3A(ii)). [Figure 3B]3B(iii) shows schematic diagrams of some embodiments of a compacted oligonucleotide, each comprising three linkage arms connected together by at least one internal intervening linker, each comprising a linkage region. In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening linker and a first linkage region arranged in a 5' to 3' orientation, with the 3' end of the first linkage region facing away from the internal intervening linker; (2) an internal intervening linker and a second linkage region arranged in a 5' to 3' orientation, with the 3' end of the second linkage region facing away from the internal intervening linker; and (3) an internal intervening linker and a third linkage region arranged in a 3' to 5' orientation, with the 5' end of the third linkage region facing away from the internal intervening linker (FIG. 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, with the 3' end of the first binding region facing away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 3' to 5' orientation, with the 5' end of the second binding region facing away from the internal intervening linker; and (3) an internal intervening linker and a third binding region arranged in a 3' to 5' orientation, with the 5' end of the third binding region facing away from the internal intervening linker (Figure 3B(iv)). [Figure 4] 1 shows a schematic diagram of one embodiment of a compacted oligonucleotide comprising three binding arms linked together by at least one internal intervening linker, each binding arm comprising a first binding region, an intervening linker, and a second binding region. In some embodiments, the compacted oligonucleotide comprises three binding arms, each binding arm comprising 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, with the 3' end of the second binding region oriented away from the internal intervening linker. [Figure 5]5(i) shows schematic diagrams of some embodiments of a compacted oligonucleotide, each comprising four linkage arms connected together by at least one internal intervening linker, each comprising a linkage region. In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening linker and a first linkage region arranged in a 5' to 3' direction, with the 3' end of the first linkage region facing away from the internal intervening linker; (2) an internal intervening linker and a second linkage region arranged in a 5' to 3' direction, with the 3' end of the second linkage region facing away from the internal intervening linker; (3) an internal intervening linker and a third linkage region arranged in a 5' to 3' direction, with the 3' end of the third linkage region facing away from the internal intervening linker; and (4) an internal intervening linker and a fourth linkage region arranged in a 5' to 3' direction, with the 3' end of the fourth linkage region facing away from the internal intervening linker (FIG. 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, with the 5' end of the first binding region facing away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 3' to 5' orientation, with the 5' end of the second binding region facing away from the internal intervening linker; (3) an internal intervening linker and a third binding region arranged in a 3' to 5' orientation, with the 5' end of the third binding region facing away from the internal intervening linker; and (4) an internal intervening linker and a fourth binding region arranged in a 3' to 5' orientation, with the 5' end of the fourth binding region facing away from the internal intervening linker (Figure 5(ii)). [Figure 6A]1 shows a schematic diagram of one embodiment of a double-comb compacted oligonucleotide comprising multiple binding arms connected to a central linker portion, each binding arm comprising a binding region. In some embodiments, the compacted oligonucleotide comprises at least three binding arms. In some embodiments, the compacted oligonucleotide comprises multiple binding arms having the same sequence. In some embodiments, each binding arm comprises a first binding region arranged in a 5' to 3' orientation, with the 3' end of the first binding region facing away from the linker portion. In some embodiments, each binding arm is connected to the linker portion by an internal intervening linker. [Figure 6B] 1 shows a schematic diagram of one embodiment of a double-comb compacted oligonucleotide comprising multiple binding arms connected to a central linker portion, each binding arm comprising a binding region. In some embodiments, the compacted oligonucleotide comprises at least three binding arms. In some embodiments, the compacted oligonucleotide comprises multiple binding arms having one of two different sequences. In some embodiments, each binding arm comprises a first binding region arranged in a 5' to 3' orientation, with the 3' end of the first binding region facing away from the linker portion. In some embodiments, each binding arm comprises a second binding region arranged in a 5' to 3' orientation, with the 3' end of the second binding region facing away from the linker portion. In some embodiments, each binding arm is connected to the linker portion by an internal intervening linker. [Figure 6C]1 shows a schematic diagram of an embodiment of a double-comb compacted oligonucleotide comprising multiple binding arms connected to a central linker portion, each binding arm comprising a binding region. In some embodiments, the compacted oligonucleotide comprises at least three binding arms. In some embodiments, the compacted oligonucleotide comprises multiple binding arms having one of three different sequences. In some embodiments, each binding arm comprises a first binding region arranged in a 5' to 3' orientation, with the 3' end of the first binding region facing away from the linker portion. In some embodiments, each binding arm comprises a second binding region arranged in a 5' to 3' orientation, with the 3' end of the second binding region facing away from the linker portion. In some embodiments, each binding arm comprises a third binding region arranged in a 5' to 3' orientation, with the 3' end of the third binding region facing away from the linker portion. In some embodiments, each binding arm is connected to the linker portion by an internal intervening linker. [Figure 7A]

[0023] Figure 1 shows a schematic diagram of one embodiment of a multi-part compaction oligonucleotide comprising a primary linear compaction oligonucleotide hybridized to 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 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 7B]

[0023] Figure 1 shows a schematic diagram of one embodiment of a multi-part compaction oligonucleotide comprising a primary linear compaction oligonucleotide hybridized to 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 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 8A]

[0023] Figure 1 shows a schematic diagram of one embodiment of a multi-part compaction oligonucleotide comprising a primary linear compaction oligonucleotide hybridized to 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 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 8B]

[0023] Figure 1 shows a schematic diagram of one embodiment of a multi-part compaction oligonucleotide comprising a primary linear compaction oligonucleotide hybridized to 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 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 9]

[0023] Figure 1 shows a schematic diagram of one embodiment of a multi-part compaction oligonucleotide comprising a primary linear compaction oligonucleotide hybridized to 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 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 10]

[0023] Figure 1 shows a schematic diagram of one embodiment of a multi-part compaction oligonucleotide comprising a primary linear compaction oligonucleotide hybridized to 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 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 11]

[0023] Figure 1 shows a schematic diagram of one embodiment of a multi-part compaction oligonucleotide comprising a primary linear compaction oligonucleotide hybridized to 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 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, a first intervening linker, a third binding region arranged in a 3' to 5' orientation, a second intervening linker, 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 12A]1 shows a schematic diagram of an embodiment of a multi-part compacted oligonucleotide comprising a primary compacted oligonucleotide having three binding arms, one of the binding arms hybridized to a secondary linear compacted oligonucleotide. In some embodiments, the primary compacted oligonucleotide comprises three binding arms linked together by at least one internal intervening linker, each of which comprises a binding region. In some embodiments, the primary compacted oligonucleotide comprises: (1) an internal intervening linker and a first binding region arranged in a 5' to 3' orientation, with the 3' end of the first binding region oriented away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 5' to 3' orientation, with the 3' end of the second binding region oriented away from the internal intervening linker; and (3) an internal intervening linker and a connector region arranged in a 5' to 3' orientation, with the 3' end of the connector region oriented away from the internal intervening linker. 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 may hybridize to the fastener region of the secondary compaction oligonucleotide. [Figure 12B]1 shows a schematic diagram of an embodiment of a multi-part compacted oligonucleotide comprising a primary compacted oligonucleotide having three binding arms, one of the binding arms hybridized to a secondary linear compacted oligonucleotide. In some embodiments, the primary compacted oligonucleotide comprises three binding arms linked together by at least one internal intervening linker, each of which comprises a binding region. In some embodiments, the primary compacted oligonucleotide comprises: (1) an internal intervening linker and a first binding region arranged in a 3' to 5' orientation, with the 5' end of the first binding region oriented away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 3' to 5' orientation, with the 5' end of the second binding region oriented away from the internal intervening linker; and (3) an internal intervening linker and a connector region arranged in a 3' to 5' orientation, with the 5' end of the connector region oriented away from the internal intervening linker. 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 may hybridize to the fastener region of the secondary compaction oligonucleotide. [Figure 13A]1 shows a schematic diagram of one embodiment of a multi-portion compacted oligonucleotide comprising 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 comprises a first binding region (110) arranged in a 5' to 3' orientation, an intervening linker, 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 linker, and a fourth binding region (220) arranged in a 3' to 5' orientation. In some embodiments, the third binding region (210) can hybridize to 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 to 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 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 or overlapping portions of the second binding region (120). [Figure 13B] 13A shows a schematic diagram of the multi-portion compacted oligonucleotide shown in FIG. 13A hybridizing to a portion of a nucleic acid concatemer. In FIG. 13B, a portion of the first binding region (110) of the first nucleic acid strand (100) hybridizes to a first portion of the concatemer, dissociating a portion of the third binding region (210) from the first binding region (110), as indicated by the two arrows. Hybridization of a portion of the first binding region (110) with 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 to the fourth binding region (220) and the fifth binding region (300). [Figure 13C]A schematic diagram of the multi-portion compacted oligonucleotide shown in Figure 13B hybridizing to different portions of the same nucleic acid concatemer is shown. In Figure 13C, a portion of the second binding region (120) of the first nucleic acid strand (100) hybridizes to a second portion of the concatemer, dissociating a portion of the fifth binding region (300) from the second binding region (120), as indicated by the two arrows. Hybridization of a portion of the second binding region (120) with 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). [Figure 13D] 13C shows a schematic diagram of a multi-portion compacted oligonucleotide in which the entire length of the first binding region (110) hybridizes to a first portion of a nucleic acid concatemer, thereby forming a first toehold duplex region, and the entire length of the second binding region (120) hybridizes to a 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). [Figure 14A] Table 1 (8 sheets) is shown containing the nucleotide sequences of the compacted oligonucleotides or portions of the compacted oligonucleotides. The sequences are listed in three sets: (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 intervening homopolymer region in bold and underlined. [Figure 14B]Table 1 (8 sheets) is shown containing the nucleotide sequences of the compacted oligonucleotides or portions of the compacted oligonucleotides. The sequences are listed in three sets: (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 intervening homopolymer region in bold and underlined. [Figure 14C] Table 1 (8 sheets) is shown containing the nucleotide sequences of the compacted oligonucleotides or portions of the compacted oligonucleotides. The sequences are listed in three sets: (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 intervening homopolymer region in bold and underlined. [Figure 14D] Table 1 (8 sheets) is shown containing the nucleotide sequences of the compacted oligonucleotides or portions of the compacted oligonucleotides. The sequences are listed in three sets: (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 intervening homopolymer region in bold and underlined. [Figure 14E] Table 1 (8 sheets) is shown containing the nucleotide sequences of the compacted oligonucleotides or portions of the compacted oligonucleotides. The sequences are listed in three sets: (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 intervening homopolymer region in bold and underlined. [Figure 14F]Table 1 (8 sheets) is shown containing the nucleotide sequences of the compacted oligonucleotides or portions of the compacted oligonucleotides. The sequences are listed in three sets: (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 intervening homopolymer region in bold and underlined. [Figure 14G] Table 1 (8 sheets) is shown containing the nucleotide sequences of the compacted oligonucleotides or portions of the compacted oligonucleotides. The sequences are listed in three sets: (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 intervening homopolymer region in bold and underlined. [Figure 14H] Table 1 (8 sheets) is shown containing the nucleotide sequences of the compacted oligonucleotides or portions of the compacted oligonucleotides. The sequences are listed in three sets: (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 intervening homopolymer region in bold and underlined. [Figure 15]Table 2 (1 sheet) is shown, containing the nucleotide sequences of various universal adapter sequences. The universal adapter sequence may be part of a concatemeric molecule having multiple copies of polynucleotide units arranged in tandem, each polynucleotide unit comprising a sequence of interest and at least one universal adapter sequence. The first binding region of the compaction oligonucleotide sequence can hybridize to at least a portion of any one of the universal adapter sequences listed in Table 2. The second binding region of the compaction oligonucleotide sequence can hybridize to at least a portion of any one of the universal adapter sequences listed in Table 2. [Figure 16A] 16A is a schematic diagram showing an exemplary linear library molecule comprising 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). In some embodiments, the linear library molecule shown in FIG. 16A is one polynucleotide unit of a concatemer having two or more tandem copies of the polynucleotide unit, each polynucleotide unit comprising a sequence of interest and at least one universal adaptor sequence. [Figure 16B]16B is a schematic diagram illustrating an exemplary linear library molecule comprising a second surface primer binding site (e.g., SP2, surface pinning primer binding site), a first sequencing primer binding site (e.g., forward sequencing primer binding site), a second index sequence, a sequence of interest (e.g., insert), a first index sequence, a second sequencing primer binding site (e.g., reverse sequencing primer binding site), and a first surface primer binding site (e.g., SP1, capture primer binding site). In some embodiments, the linear library molecule shown in FIG. 16B is one polynucleotide unit of a concatemer having two or more tandem copies of the polynucleotide unit, each polynucleotide unit comprising a sequence of interest and at least one universal adaptor sequence. [Figure 17] Schematic diagram showing an exemplary workflow for circularizing linear library molecules. A linear library molecule (A) hybridizes with a double-stranded splint molecule (B), thereby circularizing the library molecule and forming a library-splint complex (C) with two nicks. The library molecule (A) contains 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 double-stranded splint molecule contains a first splint strand (long splint strand) hybridized to a second splint strand (short splint strand). The first splint strand 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, and an internal region of the first splint strand hybridizes to the second splint strand. [Figure 18]Schematic diagram showing an exemplary workflow for generating covalently closed circular library molecules using double-stranded splint molecules. A ligation reaction is performed on the library-splint complex (A), closing the two nicks and forming a covalently closed circular library molecule (B) hybridized 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. [Figure 19] Schematic diagram showing 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 and forming a library-splint complex (C) with one nick. The library molecule (A) contains 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 contains 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. [Figure 20] Schematic diagram showing an exemplary workflow for generating covalently closed circular library molecules using single-stranded splint molecules. A ligation reaction is performed on the library-splint complex (A), closing one nick and forming a covalently closed circular library molecule (B) hybridized to the single strand. 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. [Figure 21]

[0033] Figure 2 is a schematic diagram illustrating an exemplary on-support rolling circle amplification reaction using (i) an immobilized first surface primer, (ii) a nucleic acid circular library molecule, (iii) a mixture of nucleotides including a nucleotide having a cleavable moiety that can be cleaved to generate an abasic site, (iv) a strand-displacing polymerase, and (iv) a compacting oligonucleotide. The rolling circle amplification reaction generates immobilized single-stranded nucleic acid concatemer template molecules having at least one nucleotide having a cleavable moiety that can be cleaved to generate an abasic site in the immobilized single-stranded nucleic acid concatemer template molecule. The various primer binding sequence arrangements in the nucleic acid circular library molecule are for illustrative purposes. Those skilled in the art will appreciate that many other arrangements are possible. Figures 21-28 illustrate the workflow for pairwise sequencing the immobilized concatemer template molecules shown in Figure 21. [Figure 22] 22 is a schematic diagram illustrating an exemplary immobilized nucleic acid nanostructure produced by performing a rolling circle amplification reaction on the support shown in Figure 21. A first binding region of the compacting oligonucleotide hybridizes to a first portion of the immobilized concatemeric molecule, and a second binding region of the compacting oligonucleotide hybridizes to a second portion of the concatemeric molecule, causing the concatemeric molecule to collapse or fold into the nucleic acid nanostructure. [Figure 23] 23 is a schematic diagram showing an exemplary forward sequencing reaction performed on the immobilized nucleic acid nanostructure shown in Figure 22. The forward sequencing reaction can be performed with multiple soluble forward sequencing primers to generate multiple extended forward sequencing primer strands. The immobilized nanostructure can have two or more extended forward sequencing primer strands hybridized thereto. [Figure 24]1 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, thereby generating a forward extended strand, the primer extension reaction comprising a strand-displacing polymerase, a plurality of nucleotides, and a plurality of compacting oligonucleotides. [Figure 25] 25 is a schematic diagram showing the forward extension strand produced by performing the primer extension reaction shown in Figure 24. For clarity, the compacted oligonucleotide hybridized to the forward extension strand is not shown. [Figure 26] 1 is a schematic diagram showing an exemplary method for generating an abasic site in an immobilized nucleic acid nanostructure in a nucleotide having a cleavable moiety, retaining a plurality of forward extension strands, and generating a gap at the abasic site to generate a nanostructure containing a plurality of gaps while retaining a plurality of immobilized first surface primers. For clarity, the compaction oligonucleotide hybridized to the forward extension strand is not shown. [Figure 27] FIG. 27 is a schematic diagram showing an exemplary retained forward extending strand after removal of the gap-containing nanostructure shown in FIG. 26. [Figure 28] 28 is a schematic diagram showing an exemplary reverse sequencing reaction performed on the retained forward extension strand shown in Figure 27. The reverse sequencing reaction can be performed with multiple soluble reverse sequencing primers, multiple sequencing polymerases, and multiple nucleotide reagents. The retained forward extension strand can have two or more extended reverse sequencing primer strands hybridized thereto. [Figure 29]21A and 21B are schematic diagrams showing an exemplary support having first and second surface primers immobilized thereon, and immobilized concatemers generated by the on-support RCA workflow shown in FIG. 21. A portion of the concatemers hybridize to the immobilized second surface primer. The immobilized concatemer template molecules have two or more copies of a universal binding sequence for the immobilized second surface primer. The portion of the immobilized concatemer template molecule that includes the universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer. [Figure 30] 3A-3C are schematic diagrams illustrating an exemplary in-solution rolling circle amplification reaction using (i) nucleic acid circular library molecules, (ii) soluble first amplification primers, (iii) a mixture of nucleotides including nucleotides with cleavable moieties that can be cleaved to generate abasic sites, and (iv) a strand-displacing polymerase. The rolling circle amplification reaction generates concatemer molecules in solution that have at least one nucleotide with a cleavable moiety that can be cleaved to generate abasic sites in the single-stranded nucleic acid concatemer molecules. The circular library molecules contain a sequence of interest and at least one universal adaptor sequence that includes a binding sequence for, for example, the first surface primer. Figures 30-37 illustrate the workflow for pairwise sequencing the concatemer molecules shown in Figure 30. [Figure 31] 31 is a schematic diagram illustrating an exemplary method involving distributing the rolling circle amplification reaction shown in Figure 30 onto a support having first surface primers immobilized thereon. Concatamer molecules can hybridize to the immobilized first surface primers. [Figure 32]31 is a schematic diagram illustrating an exemplary method for allowing a rolling circle amplification reaction (e.g., from FIG. 31 ) to continue on a support, thereby generating immobilized concatemeric template molecules comprising at least one nucleotide having a cleavable moiety that can be cleaved to generate an abasic site in the immobilized concatemeric template molecule. The rolling circle amplification reaction comprises a plurality of compacting oligonucleotides. A first binding region of the compacting oligonucleotide hybridizes to a first portion of the immobilized concatemeric molecule, and a second binding region of the compacting oligonucleotide hybridizes to a second portion of the concatemeric molecule, causing the concatemeric molecule to collapse or fold into a nucleic acid nanostructure. [Figure 33] 33 is a schematic diagram showing an exemplary forward sequencing reaction performed on the immobilized nucleic acid nanostructure shown in Figure 32. The forward sequencing reaction can be performed with multiple soluble forward sequencing primers. The immobilized nucleic acid nanostructure can have two or more extended forward sequencing primer strands hybridized thereto. [Figure 34] 1 is a schematic diagram showing 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 comprising a strand-displacing polymerase, a plurality of nucleotides, and a plurality of compacting oligonucleotides. [Figure 35] 35 is a schematic diagram showing the forward extension strand produced by performing the primer extension reaction shown in Figure 34. For clarity, the compacted oligonucleotide hybridized to the forward extension strand is not shown. [Figure 36]1 is a schematic diagram showing an exemplary method for generating an abasic site in an immobilized nucleic acid nanostructure in a nucleotide having a cleavable moiety, retaining a plurality of forward extension strands, and generating a gap at the abasic site to generate a nanostructure containing a plurality of gaps while retaining a plurality of immobilized first surface primers. For clarity, the compaction oligonucleotide hybridized to the forward extension strand is not shown. [Figure 37] 37 is a schematic diagram showing an exemplary reverse sequencing reaction performed on a retained forward extension strand (e.g., from FIG. 36). The reverse sequencing reaction can be performed with multiple soluble reverse sequencing primers, multiple sequencing polymerases, and multiple nucleotide reagents. The retained forward extension strand is a concatemeric molecule that can contain two or more tandem copies of a sequence of interest and various primer binding sites. Such a concatemeric molecule can have two or more extended reverse sequencing primer strands hybridized thereto. For simplicity, FIG. 37 shows an exemplary immobilized retained forward extension strand hybridized with one reverse sequencing primer and undergoing a reverse sequencing reaction to generate an extended reverse sequencing primer strand. Those skilled in the art will understand that the immobilized retained forward extension strand can hybridize to two or more extended reverse sequencing primer strands. [Figure 38] 3A-3C are schematic diagrams showing an exemplary support having first and second surface primers immobilized thereon, and immobilized concatemers produced by the in-solution RCA workflow shown in FIGS. 30-32. A portion of the concatemers is hybridized to the immobilized second surface primer. The immobilized concatemer template molecule has two or more copies of a universal binding sequence for the immobilized second surface primer. The portion of the immobilized concatemer template molecule that includes the universal binding sequence for the immobilized second surface primer can hybridize to the immobilized second surface primer. [Figure 39]

[0049] Figure 39 is a schematic diagram illustrating an exemplary on-support rolling circle amplification reaction using (i) an immobilized first surface primer, (ii) a nucleic acid circular library molecule, (iii) a mixture of nucleotides lacking nucleotides with cleavable moieties that can be cleaved to generate abasic sites, (iv) a strand-displacing polymerase, and (iv) a compacting oligonucleotide. The rolling circle amplification reaction generates immobilized single-stranded nucleic acid concatemeric template molecules. The various primer binding sequence arrangements in the nucleic acid circular library molecule are for illustrative purposes. Those skilled in the art will appreciate that many other arrangements are possible. Figures 39-45 illustrate the workflow for pairwise sequencing the immobilized concatemeric template molecules shown in Figure 39. [Figure 40] 39 is a schematic diagram illustrating an exemplary immobilized nucleic acid nanostructure produced by performing a rolling circle amplification reaction on a support as shown in Figure 39. A first binding region of the compacting oligonucleotide hybridizes to a first portion of the immobilized concatemeric molecule, and a second binding region of the compacting oligonucleotide hybridizes to a second portion of the concatemeric molecule, causing the concatemeric molecule to collapse or fold into the nucleic acid nanostructure. [Figure 41] 41 is a schematic diagram showing an exemplary forward sequencing reaction performed on the immobilized nucleic acid nanostructure shown in Figure 40. The forward sequencing reaction can be performed with multiple soluble forward sequencing primers to generate multiple extended forward sequencing primer strands. The immobilized nanostructure can have two or more extended forward sequencing primer strands hybridized thereto. [Figure 42]1 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, thereby generating a forward extended strand, the primer extension reaction comprising a strand-displacing polymerase, a plurality of nucleotides, and a plurality of compacting oligonucleotides. [Figure 43] FIG. 43 is a schematic diagram showing a continuation of the exemplary strand displacement method shown in FIG. 42, in which the polymerase-catalyzed strand displacement reaction produces forward extension strands hybridized to immobilized concatemeric molecules and partially displaced forward extension strands, as well as separated forward extension strands that are not hybridized to immobilized concatemeric molecules. [Figure 44] FIG. 1 is a schematic diagram showing an exemplary hybridization complex comprising a forward extension strand and a partially displaced forward extension strand hybridized to an immobilized concatemeric molecule, and an immobilized separated forward extension strand hybridized to the partially displaced forward extension strand via a compacting oligonucleotide. [Figure 45] 45 is a schematic diagram showing an exemplary reverse sequencing reaction performed in the hybridization complex shown in Figure 44. The reverse sequencing reaction can be performed with multiple soluble reverse sequencing primers in the partially displaced forward extension strand and the immobilized separated forward extension strand. The reverse sequencing reaction produces an extended reverse sequencing primer strand. For simplicity, Figure 45 shows one copy of the extended reverse sequencing primer strand in the partially displaced forward extension strand and one copy of the extended reverse sequencing primer strand in the immobilized separated forward extension strand. Those skilled in the art will understand that the partially displaced forward extension strand and the immobilized separated forward extension strand can include two or more extended reverse sequencing primer strands hybridized thereto. [Figure 46]

[0049] Figure 46 is a schematic diagram illustrating an exemplary in-solution rolling circle amplification reaction using (i) nucleic acid circular library molecules, (ii) soluble first amplification primers, (iii) a mixture of nucleotides lacking nucleotides with cleavable moieties that can be cleaved to generate abasic sites, and (iv) a strand-displacing polymerase. The rolling circle amplification reaction generates single-stranded nucleic acid concatemer molecules in solution. The circular library molecules contain a sequence of interest and at least one universal adaptor sequence that includes a binding sequence for, e.g., the first surface primer. Figures 46-53 illustrate the workflow for pairwise sequencing the concatemer molecules shown in Figure 46. [Figure 47] 47 is a schematic diagram illustrating an exemplary method involving distributing the rolling circle amplification reaction shown in Figure 46 onto a support having first surface primers immobilized thereon. Concatamer molecules can hybridize to the immobilized first surface primers. [Figure 48] 47 is a schematic diagram illustrating an exemplary method showing a rolling circle amplification reaction (e.g., from FIG. 47) continuing on a support, thereby generating immobilized concatemeric template molecules. The rolling circle amplification reaction includes a plurality of compacting oligonucleotides. A first binding region of the compacting oligonucleotide hybridizes to a first portion of the immobilized concatemeric molecules, and a second binding region of the compacting oligonucleotide hybridizes to a second portion of the concatemeric molecules, causing the concatemeric molecules to collapse or fold into a nucleic acid nanostructure. [Figure 49] 49 is a schematic diagram showing an exemplary forward sequencing reaction performed on the immobilized nucleic acid nanostructure shown in Figure 48. The forward sequencing reaction can be performed with multiple soluble forward sequencing primers. The immobilized nucleic acid nanostructure can have two or more extended forward sequencing primer strands hybridized thereto. [Figure 50]1 is a schematic diagram showing 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 comprising a strand-displacing polymerase, a plurality of nucleotides, and a plurality of compacting oligonucleotides. [Figure 51] FIG. 51 is a schematic diagram showing a continuation of the exemplary strand displacement method shown in FIG. 50, in which the polymerase-catalyzed strand displacement reaction produces forward extension strands hybridized to immobilized concatemeric molecules and partially displaced forward extension strands, as well as separated forward extension strands that are not hybridized to immobilized concatemeric molecules. [Figure 52] FIG. 1 is a schematic diagram showing an exemplary hybridization complex comprising a forward extension strand and a partially displaced forward extension strand hybridized to an immobilized concatemeric molecule, and an immobilized separated forward extension strand hybridized to the partially displaced forward extension strand via a compacting oligonucleotide. [Figure 53] 53 is a schematic diagram showing an exemplary reverse sequencing reaction performed in the hybridization complex shown in Figure 52. The reverse sequencing reaction can be performed with multiple soluble reverse sequencing primers in the partially displaced forward extension strand and the immobilized separated forward extension strand. The reverse sequencing reaction produces an extended reverse sequencing primer strand. For simplicity, Figure 53 shows one copy of the extended reverse sequencing primer strand in the partially displaced forward extension strand and one copy of the extended reverse sequencing primer strand in the immobilized separated forward extension strand. Those skilled in the art will understand that the partially displaced forward extension strand and the immobilized separated forward extension strand can include two or more extended reverse sequencing primer strands hybridized thereto. [Figure 54]1 is an illustrative schematic diagram of one embodiment of a low-binding support comprising alternating layers of a glass substrate and a hydrophilic coating covalently or non-covalently attached to the glass, and further comprising chemically reactive functional groups that serve as attachment sites for oligonucleotide primers (e.g., capture oligonucleotides and circularization oligonucleotides). In alternative embodiments, the support can be made from any material, such as glass, plastic, or a polymeric material. [Figure 55A] 1 is a schematic diagram of various exemplary configurations of multivalent molecules having starburst or helter-skelter configurations, where nucleotide units are represented as "N", biotin is represented as "B", and streptavidin is represented as "SA". [Figure 55B] 1 is a schematic diagram of an exemplary multivalent molecule having a dendrimer architecture, where nucleotide units are represented as "N." [Figure 55C] Schematic of multiple multivalent molecules formed by reacting streptavidin with biotin-bearing 4-arm or 8-arm PEG-NHS and dNTPs. Nucleotide units are represented as "N," biotin is represented as "B," and streptavidin is represented as "SA." [Figure 56] FIG. 1 is a schematic diagram of an exemplary multivalent molecule comprising a generic core attached to multiple nucleotide arms. [Figure 57] FIG. 1 is a schematic diagram of an exemplary multivalent molecule comprising a dendrimer core attached to multiple nucleotide arms. [Figure 58] 1 shows a schematic diagram of an exemplary multivalent molecule comprising a core attached to multiple nucleotide arms, the nucleotide arms comprising biotin, spacers, linkers, and nucleotide units. [Figure 59] FIG. 1 is a schematic diagram of an exemplary nucleotide arm comprising a core attachment moiety, a spacer, a linker, and a nucleotide unit. [Figure 60] The chemical structures of an exemplary spacer (top) and various exemplary linkers (bottom) are shown, including an 11-atom linker, a 16-atom linker, a 23-atom linker, and an N3 linker. [Figure 61] 1 shows the chemical structures of various exemplary linkers, including linkers 1-9. [Figure 62A] 1 shows the chemical structures of various exemplary linkers linked / attached to nucleotide units. [Figure 62B] 1 shows the chemical structures of various exemplary linkers linked / attached to nucleotide units. [Figure 62C] 1 shows the chemical structures of various exemplary linkers linked / attached to nucleotide units. [Figure 62D] 1 shows the chemical structures of various exemplary linkers linked / attached to nucleotide units. [Figure 63] The chemical structure of an exemplary nucleotide arm is shown. In this embodiment, the nucleotide unit is connected to the linker via a propargylamine bond at the 5-position of the pyrimidine base or the 7-position of the purine base. This nucleotide arm represents an exemplary biotinylated nucleotide arm. [Figure 64] 1 is a schematic diagram of a G-quadruplex (e.g., a G-quadruplex). [Figure 65] FIG. 1 is a schematic diagram of an exemplary intramolecular G-quadruplex structure. [Figure 66] A series of fluorescence images of nucleic acid nanostructures immobilized on a support are shown. Nanostructures were generated by on-support rolling circle amplification with various compacting oligonucleotides (100 nM) or without compacting oligonucleotides (none) as a negative control. The immobilized nanostructures were hybridized with fluorescently labeled probes, washed, and imaged. The sequence numbers of the compacting oligonucleotides are indicated in the upper left corner of each image. The images show that the shape and size of the resulting immobilized nanostructures are affected by the type of compacting oligonucleotide tested. [Figure 67]Magnifications of selected images from Figure 66 are shown. Figure 67A. Negative control: Immobilized concatemers are less compact and have a "fuzzy" appearance. Figure 67B. Compacting oligonucleotide (SEQ ID NO: 126): Immobilized nucleic acid nanostructures are more compact compared to the negative control. Figure 67C. Compacting oligonucleotide (SEQ ID NO: 57): Immobilized nucleic acid nanostructures are more compact and discrete compared to the negative control and the image shown in panel (Figure 67B). [Figure 68] Figure 1 is a box plot showing the number of immobilized nanostructures per field of view. Nanostructures were generated by on-support rolling circle amplification using various titrations of compacted oligonucleotides, or without compacted oligonucleotides as a negative control. Compacted oligonucleotides were tested at 25 nM, 100 nM, 250 nM, and 500 nM. Compacted oligonucleotides tested included SEQ ID NOs: 57, 126, and 156 (see sequences in Table 1). The data show that the compacted oligonucleotides did not inhibit the rolling circle amplification reaction. The number of immobilized nanostructures (spots) was similar for the negative control and the three different compacted oligonucleotides tested in this experiment. [Figure 69] 69 is a box plot showing the full width at half maximum (FWHM) of the immobilized nanostructures described in Figure 68. The data show that the FWHM of nanostructures generated with compacting oligonucleotides is smaller compared to the negative control. The data also show that immobilized nanostructures generated with compacting oligonucleotides comprising SEQ ID NO: 57 have a smaller FWHM compared to immobilized nanostructures generated with compacting oligonucleotides comprising SEQ ID NO: 126 or 156. [Figure 70] 69 is a box plot showing signal intensities of the immobilized nanostructures described in Figure 68. The data show a smaller (more discrete) distribution range of signals detected from nanostructures generated with compacted oligonucleotides compared to the negative control. [Figure 71]This figure shows a series of four-color fluorescence images of first-strand nanostructures immobilized on a support. The first-strand nanostructures were generated by on-support rolling circle amplification of compacted oligonucleotides, a mixture of nucleotides and titrated concentrations of dUTP, or no dUTP as a negative control. Sequencing reagents were flowed over the immobilized nanostructures to form fluorescent binding complexes on the first-strand nanostructures. Images of the resulting binding complexes were obtained. The procedure for obtaining the fluorescence images is described in Example 3. [Figure 72] A series of four-color fluorescence images of second-strand nanostructures immobilized on a support are shown. The second-strand nanostructures were generated from the first-strand nanostructures described in Figure 71. The second-strand nanostructures were generated by performing a primer extension reaction on the first-strand nanostructures using a mixture of nucleotides lacking dUTP and compacted oligonucleotides. The first strand was removed by enzymatic degradation while retaining the second-strand molecules. Sequencing reagents were flowed over the immobilized second template strand to form fluorescent binding complexes on the second-strand nanostructures. Images of the resulting binding complexes were obtained. The procedure for obtaining the fluorescence images is described in Example 3. [Figure 73] Figure 73 shows four-color fluorescence images of fluorescent binding complexes on immobilized first-strand nanostructures (top) and second-strand nanostructures (bottom), where the second strands were generated from their respective first strands on the same flow cell. The white-framed boxes in the top and bottom images of Figure 73 indicate the same field of view. The white arrows indicate the locations of nanostructures that are easily distinguishable and comparable in the top and bottom images. DETAILED DESCRIPTION OF THE INVENTION

[0026] Definition: The headings provided herein are not limitations of various aspects of the disclosure, which aspects can be understood by reference to the specification as a whole.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art. Generally, terms relating to molecular biology, nucleic acid chemistry, protein chemistry, genetics, microbiology, transgenic cell production, and hybridization techniques described herein are well known and commonly used in the art. The techniques and procedures described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 2000). See also Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992). The nomenclature used in connection with, and the experimental procedures and techniques described herein are well known and commonly used in the art.

[0028] Unless otherwise required by context herein, singular terms include pluralities and plural terms include the singular. The singular forms "a," "an," and "the," as well as use of the singular form of any word, include plural references unless expressly and unambiguously limited to a reference to one.

[0029] The use of alternative terms (eg, "or") is understood to mean either one or both of the alternatives, or any combination thereof.

[0030] The term "and / or" as used herein should be understood to mean a specific disclosure that each of the specified features or components may or may not have the other. For example, when used herein in phrases such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A" (A alone), and "B" (B alone). In a similar manner, when used in phrases such as "A, B, and / or C," the term "and / or" is intended to encompass each of the following embodiments: "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).

[0031] As used in this specification and the appended claims, the terms "comprising," "including," "having," and "containing," and grammatical variations thereof, as used herein, are intended to be open-ended, such that one or more items in a list do not exclude other items that may be substituted for or added to the listed items. Wherever an embodiment is described herein with the term "comprising," it is understood that alternative, similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0032] As used herein, the term "about" or "approximately" refers to a value or composition that is within an acceptable error range for a particular value or composition, as determined by one of ordinary skill in the art. The acceptable error range depends in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "approximately" can mean within one or more standard deviations per practice in the art. Alternatively, "about" or "approximately" can mean a range of up to 10% (i.e., ±10%) or more, depending on the limitations of the measurement system. For example, about 5 mg can include any number between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the term can mean up to one order of magnitude or up to five times the value. When a particular value or composition is provided in this disclosure, unless otherwise specified, the meaning of "about" or "approximately" should be considered to be within an acceptable error range for that particular value or composition. Additionally, when ranges and / or subranges of values ​​are provided, the ranges and / or subranges can include the endpoints of the ranges and / or subranges.

[0033] The term "biological sample" refers to a single cell, multiple cells, tissue, organ, organism, or section of any of these biological samples. Biological samples may be extracted from an organism (e.g., biopsy) or obtained from cell cultures growing in liquid or in culture dishes. Biological samples include fresh samples, frozen samples, fresh frozen samples, or archived (e.g., formalin-fixed paraffin-embedded; FFPE) samples. Biological samples may be embedded in wax, resin, epoxy, or agar. Biological samples may be fixed, for example, in any one of acetone, ethanol, methanol, formaldehyde, paraformaldehyde-Triton, or glutaraldehyde, or any combination of two or more thereof. Biological samples may or may not be sectioned. Biological samples may be stained, destained, or unstained.

[0034] Nucleic acids of interest can be extracted from biological samples using any of several techniques known to those skilled in the art. For example, a typical DNA extraction procedure involves (i) collecting a cell or tissue sample from which DNA is to be extracted, (ii) disrupting cell membranes (i.e., lysing cells) to release 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. Various suitable commercially available nucleic acid extraction and purification kits are consistent with the disclosure herein. Examples include, but are not limited to, the QIAamp kit (for isolation of genomic DNA from human samples) and the DNAeasy kit (for isolation of genomic DNA from animal or plant samples) from Qiagen (Germantown, MD), or the Maxwell® and ReliaPrep™ series of kits from Promega (Madison, WI).

[0035] As used herein, the terms "nucleic acid," "polynucleotide," and "oligonucleotide," as well as other related terms, are used interchangeably and refer to a polymer of nucleotides and are not limited to any particular length. Nucleic acids include recombinant or 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 acid (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 comprise polymers of nucleotides, where the nucleotides comprise natural or non-natural bases and / or sugars. Nucleic acids comprise naturally occurring internucleoside linkages, such as phosphodiester linkages. Nucleic acids can lack phosphate groups. Nucleic acids comprise non-natural internucleoside linkages, including phosphorothioate, phosphorothiolate, or peptide nucleic acid (PNA) linkages. In some embodiments, the nucleic acid comprises one type of polynucleotide or a mixture of two or more different types of polynucleotides.

[0036] The terms "universal sequence," "universal adapter sequence," and related terms refer to a sequence in a nucleic acid molecule that is common between two or more polynucleotide molecules. For example, an adapter having the same universal sequence can be ligated to multiple polynucleotides, such that a population of co-ligated molecules possesses 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). Exemplary universal adapter sequences are listed in Table 2 (Figure 15).

[0037] As used herein, the terms "operably linked" and "operably joined," or related terms, refer to the juxtaposition of components. Juxtaposed components can be covalently linked together. For example, two nucleic acid components can be enzymatically ligated together, where the bond linking the two components together comprises a phosphodiester bond. A first and second nucleic acid component can be linked together, where the first nucleic acid component can confer a function to the second nucleic acid component. For example, the bond between a primer binding sequence and a sequence of interest forms a nucleic acid library molecule having a portion capable of binding to a primer. In another example, a transgene (e.g., a nucleic acid encoding a polypeptide or nucleic acid sequence of interest) can be ligated into a vector, where the bond allows for expression or function of the transgene sequence contained within the vector. In some embodiments, the transgene is operably linked to a host cell regulatory sequence (e.g., a promoter sequence) that affects expression of the transgene. In some embodiments, the vector comprises at least one host cell regulatory sequence, including a promoter sequence, an enhancer, a transcriptional and / or translational initiation sequence, a transcriptional and / or translational termination sequence, a polypeptide secretion signal sequence, etc. In some embodiments, the host cell regulatory sequence controls the level, timing, and / or location of expression of the transgene.

[0038] The terms "linked," "joined," "attached," and "appended," and variations thereof, include any type of fusion, bond, adhesion, or association between any combination of compounds or molecules that is stable enough to withstand use in a particular procedure. Procedures can include, but are not limited to, nucleotide binding, nucleotide incorporation, deblocking (e.g., removal of chain-terminating moieties), washing, removal, flow, detection, imaging, and / or identification. Such bonds can include, for example, covalent bonds, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, or affinity bonds, bonds or associations involving van der Waals forces, and mechanical bonds. In some embodiments, such bonds occur intramolecularly, such as by joining the ends of a single- or double-stranded linear nucleic acid molecule together to form a circular molecule. In some embodiments, such bonds can occur between different molecular combinations or between molecules and non-molecules, including, but not limited to, bonds between nucleic acid molecules and solid surfaces, bonds between proteins and detectable reporter moieties, and bonds between nucleotides and detectable reporter moieties. Some examples of conjugation can be found, for example, in Hermanson, G., "Bioconjugate Techniques", Second Edition (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).

[0039] The term "adapter" and related terms refer to an oligonucleotide that can be operably attached to (applied to) a target polynucleotide, and the adapter confers a function to the co-ligated adapter-target molecule. Adapters include DNA, RNA, chimeric DNA / RNA, or analogs thereof. Adapters can contain at least one ribonucleoside residue. Adapters can be single-stranded or double-stranded, or can have single-stranded and / or double-stranded portions. Adapters can be configured to be linear, stem-loop, hairpin, or Y-shaped. Adapters can be any length, from 4 to 100 nucleotides or more. Adapters can have blunt ends, overhanging ends, or a combination of both. Overhanging ends include 5' overhangs and 3' overhanging ends. The 5' end of a single-stranded adapter, or one strand of a double-stranded adapter, can have a 5' phosphate group or lack a 5' phosphate group. The adapter may include a 5' tail that does not hybridize to the target polynucleotide (e.g., a tailed adapter), or the adapter may be tailless. The adapter may include a sequence 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 include a random or degenerate sequence. The adapter may include at least one inosine residue. The adapter may include at least one phosphorothioate, phosphorothiolate, and / or phosphoramidate linkage. The adapter may include a barcode sequence, which can be used to distinguish polynucleotides (e.g., insert sequences) from different sample sources in multiplex assays. The adapter may include a unique identification sequence (e.g., a unique molecular index, UMI, or unique molecular tag), which can be used to uniquely identify the nucleic acid molecule to which the adapter is attached.In some embodiments, the unique identifier 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 can comprise at least one restriction enzyme recognition sequence, wherein the at least one restriction enzyme recognition sequence comprises any one or any combination of two or more selected from the group consisting of Type I, Type II, Type III, Type IV, Hs, or Type IIB.

[0040] 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 the base nucleic acid molecule for any of the analytical methods described herein (e.g., primer extension, amplification, and / or sequencing). A template nucleic acid may be single-stranded or double-stranded, or may have single-stranded or double-stranded portions. A template nucleic acid may be obtained from a naturally occurring source or a recombinant form, or may be chemically synthesized to contain any type of nucleic acid analog. A template nucleic acid can be linear, circular, or in other forms. A template nucleic acid can include an insertion region having an insert sequence, also known as a sequence of interest. A template nucleic acid can also include at least one adapter sequence. A 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 may be isolated in any form, including from a chromosome, genome, organelle (e.g., mitochondria, chloroplast, or ribosome), recombinant molecule, cloned, amplified, cDNA, RNA such as precursor mRNA or mRNA, oligonucleotide, total 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 may be isolated from any source, including organisms such as prokaryotes, eukaryotes (e.g., human, plant, and animal), fungi, viral cells, tissues, normal or diseased cells or tissues, bodily 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. The insertion region 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, thymus, skin, heart, larynx, or other organ. The template nucleic acid can be subjected to nucleic acid analysis, including sequencing and compositional analysis.

[0041] As used herein, the term "polymerase" and variations thereof include enzymes that contain a nucleotide (or nucleoside)-binding domain, and the polymerase can form a complex with a template nucleic acid and a complementary nucleotide. A 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. A polymerase can be any enzyme that can catalyze the polymerization of nucleotides (including their analogs) into a nucleic acid strand. Typically, but not necessarily, such nucleotide polymerization can occur in a template-dependent manner. Typically, a polymerase contains one or more active sites, and nucleotide binding and / or catalysis of nucleotide polymerization can occur in one or more active sites. In some embodiments, a polymerase includes other enzymatic activities, such as 3' to 5' exonuclease activity or 5' to 3' exonuclease activity. In some embodiments, a polymerase has strand displacement activity. Polymerases can include, but are not limited to, naturally occurring polymerases and any subunits and truncations thereof, 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 that contain a nucleotide-binding domain. In some embodiments, polymerases may be isolated from cells or produced using recombinant DNA technology or chemical synthesis methods. In some embodiments, polymerases may be expressed in prokaryotic, eukaryotic, viral, or phage organisms. In some embodiments, polymerases may be post-translationally modified proteins or fragments thereof. Polymerases may be derived from prokaryotic, eukaryotic, viral, or phage organisms.Polymerases include DNA-directed DNA polymerases and RNA-directed DNA polymerases.

[0042] The term "strand displacement" refers to the ability of a polymerase to locally separate strands of double-stranded nucleic acid and synthesize a new strand in a template-based manner. Strand-displacing polymerases displace a complementary strand from the template strand and catalyze new strand synthesis. Strand-displacing polymerases include mesophilic and thermophilic polymerases. Strand-displacing polymerases include wild-type enzymes and variants, including exonuclease-minus mutants, mutated versions, chimeric enzymes, and truncated enzymes. Examples of strand-displacing polymerases include phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase (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 can be a wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon™), or a variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific™), or a chimeric QualiPhi DNA polymerase (e.g., from 4basebio™).

[0043] As used herein, the term "fidelity" refers to the accuracy of DNA polymerization by a template-dependent DNA polymerase. The fidelity of a DNA polymerase is typically measured by the error rate (the frequency of incorporating an incorrect nucleotide, i.e., a nucleotide that is not complementary to the template nucleotide). The accuracy or fidelity of DNA polymerization is maintained by both the polymerase activity and the 3' to 5' exonuclease activity of the DNA polymerase.

[0044] As used herein, the term "bound complex" refers to a complex formed by binding together a nucleic acid duplex, a polymerase, and a free nucleotide or nucleotide unit of a multivalent molecule, where the nucleic acid duplex comprises a nucleic acid template molecule hybridized to a nucleic acid primer. In the bound complex, the free nucleotide or nucleotide unit may or may not be bound to the 3' end of the nucleic acid primer, opposite its complementary nucleotide in the nucleic acid template molecule. A "ternary complex" is an example of a bound complex formed by binding together a nucleic acid duplex, a polymerase, and a free nucleotide or nucleotide unit of a multivalent molecule, where the free nucleotide or nucleotide unit is bound to the 3' end of the nucleic acid primer (as part of the nucleic acid duplex) opposite its complementary nucleotide in the nucleic acid template molecule.

[0045] The term "duration" and related terms refer to the length of time during which a binding complex remains stable without any of its components dissociating, and the components of the binding complex include a nucleic acid template and nucleic acid primer, a polymerase, a nucleotide unit of a multivalent molecule, or a free (e.g., unconjugated) nucleotide. The nucleotide unit or free nucleotide may be complementary or non-complementary to a nucleotide residue in the template molecule. The nucleotide unit or free nucleotide may be attached to the 3' end of the nucleic acid primer at a position opposite the complementary nucleotide residue in the nucleic acid template molecule. The duration indicates the stability of the binding complex and the strength of the binding interaction. The duration can be measured by observing the onset and / or duration of the binding complex, for example, by observing a signal from a labeled component of the binding complex. For example, a labeled nucleotide or a labeled reagent comprising one or more nucleotides can be present in the binding complex, thus allowing a signal from the label to be detected during the duration of the binding complex. One exemplary label is a fluorescent label. The bound complex (e.g., ternary complex) remains stable until subjected to conditions that cause dissociation of interactions between the polymerase, template molecule, primer, and / or any of the 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.

[0046] As used herein, the term "primer" and related terms refer to an oligonucleotide capable of hybridizing to a DNA and / or RNA polynucleotide template to form a duplex molecule. A primer contains natural nucleotides and / or nucleotide analogs. A primer can be a recombinant nucleic acid molecule. A primer can be of any length, but typically ranges from 4 to 50 nucleotides. A typical primer contains a 5' end and a 3' end. The 3' end of a primer can contain a 3' OH moiety that functions as a nucleotide polymerization initiation site in a polymerase-catalyzed primer extension reaction. Alternatively, the 3' end of a primer can lack a 3' OH moiety or contain a terminal 3' blocking group that inhibits nucleotide polymerization in a polymerase-catalyzed reaction. Any one or more nucleotides along the length of a primer can be labeled with a detectable reporter moiety. A primer can be in solution (e.g., a soluble primer) or immobilized on a support (e.g., a capture primer).

[0047] When used in reference to nucleic acid molecules, the terms "hybridize" or "hybridizing" or "hybridization," or other related terms, refer to hydrogen bonding between two different nucleic acids to form a double-stranded nucleic acid. Hybridization also includes hydrogen bonding between two different regions of a single nucleic acid molecule to form a self-hybridizing molecule having a double-stranded region. Hybridization can involve Watson-Crick or Hoogstein binding to form a double-stranded double-stranded nucleic acid or a double-stranded region within a nucleic acid molecule. A double-stranded nucleic acid, or two different regions of a single nucleic acid, can be fully complementary or partially complementary. Complementary nucleic acid strands need not hybridize to each other throughout their entire length. Complementary base pairing can be standard AT or CG base pairing, or other forms of base-pairing interactions. A double-stranded nucleic acid can contain mismatched base-pairing nucleotides.

[0048] When used with respect to nucleic acids, the terms "extend," "extending," "extension," and other variations refer to the incorporation of one or more nucleotides into a nucleic acid molecule. Nucleotide incorporation involves the polymerization of one or more nucleotides into the terminal 3' OH terminus of a nucleic acid strand (e.g., a nucleic acid primer), resulting in the extension of the nucleic acid strand (e.g., an extended primer). Nucleotide incorporation can be performed with natural nucleotides and / or nucleotide analogs. Typically, although not necessarily, nucleotide incorporation occurs in a template-dependent manner. Any suitable method for extending a nucleic acid molecule may be used, including primer extension catalyzed by DNA polymerase or RNA polymerase.

[0049] 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, or about 5 to 40 nucleotides in length, or about 5 to 25 nucleotides in length.

[0050] The term "nucleotide" and related terms refer to a molecule comprising an aromatic base, a five-carbon sugar (e.g., ribose or deoxyribose), and at least one phosphate group. Standard or non-standard nucleotides are consistent with the use of this term. In some embodiments, the phosphate comprises a monophosphate, diphosphate, or triphosphate, or a corresponding phosphate analog. 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.

[0051] Nucleotides (and nucleosides) typically contain a heterocyclic base containing a substituted or unsubstituted nitrogen-containing parent heteroaromatic ring, 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 Hoogstein hydrogen bonds with an appropriate complementary base. Exemplary bases are purines and pyrimidines, such as 2-aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N, N-acetyl-p-tolyl-2-one ... 6 -Δ 2 -Isopentenyladenine (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-deaza-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 Examples of bases include, but are not limited to, methylthymine, uracil (U), 4-thiouracil (4sU), and 5,6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and 4-methylindole; pyrroles such as nitropyrrole; nebularine; inosine; hydroxymethylcytosine; 5-methycytosine; base (Y); and methylated, glycosylated, and acylated base moieties. Additional exemplary bases can be found in Fasman, 1989, "Practical Handbook of Biochemistry and Molecular Biology," pp. 385-394, CRC Press, Boca Raton, Fla.

[0052] Nucleotides (and nucleosides) typically include a sugar moiety, e.g., 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 another sugar moiety (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'-mercaptoriboxyl; 2'-alkylthioribosyl; 3'-alkoxyribosyl; 3'-azidoribosyl; 3'-aminoribosyl; 3'-fluororibosyl; 3'-mercaptoriboxyl; 3'-alkylthioribosyl carbocyclic; acyclic, or other modified sugars.

[0053] In some embodiments, the nucleotide comprises a chain of one, two, or three phosphorus atoms, typically attached to the 5' carbon of the sugar moiety via an ester or phosphoramido linkage. In some embodiments, the nucleotide is an analog having a phosphorus chain in which the phosphorus atoms are linked together with intervening O, S, NH, methylene, or ethylene. In some embodiments, the phosphorus atoms in the chain comprise substituted side chain groups including O, S, or BH. In some embodiments, the chain comprises phosphate groups substituted with analogs including phosphoramidate, phosphorothioate, phosphordithioate, and O-methylphosphoramidite groups.

[0054] The terms "reporter moiety," "reporter moieties," or related terms refer to a compound that produces or can be caused to produce a detectable signal. Reporter moieties are often referred to as "labels." Any suitable reporter moiety can be used, and suitable reporter moieties include luminescence, photoluminescence, electroluminescence, bioluminescence, chemiluminescence, fluorescence, phosphorescence, chromophores, radioisotopes, electrochemistry, mass spectrometry, Raman, haptens, affinity tags, atoms, or enzymes. A reporter moiety produces a detectable signal that results from a chemical or physical change (e.g., heat, light, electricity, pH, salt concentration, enzymatic activity, or a proximity event). A proximity event involves two reporter moieties coming into close proximity with, associating with, or binding to each other. It is well known to those skilled in the art to select reporter moieties so that each absorbs excitation radiation and / or emits fluorescence at a wavelength distinguishable from other reporter moieties, allowing for the monitoring of the presence of different reporter moieties in the same or different reactions. Two or more different reporter moieties may be selected that have spectrally distinct emission profiles or that have minimal overlapping spectral emission profiles. The reporter moiety may be bound (e.g., operably bound) to a nucleotide, a nucleoside, a nucleic acid, an enzyme (e.g., a polymerase or reverse transcriptase), or a support (e.g., a surface).

[0055] As used herein, "nucleotide unit" or "nucleotide moiety" refers to a nucleotide (e.g., dATP, dTTP, dGTP, dCTP, or dUTP), or an analog thereof, that comprises a base, a sugar, and at least one phosphate group. The nucleotide unit can be attached to a multivalent molecule used in the sequencing reactions described herein. Generally, all nucleotide units attached to the same multivalent molecule will have the same identity (e.g., all A's, all T's, all C's, or all G's), although one of skill in the art will understand that there may be situations in which multivalent molecules comprising nucleotide units of different identities are advantageous.

[0056] The reporter moiety (or label) comprises a fluorescent label or fluorophore. Exemplary fluorescent moieties that can function as fluorescent labels or fluorophores include fluorescein and fluorescein derivatives, such as carboxyfluorescein, tetrachlorofluorescein, hexachlorofluorescein, carboxynapthofluorescein, fluorescein isothiocyanate, NHS-fluorescein, iodoacetamidofluorescein, fluorescein maleimide, SAMSA-fluorescein, fluorescein thiosemicarbazide, carbohydrazinomethylthioacetyl-aminofluorescein, 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 sulfonylhydrazine, 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, BODIPY 530 / 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 azide, 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-based cyanine dyes, benzo-indolium-based cyanine dyes, pyridium-based cyanine dyes, thiozolium-based cyanine dyes, quinolinium-based cyanine dyes, imidazolium-based cyanine dyes, Cy3, 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 dyes, WellRED dyes, IRD dyes, phycoerythrin and phycobilin dyes, malachite green, stilbenes, DEG dyes, NR dyes, near-infrared dyes, and others known in the art, such as those described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition, Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999), or Hermanson, Bioconjugate Techniques, 2nd Edition, or derivatives thereof, or any combination thereof. Cyanine dyes can exist in either sulfonated or non-sulfonated form and consist of two indolenine, benzoindolium, pyridium, thiozolium, and / or quinolinium groups separated by a polymethine bridge between the two nitrogen atoms. Commercially available cyanine fluorophores include, for example, Cy3 (which is 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, may include 1-[6-(2,5-dioxopyrrolidin-1-yloxy)-6-oxohexyl]-2-(3-{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), Cy5 (which may include1-(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-indolin-2-ylidene)penta-1,3-dien-1-yl)-3,3-dimethyl-3H-yne dol-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-sulfoindolin-2-ylidene)penta-1,3-dien-1-yl) Cy7 (which may include 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-5-sulfonate), and Cy8 (which may include 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 number identifies the number of carbon atoms between the two indolenine groups. Cy2, which is an oxazole derivative rather than an indolenine, and benzo-derivatized Cy3.5, Cy5.5, and Cy7.5 are exceptions to this rule.

[0057] In some embodiments, the reporter moieties can be FRET pairs, allowing multiple classifications to be performed under a single excitation and imaging step. As used herein, FRET can include excitation exchange (Förster) transfer or electron exchange (Dexter) transfer.

[0058] As used herein, the term "support" refers to a substrate designed for the deposition of biomolecules or biological samples for assay and / or analysis. Examples of biomolecules deposited on a support include nucleic acids (e.g., DNA, RNA), polypeptides, sugars, 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.

[0059] In some embodiments, the support is solid, semi-solid, or a combination of both. In some embodiments, the support is porous, semi-porous, non-porous, or any combination of porous. 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.

[0060] In some embodiments, the surface of the support can be substantially smooth, hi some embodiments, the support can be regularly or irregularly textured, including ridges, etchings, pores, three-dimensional scaffolds, or any combination thereof.

[0061] In some embodiments, the support comprises beads having any shape, including spherical, hemispherical, cylindrical, barrel-shaped, toroidal, disk-shaped, rod-shaped, conical, triangular, cubic, polygonal, tubular, or wire-shaped.

[0062] The support can be made of any material, including, but not limited to, glass, fused silica, silicon, polymer (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.

[0063] The support can have a plurality (e.g., two or more) of nucleic acid templates immobilized thereon. The plurality of immobilized nucleic acid templates have the same sequence or different sequences. In some embodiments, individual nucleic acid template molecules in the plurality of nucleic acid templates are immobilized at different sites on the support. In some embodiments, two or more individual nucleic acid template molecules in the plurality of nucleic acid templates are immobilized at sites on the support.

[0064] The term "array" refers to a support comprising a plurality of sites located at predetermined locations on the support, forming an array of sites. The sites may be dispersed and separated by interstitial regions. In some embodiments, the predetermined sites on the support may be arranged in rows or columns in one dimension, or in rows or columns in two dimensions. In some embodiments, the 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 linear, hexagonal, lattice, patterns with reflection symmetry, patterns with rotational symmetry, etc. The pitch between different pairs of sites may be the same or may vary. In some embodiments, the support has a pitch of at least 10 2 at least 10 sites 3 at least 10 sites 4 at least 10 sites5 at least 10 sites 6 at least 10 sites 7 at least 10 sites 8 at least 10 sites 9 at least 10 sites 10 at least 10 sites 11 at least 10 sites 12 at least 10 sites 13 at least 10 sites 14 sites, or at least 10 15 In some embodiments, the substrate comprises a plurality of predetermined sites (e.g., 10 or more sites), the sites being located at predetermined locations on the substrate. 2 ~10 15 At a plurality of predetermined sites (e.g., 10 or more sites), a nucleic acid template is immobilized, forming a nucleic acid template array. In some embodiments, the nucleic acid template is immobilized at a plurality of predetermined sites by hybridization to an immobilized surface capture primer, or the nucleic acid template is covalently attached to the surface capture primer. In some embodiments, the plurality of predetermined sites, e.g., 10 2 ~10 15 sites (e.g., at least 10 2 at least 10 sites 3 at least 10 sites 4 at least 10 sites 5 at least 10 sites 6 at least 10 sites 7 at least 10 sites 8 at least 10 sites 9 at least 10 sites 10 at least 10 sites 11 at least 10 sites 12 at least 10 sites 13 at least 10 sites 14 at least 10 sites 15A nucleic acid template molecule that is immobilized at multiple predetermined sites. In some embodiments, the immobilized nucleic acid template is clonally amplified to generate immobilized nucleic acid polonies at multiple predetermined sites. In some embodiments, each immobilized nucleic acid polony comprises a single-stranded or double-stranded concatemer.

[0065] 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 located sites thereon. The locations of the randomly located sites on the support are not predetermined locations. The plurality of randomly located sites are arranged on the support in a non-ordered and / or unpredictable manner. In some embodiments, the support has at least 10 2 at least 10 sites 3 at least 10 sites 4 at least 10 sites 5 at least 10 sites 6 at least 10 sites 7 at least 10 sites 8 at least 10 sites 9 at least 10 sites 10 at least 10 sites 11 at least 10 sites 12 at least 10 sites 13 at least 10 sites 14 sites, or at least 10 15 In some embodiments, the substrate comprises a plurality of randomly positioned sites (e.g., 10 sites, 100 sites, or more), where the sites are randomly located on the substrate. 2 ~10 15In some embodiments, the nucleic acid template is immobilized at a plurality of randomly located sites by hybridization to the 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, e.g., 10 2 ~10 15 In some embodiments, the immobilized nucleic acid template is clonally amplified to generate a nucleic acid polony immobilized at multiple randomly located sites. In some embodiments, each immobilized nucleic acid polony comprises a single-stranded or double-stranded concatemer.

[0066] The term "immobilized" and related terms, when used in reference to immobilized nucleic acids, refer to nucleic acid molecules that are attached to a support via covalent or non-covalent interactions, or that are attached to a coating on a support, or that are embedded within a matrix formed by a coating on a support, the nucleic acid molecules comprising a surface capture primer, a nucleic acid template molecule, and an extension product of the capture primer. The extension product of the capture primer comprises a nucleic acid concatemer that can form a nucleic acid polony.

[0067] In some embodiments, one or more nucleic acid templates are immobilized on a support, for example, at a site on the support. In some embodiments, one or more nucleic acid templates are clonally amplified. In some embodiments, one or more nucleic acid templates are clonally amplified off the support (e.g., in solution) and then deposited on the support and immobilized thereon. In some embodiments, a clonal amplification reaction of one or more nucleic acid templates is performed on the support, resulting in immobilization on the support. In some embodiments, one or more nucleic acid templates are clonally amplified (e.g., in solution or on the support) using a nucleic acid amplification reaction, the nucleic acid amplification reaction comprising any one of polymerase chain reaction (PCR), multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification (RCA), circle-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, and / or single-strand binding (SSB) protein-dependent amplification, or any combination thereof.

[0068] The terms "surface primer," "surface capture primer," and related terms refer to a single-stranded oligonucleotide immobilized on a support and comprising a sequence capable of hybridizing to at least a portion of a nucleic acid template molecule. Surface primers can be used to immobilize template molecules to a support via hybridization. Surface primers can be immobilized to a support in a manner that resists primer removal during flow, washing, aspiration, and changes in temperature, pH, salt, chemical, and / or enzyme conditions. Typically, although not necessarily, the 5' end of a surface primer can be immobilized to the support. Alternatively, an internal portion or the 3' end of a surface primer can be immobilized to the support.

[0069] In some embodiments, surface primers comprise DNA, RNA, or analogs thereof. Surface primers can comprise a combination of DNA and RNA. The sequences of surface primers can be fully or partially complementary along their length to at least a portion of a nucleic acid template molecule (e.g., a linear or circular template molecule). The support can comprise multiple immobilized surface primers having the same sequence or two or more different sequences. Surface primers can be any length, for example, 4-50 nucleotides, or 50-100 nucleotides, or 100-150 nucleotides long, or longer, or any range therebetween.

[0070] A surface primer can comprise a terminal 3' nucleotide having a sugar 3' OH moiety that is extendable for nucleotide polymerization (e.g., polymerase-catalyzed polymerization). A surface primer can comprise a terminal 3' nucleotide having a moiety that blocks polymerase-catalyzed extension. A surface primer can comprise a terminal 3' nucleotide having a 3' sugar moiety attached to a chain-terminating moiety that inhibits nucleotide polymerization. The 3' chain-terminating moiety can be removed (e.g., deblocked) using a deblocking agent to convert the 3' end to an extendable 3' OH end. Examples of chain-terminating moieties include alkyl, alkenyl, alkynyl, allyl, aryl, benzyl, azide, amine, amide, keto, isocyanate, phosphate, thio, disulfide, carbonate, urea, or silyl groups. Azido-type chain-terminating moieties include azide, azido, and azidomethyl groups. Examples of deblocking agents include phosphine compounds, such as tris(2-carboxyethyl)phosphine (TCEP) and bis-sulfotriphenylphosphine (BS-TPP), for chain-terminating azide, azido, and azidomethyl groups. Examples of deblocking agents include tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) with piperidine or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) for chain-terminating alkyl, alkenyl, alkynyl, and aryl groups. Examples of deblocking agents include Pd / C for chain-terminating aryl and benzyl groups. Examples of deblocking agents include phosphines, beta-mercaptoethanol, or dithiothreitol (DTT) for chain-terminating amine, amide, keto, isocyanate, phosphate, thio, and disulfide groups. Examples of deblocking agents include potassium carbonate (K2CO3) in MeOH, triethylamine in pyridine, or 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 urea and silyl chain terminating groups.

[0071] In some embodiments, multiple immobilized surface capture primers on a support are in fluid communication with each other, allowing solutions of reagents (e.g., linear or circular nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, and reagents, etc.) to flow over the support, thereby allowing multiple immobilized surface capture primers on a support to react with reagents essentially simultaneously in a massively parallel manner. In some embodiments, the fluid communication of multiple immobilized surface capture primers can be used to perform nucleic acid amplification reactions (e.g., RCA, MDA, PCR, and bridge amplification) essentially simultaneously on multiple immobilized surface capture primers.

[0072] In some embodiments, the plurality of immobilized single-stranded nucleic acid concatemer template molecules on the support are in fluid communication with each other, allowing solutions of reagents (e.g., soluble primers, enzymes, nucleotides, divalent cations, buffers, and reagents, etc.) to flow over the support, thereby allowing the plurality of immobilized single-stranded nucleic acid concatemer template molecules on the support to react with the reagents essentially 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) essentially simultaneously on the plurality of immobilized single-stranded nucleic acid concatemer template molecules, and optionally, can be used to perform detection and imaging for massively parallel sequencing.

[0073] The terms "amplify," "amplifying," "amplification," and other related terms, when used with respect to nucleic acids, include producing multiple copies of an original polynucleotide template molecule, where the copies contain a sequence that is complementary to the template sequence or where the copies contain a sequence that is identical to the template sequence. In some embodiments, the copies contain a sequence that is substantially identical to the template sequence or a sequence that is substantially identical to the sequence that is complementary to the template sequence.

[0074] 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 "TrisHCl" 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-Trispropane" refers to the pH buffer 1,3 bis[tris(hydroxymethyl)methylamino]propane. The term "HEPES" refers to the pH buffer 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. The term "MES" refers to the pH buffer 2-(N-morpholino)ethanesulfonic acid. The term "MOPS" refers to the pH buffer 3-(N-morpholino)propanesulfonic 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 piperazine-1,4-bis(2-ethanesulfonic acid), a pH buffering agent.

[0075] Introduction The present disclosure provides compacted oligonucleotides and methods of using compacted oligonucleotides for preparing nucleic acid nanostructures having compact sizes and shapes.

[0076] In some embodiments, each compacting oligonucleotide comprises two or more binding regions designed to hybridize to at least two regions of a nucleic acid molecule. In some embodiments, different binding regions of the compacting oligonucleotide are designed to hybridize to distal portions of the same nucleic acid molecule, drawing the distal portions together and causing compaction of the nucleic acid molecule to form a compact nanostructure. For example, but not limited to, different binding regions of the compacting oligonucleotide are designed to hybridize to distal portions of the same nucleic acid concatemer. In some embodiments, different binding regions of the compacting oligonucleotide hybridize to portions of universal adapter sequences located at distal positions on the same concatemer, drawing the distal portions together and causing the concatemer to form a DNA nanostructure with a more compact shape and size than a concatemer that is not hybridized to a compacting oligonucleotide.

[0077] In some embodiments, each compaction oligonucleotide contains two or more binding regions, each designed to hybridize to a region of two different nucleic acid molecules. In some embodiments, different binding regions of the compaction oligonucleotide are designed to hybridize to two different nucleic acid molecules and form a compact nanostructure. For example, but not limited to, different binding regions of the compaction oligonucleotide are designed to hybridize to a region 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, different binding regions of the compaction oligonucleotide hybridize to portions of universal adapter sequences on two different library molecules, forming DNA nanostructures with more compact shapes and sizes compared to library molecules not hybridized to the compaction oligonucleotide.

[0078] 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.

[0079] In some embodiments, the compacted oligonucleotide can include two, three, four, or more binding regions (e.g., Figures 1-13). In some embodiments, the compacted oligonucleotide can be modified to increase its resistance to exonuclease degradation.

[0080] In some embodiments, including a compacting oligonucleotide in a rolling circle amplification reaction can promote the formation of nanostructures with a denser size and shape compared to concatemers generated in the absence of the compacting oligonucleotide. The compact and stable characteristics of nucleic acid nanostructures improve sequencing accuracy by increasing signal intensity, and they retain their shape and size and resist disintegration over multiple sequencing cycles.

[0081] Rolling circle amplification (RCA) can be performed in the presence of compacting oligonucleotides to generate single-stranded concatemeric molecules having multiple copies of tandemly arranged polynucleotide units, each polynucleotide unit comprising a sequence of interest and at least one universal adapter sequence (e.g., at least one universal primer binding site). In some embodiments, individual compacting oligonucleotides hybridize to two or more regions of the same concatemeric molecule.

[0082] Rolling circle amplification (RCA) can be performed using compaction oligonucleotides to generate single-stranded concatemeric molecules containing multiple copies of tandemly arranged polynucleotide units, each containing a sequence of interest and at least one universal adapter sequence (e.g., at least one universal primer binding site). In some embodiments, the compaction oligonucleotides can have any shape (e.g., linear, branched, star, dendrimer, or other shape) and can contain two, three, four, or more binding regions (e.g., Figures 1-13). In some embodiments, the binding region of the compaction oligonucleotide is designed to hybridize to at least one universal adapter sequence in a linear library molecule.

[0083] In some embodiments, the binding region of the compacting oligonucleotide is designed to hybridize to at least one universal binding sequence in the concatemer molecule. In some embodiments, the binding region of the compacting oligonucleotide can be modified to increase resistance to exonuclease degradation. Different binding regions of the compacting oligonucleotide can be designed to hybridize to distal portions of the same concatemer molecule, pulling the distal portions together and causing the concatemer to compact to form a compact nanostructure. Inclusion of the compacting oligonucleotide in RCA can promote the formation of nanostructures with a more compact size and shape compared to concatemers generated in the absence of the compacting oligonucleotide. Without wishing to be bound by theory, it is hypothesized that the compact and stable characteristics of nucleic acid nanostructures improve sequencing accuracy, for example, by increasing signal intensity, and that the nanostructures retain their shape and size during multiple sequencing cycles.

[0084] As used herein, a "nanostructure" or "nucleic acid nanostructure" is a compacted concatemeric molecule, each nanostructure carrying multiple copies of a polynucleotide unit along its length. Each polynucleotide unit can bind to a sequencing primer, a sequencing polymerase, and a detectably labeled nucleotide reagent to form a detectable sequencing complex. Each nanostructure can bind to multiple detectable sequencing complexes. Thus, the compact size of a nanostructure can improve sequencing accuracy by increasing the local concentration of the detectably labeled nucleotide reagent used during the sequencing workflow, thereby increasing the signal intensity emitted from a given nanostructure and providing a distinct, detectable signal.

[0085] Massively parallel sequencing workflows typically involve multiple steps, including reagent flow, microscopic imaging of detectable sequencing complexes, washing of template strands, and repeating these steps for hundreds of cycles. DNA template molecules immobilized on a flow cell may be subjected to multiple reagent flow cycles, each intended to alter the reaction environment of the immobilized template molecules through changes in temperature, pH, salt, and enzymes. DNA template molecules may appear to unwind or shift position during later sequencing cycles, resulting in reduced base calling accuracy. The inclusion of compacting oligonucleotides in RCA generates stable, compact nanostructures. The nanostructures described in this disclosure resist unwinding and shifting and retain their compact shape and size throughout multiple sequencing cycles, thereby improving base calling accuracy in later sequencing cycles.

[0086] Using the compositions and methods described herein, compact nanostructures can be prepared that are immobilized at random locations on a flow cell at high densities. The compact nanostructures are tightly packed, filling most of the space on the flow cell. However, the compact nanostructures are distinguishable from their neighbors, facilitating the preparation of high-density nanostructures for high-throughput massively parallel nucleic acid sequencing. For example, fluorescently labeled nanostructures can be prepared at approximately 6 x 10 5 / mm 2 Even nanowells with a density of 1000 s can be imaged as distinct nanoballs and placed at random locations on the flow cell. The inclusion of compacted oligonucleotides in the RCA may eliminate the need to fabricate sequencing flow cells with organized, patterned arrays of nanowells.

[0087] In some embodiments, the compacted oligonucleotide may be a linear oligonucleotide molecule that can be adapted to be incorporated into existing library preparation workflows using rolling circle amplification. The first and second regions of the linear compacted oligonucleotide may be designed to hybridize to universal binding sequences within the concatemer molecule. The linear compacted oligonucleotide described herein may be simpler than the multi-branched dendrimers described in, for example, U.S. Patent No. 8,445,194.

[0088] In some embodiments, the workflow described herein does not require special conditions or additives to generate stable, compact nanostructures. For example, the workflow described herein does not require the addition of proteins (e.g., streptavidin or histones) to generate stable nanostructures. Inorganic cation Co(NH3)6 3+ Additives such as cellulose acetate, polyvinylpyrrolidone, or cationic liposomes are not required to generate stable nanostructures (see, e.g., DNA condensation in U.S. Pat. No. 9,982,293). These additives may interfere with downstream reactions, including DNA sequencing reactions.

[0089] composition The present disclosure provides a plurality of nucleic acid nanostructures, each comprising a nucleic acid concatemer molecule hybridized to at least one compacting oligonucleotide. In some embodiments, the nucleic acid nanostructures are in solution, immobilized on a support, or a mixture of nanostructures in solution and immobilized on a support.

[0090] In some embodiments, an individual compacting oligonucleotide comprises at least a first binding region capable of hybridizing to a first portion of a concatemer molecule, and the compacting oligonucleotide comprises at least a second binding region capable of hybridizing to a second portion of the concatemer molecule (e.g., the same concatemer molecule) (e.g., Figures 1-13). In some embodiments, the first and second regions of an individual compacting oligonucleotide can hybridize to two portions of the same concatemer, pulling distal portions of the concatemer together and causing compaction of the concatemer to form a nucleic acid nanostructure.

[0091] In some embodiments, hybridization of compacting oligonucleotides to individual concatemer molecules causes the concatemer molecules to collapse or fold into a nucleic acid nanostructure. In some embodiments, the nucleic acid nanostructure may include one or more loops, or may have a spherical shape (e.g., nanoball), an elongated shape (e.g., nanorod), a prototoroid shape, or a toroid shape (e.g., nanotoroid). A spot image of a nucleic acid nanostructure can be represented as a Gaussian spot, and its size can be measured as a full width half maximum (FWHM). A smaller spot size, indicated by a smaller FWHM, typically correlates with an improved image of the spot. In some embodiments, the FWHM of the nanostructure spot can be about 10 μm or less. The nucleic acid nanostructure can be a compact nucleic acid structure with a smaller full width half maximum (FWHM) compared to concatemers that have not collapsed / folded into a nanostructure.

[0092] In some embodiments, each compacted oligonucleotide in the plurality of compacted oligonucleotides comprises a nucleic acid and can have any shape, including linear, branched, star, or dendrimer shapes (e.g., bottle-brush shapes). In some embodiments, the compacted oligonucleotide can fold by forming intramolecular base pairs with the duplex portion via Watson-Crick base pairing, Hoogstein base pairing, and / or a G-quadruplex structure. In some embodiments, the compacted oligonucleotide comprises a nucleic acid that can fold into any shape, including at least one hairpin, at least one stem-loop, and / or at least one star shape.

[0093] In some embodiments, each compacted oligonucleotide in the plurality of compacted oligonucleotides comprises DNA, RNA, or a combination of DNA and RNA. The compacted oligonucleotides can be any length, including 20-200 nucleotides, 20-150 nucleotides, 30-100 nucleotides, 40-80 nucleotides, or any range therebetween.

[0094] In some embodiments, each compacted oligonucleotide comprises a linear nucleic acid having a first binding region and a second binding region, optionally with an intervening linker between the first and second binding regions (e.g., FIG. 1). In some embodiments, the first binding region of the compacted oligonucleotide hybridizes to a first portion of a concatemeric molecule. In some embodiments, the second binding region of the same compacted oligonucleotide hybridizes to a second portion of the same concatemeric 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 concatemeric 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 concatemeric molecule. In some embodiments, the first and second binding regions 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 compacting oligonucleotide is 5' to 3' or 3' to 5', hi some embodiments, the orientation of the second binding region of the compacting oligonucleotide is 5' to 3' or 3' to 5' (Figure 1).

[0095] 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)).

[0096] In some embodiments, the intervening linker of the compacted oligonucleotide is designed to be flexible. In some embodiments, the intervening linker of the compacted oligonucleotide is designed to be rigid. In some embodiments, the intervening linker of the compacted oligonucleotide comprises any one or any combination of nucleotides, nucleotide analogs, and / or non-nucleotide linkers. In some embodiments, the intervening linker of the compacted oligonucleotide exhibits little or no hybridization to any portion of the concatemeric molecule.

[0097] 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 linkers. In some embodiments, the first intervening linker is located between the first binding region and the second binding region. In some embodiments, the second intervening linker is located between the second binding region and the third binding region (e.g., Figures 2A-2C). In some embodiments, the first binding region of the compacted oligonucleotide hybridizes to a first portion of a concatemeric molecule. In some embodiments, the second binding region of the compacted oligonucleotide hybridizes to a second portion of the same concatemeric molecule. In some embodiments, the third binding region of the compacted oligonucleotide hybridizes to a third portion of the same concatemeric molecule.

[0098] 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)).

[0099] 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)).

[0100] 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)).

[0101] 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)).

[0102] 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 second binding region of the compacted oligonucleotide hybridizes to at least a portion of the 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 the third universal binding sequence in the same concatemer molecule.

[0103] In some embodiments, the first binding region, the second binding region, and the third binding region 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.

[0104] In some embodiments, the third binding region of the compacting oligonucleotide comprises the reverse sequence of the first binding region of the compacting oligonucleotide, hi some embodiments, the second binding region of the compacting oligonucleotide comprises a sequence that is the reverse of the first binding region.

[0105] In some embodiments, the intervening linker of the compacted oligonucleotide is designed to be flexible. In some embodiments, the intervening linker of the compacted oligonucleotide is designed to be rigid. In some embodiments, the intervening linker of the compacted oligonucleotide comprises any one or any combination of nucleotides, nucleotide analogs, and / or non-nucleotide linkers. In some embodiments, the intervening linker of the compacted oligonucleotide exhibits little or no hybridization to any portion of the concatemeric molecule.

[0106] In some embodiments, the compacted oligonucleotide comprises a star-shaped nucleic acid having a first binding region, an internal region, a second binding region, and optionally two intervening linkers. In some embodiments, the first intervening linker is located between the first binding region and the internal region. In some embodiments, the second intervening linker is located between the internal region and the second binding region (e.g., Figures 3A and 3B). In some embodiments, the first binding region of the compacted oligonucleotide hybridizes to a first portion of a concatemeric molecule. In some embodiments, the internal region of the compacted oligonucleotide hybridizes to a second portion of the same concatemeric molecule. In some embodiments, the second binding region of the compacted oligonucleotide hybridizes to a third portion of the same concatemeric molecule.

[0107] In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening linker and a first binding region arranged in a 5' to 3' orientation, with the 3' end of the first binding region facing away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 5' to 3' orientation, with the 3' end of the second binding region facing away from the internal intervening linker; and (3) an internal intervening linker and a third binding region arranged in a 5' to 3' orientation, with the 3' end of the third binding region facing away from the internal intervening linker (Figure 3A(i)).

[0108] In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening linker and a first binding region arranged in a 3' to 5' orientation, with the 5' end of the first binding region facing away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 3' to 5' orientation, with the 5' end of the second binding region facing away from the internal intervening linker; and (3) an internal intervening linker and a third binding region arranged in a 3' to 5' orientation, with the 5' end of the third binding region facing away from the internal intervening linker (Figure 3A(ii)).

[0109] In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening linker and a first binding region arranged in a 5' to 3' orientation, with the 3' end of the first binding region facing away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 5' to 3' orientation, with the 3' end of the second binding region facing away from the internal intervening linker; and (3) an internal intervening linker and a third binding region arranged in a 3' to 5' orientation, with the 5' end of the third binding region facing away from the internal intervening linker (Figure 3B(iii)).

[0110] In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening linker and a first binding region arranged in a 5' to 3' orientation, with the 3' end of the first binding region facing away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 3' to 5' orientation, with the 5' end of the second binding region facing away from the internal intervening linker; and (3) an internal intervening linker and a third binding region arranged in a 3' to 5' orientation, with the 5' end of the third binding region facing away from the internal intervening linker (Figure 3B(iv)).

[0111] 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 concatemeric 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 concatemeric 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 concatemeric molecule. In some embodiments, the first binding region, internal region, and second binding region of the compacted oligonucleotide comprise the same or different sequences. In some embodiments, the internal and second binding regions 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 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 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 compacting oligonucleotide comprises the reverse sequence of the first binding region of the compacting oligonucleotide, hi some embodiments, the internal region of the compacting oligonucleotide comprises a sequence that is the reverse of the first binding region.

[0112] In some embodiments, the intervening linker of the compacted oligonucleotide is designed to be flexible or rigid. In some embodiments, the intervening linker of the compacted oligonucleotide comprises any one or any combination of nucleotides, nucleotide analogs, and / or non-nucleotide linkers. In some embodiments, the intervening linker of the compacted oligonucleotide exhibits little or no hybridization to any portion of the concatemeric molecule.

[0113] In some embodiments, the compacted oligonucleotide comprises three binding arms, each binding arm comprising 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, with the 3' end of the second binding region oriented away from the internal intervening linker (Figure 4).

[0114] In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening linker and a first binding region arranged in a 5' to 3' direction, with the 3' end of the first binding region facing away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 5' to 3' direction, with the 3' end of the second binding region facing away from the internal intervening linker; (3) an internal intervening linker and a third binding region arranged in a 5' to 3' direction, with the 3' end of the third binding region facing away from the internal intervening linker; and (4) an internal intervening linker and a fourth binding region arranged in a 5' to 3' direction, with the 3' end of the fourth binding region facing away from the internal intervening linker (Figure 5(i)).

[0115] In some embodiments, the compacted oligonucleotide comprises: (1) an internal intervening linker and a first binding region arranged in a 3' to 5' orientation, with the 5' end of the first binding region facing away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 3' to 5' orientation, with the 5' end of the second binding region facing away from the internal intervening linker; (3) an internal intervening linker and a third binding region arranged in a 3' to 5' orientation, with the 5' end of the third binding region facing away from the internal intervening linker; and (4) an internal intervening linker and a fourth binding region arranged in a 3' to 5' orientation, with the 5' end of the fourth binding region facing away from the internal intervening linker (Figure 5(ii)).

[0116] In some embodiments, the compacted oligonucleotide comprises at least three binding arms. In some embodiments, the compacted oligonucleotide comprises multiple binding arms having the same sequence. In some embodiments, each binding arm comprises a first binding region arranged in a 5' to 3' orientation, with the 3' end of the first binding region facing away from the linker moiety. In some embodiments, each binding arm is connected to the linker moiety by an internal intervening linker (Figure 6A).

[0117] In some embodiments, the compacted oligonucleotide comprises at least three binding arms. In some embodiments, the compacted oligonucleotide comprises multiple binding arms having one of two different sequences. In some embodiments, each binding arm comprises a first binding region arranged in a 5' to 3' orientation, with the 3' end of the first binding region facing away from the linker moiety. In some embodiments, each binding arm comprises a second binding region arranged in a 5' to 3' orientation, with the 3' end of the second binding region facing away from the linker moiety. In some embodiments, each binding arm is connected to the linker moiety by an internal intervening linker (Figure 6B).

[0118] In some embodiments, the compacted oligonucleotide comprises at least three binding arms. In some embodiments, the compacted oligonucleotide comprises multiple binding arms having one of three different sequences. In some embodiments, each binding arm comprises a first binding region arranged in a 5' to 3' orientation, with the 3' end of the first binding region facing away from the linker moiety. In some embodiments, each binding arm comprises a second binding region arranged in a 5' to 3' orientation, with the 3' end of the second binding region facing away from the linker moiety. In some embodiments, each binding arm comprises a third binding region arranged in a 5' to 3' orientation, with the 3' end of the third binding region facing away from the linker moiety. In some embodiments, each binding arm is connected to the linker moiety by an internal intervening linker (Figure 6C).

[0119] 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).

[0120] 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 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 7B).

[0121] 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).

[0122] 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).

[0123] 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).

[0124] 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).

[0125] 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, a first intervening linker, a third binding region arranged in a 3' to 5' orientation, a second intervening linker, 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).

[0126] In some embodiments, the primary compaction oligonucleotide comprises three binding arms linked together by at least one internal intervening linker, each binding arm comprising a binding region. In some embodiments, the primary compaction oligonucleotide comprises: (1) an internal intervening linker and a first binding region arranged in a 5' to 3' orientation, with the 3' end of the first binding region oriented away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 5' to 3' orientation, with the 3' end of the second binding region oriented away from the internal intervening linker; and (3) an internal intervening linker and a connector region arranged in a 5' to 3' orientation, with the 3' end of the connector region oriented away from the internal intervening linker. 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 a primary compaction oligonucleotide can hybridize to the fastener region of a secondary compaction oligonucleotide (Figure 12A).

[0127] In some embodiments, the primary compaction oligonucleotide comprises three binding arms linked together by at least one internal intervening linker, each binding arm comprising a binding region. In some embodiments, the primary compaction oligonucleotide comprises: (1) an internal intervening linker and a first binding region arranged in a 3' to 5' orientation, with the 5' end of the first binding region oriented away from the internal intervening linker; (2) an internal intervening linker and a second binding region arranged in a 3' to 5' orientation, with the 5' end of the second binding region oriented away from the internal intervening linker; and (3) an internal intervening linker and a connector region arranged in a 3' to 5' orientation, with the 5' end of the connector region oriented away from the internal intervening linker. 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 a primary compaction oligonucleotide can hybridize to the fastener region of a secondary compaction oligonucleotide (Figure 12B).

[0128] In some embodiments, the first nucleic acid strand comprises a first binding region (110) arranged in a 5' to 3' orientation, an intervening linker, 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 linker, and a fourth binding region (220) arranged in a 3' to 5' orientation. In some embodiments, the third binding region (210) can hybridize to 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 to 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 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 or overlapping portions of the second binding region (120) (FIG. 13A).

[0129] In Figure 13B, a portion of the first binding region (110) of the first nucleic acid strand (100) hybridizes to a first portion of the concatemer, dissociating a portion of the third binding region (210) from the first binding region (110), as indicated by the two arrows. Hybridization of a portion of the first binding region (110) with 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 to the fourth binding region (220) and the fifth binding region (300).

[0130] In Figure 13C, a portion of the second binding region (120) of the first nucleic acid strand (100) hybridizes to a second portion of the concatemer, dissociating a portion of the fifth binding region (300) from the second binding region (120), as indicated by the two arrows. Hybridization of a portion of the second binding region (120) with 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).

[0131] In Figure 13D, the second nucleic acid strand (200) is completely dissociated from the first binding region (110) of the first nucleic acid strand (100), and the third nucleic acid strand (300) is completely dissociated from the second binding region (120) of the first nucleic acid strand (100).

[0132] In some embodiments, the intervening linker of any of the compacted oligonucleotides described herein can be any length, e.g., about 2 to 20 nucleotides in length. The intervening linker comprises a homopolymer having consecutive identical bases (e.g., AAA, GGG, CCC, TTT, or UUU). The intervening linker comprises a non-homopolymer sequence. In some embodiments, the intervening linker comprises at least one inosine. In some embodiments, the intervening linker comprises a homopolymer having consecutive identical bases (e.g., inosine).

[0133] 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 a spacer 9, including an 18-carbon spacer (e.g., including a hexa-ethylene glycol spacer), multiple C3 spacer phosphoramidites, or a trimethylene glycol spacer. In some embodiments, the spacer comprises a polyethylene glycol spacer, including a PEG2, PEG3, or PEG4 spacer.

[0134] In some embodiments, the intervening linker comprises at least one non-nucleotidic linker and at least one PEG spacer, in any configuration. For example, the intervening linker can be 5'-right arm-([PEG-spacer]-[C18-spacer]). n -left arm-3', and "n" is 1 to 10. In another example, the intervening linker is 5'-right arm-([C18-spacer]-[PEG-spacer]) n -Left Arm-3', and "n" is 1 to 10.

[0135] Any of the binding regions of the compacted oligonucleotide may be fully or partially complementary to a portion of the concatemeric molecule along its length, hi some embodiments, the binding region of the compacted oligonucleotide is designed to hybridize to a universal binding sequence in the concatemeric molecule.

[0136] 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 adapter sequence according to any one of SEQ ID NOs: 157-176, or a complementary sequence thereof (see Table 2).

[0137] 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 adapter sequence according to any one of SEQ ID NOs: 157-176, or a complementary sequence thereof (see Table 2).

[0138] In some embodiments, any binding region of the compacted oligonucleotide (e.g., the first, second, third, fourth, fifth, sixth, or other binding region) can hybridize to a portion of a concatemer molecule, where that 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).

[0139] In some embodiments, the compacted oligonucleotide comprises two or more binding regions, and all of the 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 of the binding regions have different sequences.

[0140] The first binding region of the compacted oligonucleotide may have the same sequence as the second binding region.

[0141] The first binding region of the compacted oligonucleotide may have a different sequence than the second binding region.

[0142] In some embodiments, the first binding region of the compacting oligonucleotide comprises a sequence according to any of SEQ ID NOs: 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).

[0143] In some embodiments, the second, third, fourth, fifth, or any subsequent binding region of the compacting oligonucleotide comprises a sequence that is the reverse sequence of the first binding region (e.g., a reverse sequence according to any one of SEQ ID NOs: 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).

[0144] In some embodiments, the first binding region of the compacting oligonucleotide comprises a sequence according to any of SEQ ID NOs: 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).

[0145] In some embodiments, the second, third, fourth, fifth, or any subsequent binding region of the compacting oligonucleotide comprises a sequence that is the reverse sequence of the first binding region, and the second, third, fourth, fifth, or any subsequent binding region comprises any one of SEQ ID NOs: 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).

[0146] In some embodiments, the first binding region of the compacting oligonucleotide may have a sequence that is the reverse sequence of the second binding region (e.g., a reverse sequence according to any one of SEQ ID NOs: 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).

[0147] In some embodiments, the second binding region of the compacting oligonucleotide comprises a sequence according to any of SEQ ID NOs: 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).

[0148] In some embodiments, the third binding region of the compacting oligonucleotide comprises a sequence according to any of SEQ ID NOs: 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).

[0149] In some embodiments, the fourth binding region of the compacting oligonucleotide comprises a sequence according to any of SEQ ID NOs: 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).

[0150] In some embodiments, the fifth binding region of the compacting oligonucleotide comprises a sequence according to any of SEQ ID NOs: 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).

[0151] In some embodiments, the subsequent binding region(s) of the compacting oligonucleotide comprise a sequence according to any of SEQ ID NOs: 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).

[0152] In some embodiments, the compacted oligonucleotide comprises a full-length sequence according to any one of SEQ ID NOs: 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).

[0153] In some embodiments, the terminal 3' region of either of the compacted oligonucleotides can include at least one additional base that includes one or more 2'-O-methyl RNA bases (e.g., designated mUmUmU), or the terminal 3' end lacks additional 2'-O-methyl RNA bases.

[0154] In some embodiments, the compacted oligonucleotide contains one or more modified bases or linkages at its 5'- or 3'-end to confer specific functionality. In some embodiments, the compacted oligonucleotide contains at least one phosphorothioate linkage at its 5'- and / or 3'-end to confer exonuclease resistance. In some embodiments, at least one nucleotide at or near the 3'-end contains a 2'-fluoro base, which confers exonuclease resistance. In some embodiments, the 3'-end of the compacted oligonucleotide contains at least one 2'-O-methyl RNA base that blocks polymerase-catalyzed extension. For example, the 3'-end of the compacted oligonucleotide contains at least one base that includes a 2'-O-methyl RNA base (e.g., designated mUmUmU). In some embodiments, the compacted oligonucleotide contains a 3' inverted dT at its 3'-end to block polymerase-catalyzed extension. In some embodiments, the compacted oligonucleotide contains a 3' phosphorylation that blocks polymerase-catalyzed extension. In some embodiments, the compacted oligonucleotide comprises at least one locked nucleic acid (LNA), which increases the thermal stability of the duplex formed by hybridizing the compacted oligonucleotide to the concatemeric molecule.

[0155] The compacted oligonucleotide may comprise at least one region (e.g., hybridization / binding region) with consecutive guanines. For example, the compacted oligonucleotide may comprise at least one region with 2, 3, 4, 5, or more consecutive guanines. In some embodiments, the compacted oligonucleotide comprises four consecutive guanines that can form a G-quadruplex structure (see Figure 64). The G-quadruplex structure may be stabilized via Hoogsteen hydrogen bonding. The G-quadruplex structure may be stabilized by a central cation including potassium, sodium, lithium, rubidium, or cesium.

[0156] At least one compacting oligonucleotide can form a G-quadruplex (Figure 64) and hybridize to the universal binding sequence in the concatemer, which allows the concatemer to fold and form an intramolecular G-quadruplex structure (Figure 65). The concatemer can self-collapse to form a compact nanostructure. The formation of G-quadruplexes and G-quadruplexes in the nanostructures can increase the stability of the nanostructures and retain their compact size and shape, which can withstand changes in pH, temperature, and / or repeated flow of reagents.

[0157] In some embodiments, the plurality of compacting oligonucleotides comprises the same sequence. In some embodiments, the plurality of compacting oligonucleotides comprises a sequence according to any one of SEQ ID NOs: 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, a plurality of compacted oligonucleotides comprise a sequence according to any one of SEQ ID NOs: 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 also comprises three bases including a 2'-O-methyl RNA base (e.g., designated mUmUmU).

[0158] In some embodiments, the plurality of compacted oligonucleotides comprises a mixture of two or more different populations of compacted oligonucleotides having different sequences, hi 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 of 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 NOs: 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.

[0159] In some embodiments, individual nucleic acid concatemers comprise single-stranded nucleic acid molecules. Individual concatemer molecules comprise at least two copies of a polynucleotide unit arranged in tandem. In some embodiments, each polynucleotide unit comprises a sequence of interest. In some embodiments, each polynucleotide unit comprises at least one universal adaptor sequence. In some embodiments, each polynucleotide unit comprises a sequence of interest and at least one universal adaptor sequence. In some embodiments, individual concatemer molecules comprise 2 to 100 copies of a polynucleotide unit, or 100 to 250 copies of a polynucleotide unit, or 250 to 500 copies of a polynucleotide unit, or 500 to 750 copies of a polynucleotide unit, or 750 to 1000 copies of a polynucleotide unit, or more than 1000 copies of a polynucleotide unit. In some embodiments, individual concatemer molecules comprise 1000 to 10,000 copies of a polynucleotide unit. In some embodiments, concatemer molecules can be generated by performing a rolling circle amplification reaction using circular library molecules as library template molecules, amplification primers (e.g., immobilized or soluble primers), an amplification polymerase (e.g., having strand displacement activity), and multiple nucleotides. In some embodiments, concatemers comprise multiple tandem polynucleotide units, and the sequence of each polynucleotide unit of a given concatemer molecule is complementary to the sequence of the circular library molecule that served as the template library molecule.

[0160] In some embodiments, concatemers can be generated by performing a rolling circle amplification reaction using a plurality of nucleotides containing any combination of two or more nucleotides, including dATP, dGTP, dCTP, dTTP, and / or dUTP. In some embodiments, each concatemer in the plurality of concatemer molecules contains at least two uracil bases distributed at random positions along each concatemer template molecule, and the uracil bases can be distributed at different positions in different concatemer molecules.

[0161] In some embodiments, the 5' or 3' ends of individual concatemers may be immobilized to a support or coating. In some embodiments, interior regions of individual concatemers may be immobilized to a support or coating. In some embodiments, the concatemers remain immobilized to a support or coating upon collapse / folding into the nanostructure.

[0162] In some embodiments, the coating comprises at least one hydrophilic polymer layer and a plurality of surface capture primers having sequences capable of hybridizing to at least a portion of the cyclic library molecules. The surface capture primers may be immobilized on the coating and / or embedded within the coating. The surface capture primers may be covalently attached to the monomer compounds that form the polymer layer. In some embodiments, the density of the surface capture primers is greater than 1 mm 2 Approximately 10 per 2 ~10 15 The coating may include a plurality of one or more types of surface capture primers. One type of surface capture primer may be used to perform an on-support rolling circle amplification workflow. Another type of surface capture primer may be used to perform an in-solution rolling circle amplification workflow.

[0163] In some embodiments, individual concatemer molecules can be generated by hybridizing circular library molecules with immobilized surface capture primers and performing an on-support rolling circle amplification reaction with an amplifying polymerase (e.g., having strand displacement activity) and a plurality of nucleotides to generate concatemers that are covalently linked to the surface primers immobilized or embedded in the coating (e.g., Figures 21-23, 39-40). The surface capture primers used to perform on-support rolling circle amplification can hybridize to at least a portion of the circular library molecules.

[0164] In some embodiments, individual concatemer molecules can be generated by hybridizing circular library molecules with soluble amplification primers and performing a solution-based rolling circle amplification reaction with an amplification polymerase (e.g., having strand displacement activity) and a plurality of nucleotides to generate duplexes containing concatemers hybridized to the circular library molecules. The duplexes can be distributed onto a support that includes at least a hydrophilic polymer layer and a plurality of surface capture primers having sequences that can hybridize to at least a portion of the concatemer molecules. A portion of the concatemers can hybridize to the surface capture primers to generate immobilized concatemers by hybridizing to the surface primers. The rolling circle amplification reaction can continue after distribution onto the coated support (e.g., Figures 30-32, 46-48).

[0165] 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 polymeric material.

[0166] In some embodiments, the surface of the support can be coated with one or more compounds to create a passivated layer on the support. In some embodiments, the passivated layer forms a porous or semi-porous layer. In some embodiments, individual concatemer molecules can be attached to the support or to the passivated layer to immobilize the concatemer molecules on the support. In some embodiments, the support comprises a low-nonspecific binding surface, which enables improved nucleic acid hybridization, amplification, and sequencing performance on the support. Generally, the support can comprise a covalently or non-covalently bound low-binding chemically modified layer, such as a silane layer, a polymer film, and one or more layers of one or more covalently or non-covalently bound oligonucleotides that can be used to immobilize multiple nucleic acid template molecules on the support. In some embodiments, the support can comprise, at least in part, a functionalized polymer coating layer covalently bound via chemical groups on the support, a primer grafted to 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-azidoacetamidylpentyl)acrylamide-co-acrylamide (PAZAM). In some embodiments, the support comprises a surface coating having at least one hydrophilic polymer coating layer. The surface coating may further comprise at least one layer of a plurality of oligonucleotides (e.g., a surface primer). The hydrophilic polymer coating layer may comprise polyethylene glycol (PEG). The hydrophilic polymer coating layer may comprise a branched PEG having at least four 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 is 45 degrees or less.

[0167] In some embodiments, the plurality of concatemeric molecules is immobilized to a support or to a coating on a support. 2Approximately 10 per 2 ~10 15 In some embodiments, the plurality of concatemers remains immobilized on the support when collapsed or folded into the nanostructure. Thus, the support may have a density of 1 mm 2 Approximately 10 per 2 ~10 15 The nanostructures are immobilized at a density of 1000 .mu.m.

[0168] In some embodiments, the concatemers are immobilized at different sites on the support. In some embodiments, multiple concatemer molecules are immobilized at predetermined sites (e.g., locations) on the support. Multiple concatemers can be arranged in an organized, predetermined pattern on the support. In some embodiments, multiple concatemer molecules are immobilized at random, non-predetermined sites (e.g., locations) on the support. In some embodiments, multiple concatemers remain immobilized on the support when they collapse or fold into nanostructures. Thus, multiple nanostructures can be immobilized on the support at predetermined sites on the support or random sites on the support.

[0169] In some embodiments, the multiple immobilized concatemeric molecules are in fluid communication with each other, allowing a solution of reagents (e.g., enzymes including polymerases, multivalent molecules, nucleotides, and / or divalent cations, etc.) to flow over the support such that the multiple immobilized concatemeric molecules on the support can react with the solution of reagents in a massively parallel manner.

[0170] In some embodiments, the multiple immobilized nanostructures are in fluid communication with one another, allowing a solution of reagents (e.g., enzymes including polymerases, multivalent molecules, nucleotides, and / or divalent cations, etc.) to flow over the support such that the multiple immobilized nanostructures on the support can react with the solution of reagents in a massively parallel manner.

[0171] In some embodiments, the plurality of immobilized 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 not hybridized to the immobilized nucleic acid nanostructures. In some embodiments, at least one of the immobilized nucleic acid nanostructures is hybridized to a circular nucleic acid library molecule. In some embodiments, the nucleic acid nanostructure comprises a plurality of tandem polynucleotide units, where the sequence of each polynucleotide unit of a given nanostructure molecule is complementary to the sequence of the circular library molecule.

[0172] In some embodiments, the plurality of immobilized nucleic acid nanostructures further comprise a plurality of circular nucleic acid library molecules, a plurality of amplification polymerases (e.g., having strand displacement activity), and a plurality of nucleotides. In some embodiments, the plurality of circular nucleic acid library molecules are soluble and not hybridized to the immobilized nucleic acid nanostructures. In some embodiments, at least one of the immobilized nucleic acid nanostructures is hybridized to the circular nucleic acid library molecule to form a nucleic acid amplification duplex having a template molecule (e.g., a circular library molecule) and a 3' primer start site (e.g., the 3' end of the nucleic acid nanostructure). In some embodiments, the nucleic acid amplification duplex binds with the amplification polymerase to form a multiplex amplification polymerase. In some embodiments, in the multiplex amplification polymerase, a complementary nucleotide can bind to the 3' primer start site at a position opposite the complementary nucleotide in the template molecule (e.g., the circular library molecule). In some embodiments, the multiplex nucleotides comprise any combination of two or more nucleotides, including dATP, dGTP, dCTP, dTTP, and / or dUTP.

[0173] In some embodiments, the plurality of immobilized 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 are not hybridized to the immobilized nucleic acid nanostructures. In some embodiments, at least one of the immobilized nucleic acid nanostructures is hybridized to 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 multiplexed sequencing polymerase. In some embodiments, the multiplexed sequencing polymerase is combined with a nucleotide reagent comprising a standard nucleotide, a nucleotide analog, or a multivalent molecule.

[0174] In some embodiments, a standard nucleotide can comprise an aromatic base, a five-carbon sugar, and at least one phosphate group. A standard nucleotide can be unlabeled or labeled with a detectable reporter moiety (e.g., a fluorophore). In some embodiments, a complementary standard nucleotide can be attached to the 3' primer initiation site at a position opposite the complementary nucleotide in the template molecule (e.g., nanostructure molecule). In some embodiments, the plurality of immobilized nucleic acid nanostructures further comprise catalytic divalent cations or non-catalytic divalent cations. Exemplary catalytic divalent cations include magnesium and / or manganese, which promote polymerase-catalyzed nucleotide incorporation. Exemplary non-catalytic divalent cations include strontium, barium, and / or calcium, which inhibit polymerase-catalyzed nucleotide incorporation.

[0175] In some embodiments, the nucleotide analog may comprise an aromatic base, a 5-carbon sugar having a 3' chain-terminating moiety that inhibits polymerase-catalyzed nucleotide incorporation, and at least one phosphate group. The nucleotide analog may be unlabeled or may be labeled with a detectable reporter moiety (e.g., a fluorophore). In some embodiments, the complementary nucleotide analog can bind to the 3' primer initiation site at a position opposite the complementary nucleotide in the template molecule (e.g., nanostructure molecule). In some embodiments, the plurality of immobilized nucleic acid nanostructures further comprise catalytic or non-catalytic divalent cations. Exemplary catalytic divalent cations include magnesium and / or manganese, which promote polymerase-catalyzed nucleotide incorporation. Exemplary non-catalytic divalent cations include strontium, barium, and / or calcium, which inhibit polymerase-catalyzed nucleotide incorporation.

[0176] In some embodiments, a multivalent molecule can comprise (1) a core and (2) a plurality of nucleotide arms, each of which comprises (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, the spacer is attached to the linker, and the linker is attached to the nucleotide unit (see Figures 56-59). In some embodiments, the nucleotide unit comprises an aromatic base, a five-carbon sugar, and at least one phosphate group, and the linker is attached to the nucleotide unit via the base. The multivalent molecule can be unlabeled or labeled with a detectable reporter moiety (e.g., a fluorophore). In some embodiments, the complementary nucleotide unit of the multivalent molecule can bind to a 3' primer initiation site at a position opposite the complementary nucleotide in the template molecule (e.g., nanostructure molecule). In some embodiments, the plurality of immobilized nucleic acid nanostructures further comprises catalytic or non-catalytic divalent cations. Exemplary catalytic divalent cations include magnesium and / or manganese, which promote polymerase-catalyzed nucleotide incorporation. Exemplary non-catalytic divalent cations include strontium, barium, and / or calcium, which inhibit polymerase-catalyzed nucleotide incorporation.

[0177] In some embodiments, the plurality of immobilized nucleic acid nanostructures further comprise a plurality of binding complexes comprising at least first and second binding complexes, wherein (i) the first binding complex comprises a first nucleic acid primer, a first polymerase, and a first multivalent molecule bound to a first portion of the nucleic acid nanostructure, thereby forming 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 bound to a second portion of the same nucleic acid nanostructure, thereby forming a second binding complex, wherein a second nucleotide unit of the multivalent molecule is bound to the second polymerase, and the first and second binding complexes comprising the same multivalent molecule form an avidity complex. In some embodiments, the first multivalent molecule is unlabeled or labeled with a detectable reporter moiety.

[0178] 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.

[0179] In some embodiments, the first nucleic acid primer comprises a first amplification primer and the second nucleic acid primer comprises a second amplification primer, hi some embodiments, the first polymerase comprises a first amplification polymerase and the second polymerase comprises a second amplification polymerase.

[0180] In some embodiments, the plurality of immobilized nucleic acid nanostructures further comprises a cellular biological sample located on the immobilized nanostructures.

[0181] In some embodiments, the cellular biological sample comprises a single cell, multiple cells, tissues, organs, organisms, or sections from any of these cellular biological samples. The cellular biological sample includes a fresh sample, a frozen sample, a fresh frozen sample, or an archived (e.g., formalin-fixed, paraffin-embedded, FFPE) sample. The cellular biological sample may be embedded in a matrix material. The cellular biological sample may be stained, destained, or unstained. The cellular biological sample may be permeabilized to allow nucleic acids within the cellular sample to transfer from the cell(s) to the multiple immobilized nanostructures.

[0182] On-support methods Generate high-density immobilized nanostructures The present disclosure provides a method for generating high density nucleic acid nanostructures immobilized on a support, 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 greater than or equal to 1 mm 2 Approximately 10 per 2 ~10 15and (b) generating a plurality of immobilized single-stranded nucleic acid concatemeric template molecules, wherein generating is performed 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 with (i) a plurality of strand-displacing polymerases, (ii) a plurality of nucleotides, and (iii) a plurality of compacting oligonucleotides, thereby generating a plurality of immobilized single-stranded nucleic acid concatemeric template molecules (Figures 21 and 39). In some embodiments, each compacting oligonucleotide comprises a single-stranded linear oligonucleotide having a 5' region capable of hybridizing to a first portion of a concatemer molecule and a compacting oligonucleotide having a 3' region capable of hybridizing to a second portion of the concatemer molecule (the same concatemer molecule) (Figures 23 and 40), wherein the plurality of immobilized concatemer molecules collapse or fold into a compact nucleic acid nanostructure upon binding to the compacting oligonucleotide, and the plurality of concatemers remain immobilized on the support as they collapse or fold into the nanostructure, thereby forming a nanostructure 1 mm immobilized on the support. 2 Approximately 10 per 2 ~10 15 This produces a support having a density of

[0183] In some embodiments, nucleic acid nanostructures may include one or more loops, or may have a spherical shape (e.g., a nanoball), an elongated shape (e.g., a nanorod), a prototoroid shape, or a toroid shape (e.g., a nanotoroid).

[0184] Inclusion of multiple compacted oligonucleotides in the on-support rolling circle amplification reaction can improve the FWHM (full width at half maximum) of the nanostructured spot image. The spot image can be represented as a Gaussian spot, and the size can be measured as the FWHM. A smaller spot size, indicated by a smaller FWHM, typically correlates with an improved image of the spot. In some embodiments, the FWHM of the nanostructured spot can be about 10 μm or less.

[0185] Nucleic acid nanostructures can be compact nucleic acid structures that have a smaller full width at half maximum (FWHM) compared to concatemers that have not collapsed / folded into nanostructures.

[0186] 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 in length, or 30-100 nucleotides in length, or 40-80 nucleotides in length, or any range therebetween.

[0187] In some embodiments, the compacted oligonucleotide comprises a first binding region, a second binding region, and optionally an intervening linker between the 5' and 3' regions. The intervening linker can be, for example, of any length, about 2 to 20 nucleotides in length. The intervening linker can comprise a homopolymer having consecutive identical bases (e.g., AAA, GGG, CCC, TTT, or UUU). The intervening linker can comprise a non-homopolymer sequence.

[0188] The first binding region of the compacting oligonucleotide may be fully or partially complementary to a first portion of the concatemer molecule along its length. The second binding region of the compacting oligonucleotide may be fully or partially complementary to a second portion of the concatemer molecule along its length. The first binding region of the compacting oligonucleotide can hybridize to a first universal sequence portion of a concatemer molecule having a sequence of any one of SEQ ID NOS: 157-176 (see Table 2). The second binding region of the compacting oligonucleotide can hybridize to a second universal sequence portion of a concatemer molecule having a sequence of any one of SEQ ID NOS: 157-176 (see Table 2). The first and second binding regions of the compacting oligonucleotide can hybridize to the concatemer and pull the distal portions of the concatemer together, causing compaction of the concatemer to form a nanostructure.

[0189] In some embodiments, any binding region of the compacted oligonucleotide (e.g., the first, second, third, fourth, fifth, sixth, or other binding region) can hybridize to a portion of a concatemer molecule, where that 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).

[0190] In some embodiments, in step (b), the first binding region of the compacting oligonucleotide comprises a sequence according to any of SEQ ID NOs: 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).

[0191] In some embodiments, in step (b), the second, third, fourth, fifth, or any subsequent binding region of the compacting oligonucleotide comprises a sequence that is the reverse sequence of the first binding region (e.g., a reverse sequence according to any one of SEQ ID NOs: 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).

[0192] In some embodiments, in step (b), the first binding region of the compacting oligonucleotide comprises a sequence according to any of SEQ ID NOs: 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).

[0193] 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, and the second, third, fourth, fifth, or any subsequent binding region is selected from the group consisting of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, , 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).

[0194] In some embodiments, in step (b), the first binding region of the compacted oligonucleotide may have a sequence that is the reverse sequence of the second binding region (e.g., a reverse sequence according to any one of SEQ ID NOs: 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).

[0195] In some embodiments, in step (b), the second binding region of the compacting oligonucleotide comprises a sequence according to any of SEQ ID NOs: 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).

[0196] In some embodiments, in step (b), the third binding region of the compacting oligonucleotide comprises a sequence according to any of SEQ ID NOs: 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).

[0197] In some embodiments, in step (b), the fourth binding region of the compacting oligonucleotide comprises a sequence according to any of SEQ ID NOs: 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).

[0198] In some embodiments, in step (b), the fifth binding region of the compacting oligonucleotide comprises a sequence according to any of SEQ ID NOs: 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).

[0199] In some embodiments, in step (b), the subsequent binding region(s) of the compacting oligonucleotide comprise a sequence according to any of SEQ ID NOs: 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).

[0200] In some embodiments, in step (b), the compacted oligonucleotide comprises a full-length sequence according to any one of SEQ ID NOs: 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).

[0201] In some embodiments, the 3' region of either of the compacted oligonucleotides can include an additional three bases at the 3' end that include 2'-O-methyl RNA bases (e.g., designated mUmUmU), or the 3' end lacks additional 2'-O-methyl RNA bases.

[0202] In some embodiments, the compacted oligonucleotide contains one or more modified bases or linkages at its 5'- or 3'-end to confer specific functionality. In some embodiments, the compacted oligonucleotide contains at least one phosphorothioate linkage at its 5'- and / or 3'-end to confer exonuclease resistance. In some embodiments, at least one nucleotide at or near the 3'-end contains a 2'-fluoro base, which confers exonuclease resistance. In some embodiments, the 3'-end of the compacted oligonucleotide contains at least one 2'-O-methyl RNA base that blocks polymerase-catalyzed extension. For example, the 3'-end of the compacted oligonucleotide can contain three bases containing a 2'-O-methyl RNA base (e.g., designated mUmUmU). In some embodiments, the compacted oligonucleotide contains a 3' inverted dT at its 3'-end to block polymerase-catalyzed extension. In some embodiments, the compacted oligonucleotide contains a 3' phosphorylation that blocks polymerase-catalyzed extension. In some embodiments, the internal region of the compacted oligonucleotide comprises at least one locked nucleic acid (LNA), which increases the thermal stability of the duplex formed by hybridizing the compacted oligonucleotide to the concatemeric molecule.

[0203] 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, or more consecutive guanines. In some embodiments, the compacted oligonucleotide comprises four consecutive guanines that can form a G-quadruplex structure (see Figure 64). The G-quadruplex structure may be stabilized via Hoogsteen hydrogen bonding. The G-quadruplex structure may be stabilized by a central cation including potassium, sodium, lithium, rubidium, or cesium.

[0204] A rolling circle amplification reaction can be performed in the presence of multiple compacted oligonucleotides with at least four consecutive guanines. The resulting concatemers contain repeated copies of the universal binding sequence for the compacted oligonucleotides. At least one compacted oligonucleotide can form a G-quadruplex (Figure 64) and hybridize to the universal binding sequence for the compacted oligonucleotide, and the resulting concatemers can fold and form an intramolecular G-quadruplex structure (Figure 65). The concatemers can self-collapse to form compact nanostructures. The formation of G-quadruplexes and G-quadruplexes in the nanostructures increases the stability of the nanostructures, allowing them to retain their compact size and shape, which can withstand changes in pH, temperature, and / or repeated flow of reagents.

[0205] In some embodiments, the plurality of compacted oligonucleotides in step (b) comprise the same sequence. In some embodiments, the plurality of compacted oligonucleotides in step (b) comprise a sequence according to any one of SEQ ID NOs: 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, a plurality of compacted oligonucleotides comprise a sequence according to any one of SEQ ID NOs: 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 also comprises three bases including a 2'-O-methyl RNA base (e.g., designated mUmUmU).

[0206] In some embodiments, the plurality of compacted oligonucleotides in step (b) comprises a mixture of two or more different populations of compacted oligonucleotides having different sequences, hi some embodiments, the plurality of compacted oligonucleotides in step (b) comprises a mixture of 2, 3, 4, 5, 6, 7, 8, 9, or 10 different populations of compacted oligonucleotides, wherein the compacted oligonucleotides of the different populations have different sequences. In some embodiments, in the mixture of different compacted oligonucleotides in step (b), any given population of compacted oligonucleotides comprises a sequence according to any one of SEQ ID NOs: 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.

[0207] The substrate may be passivated with at least one layer of a hydrophilic polymer coating that includes a plurality of first universal surface primers (Figure 54).

[0208] In some embodiments, the multiple immobilized first universal surface primers are positioned on the support or coating at random locations. When the immobilized universal surface primers are positioned on the support or coated support at random locations, the nucleic acid nanostructures are also positioned at random locations. The randomly positioned nucleic acid nanostructures can be tightly packed and fill most of the space on the coated support. When the immobilized nanostructures are bound to detectably labeled oligonucleotide or nucleotide reagents, the signal intensity can be concentrated into a smaller space, significantly increasing signal intensity and color differentiation compared to when the detectably labeled oligonucleotide or nucleotide reagents are bound to concatemers that have not collapsed / folded into the nanostructures. Thus, the nanostructures described herein and the methods used to generate them improve signal intensity without the need to prepare a support (e.g., a flow cell) with a predetermined, organized pattern array.

[0209] In some embodiments, a plurality of immobilized first universal surface primers are located on a support or coating at predetermined locations. For example, the immobilized first universal surface primers are arranged in an organized pattern. When the immobilized universal surface primers are located on a support or coated support at predetermined locations, nucleic acid nanostructures are also located at predetermined locations.

[0210] In some embodiments, the first universal surface primer lacks a cleavable moiety (e.g., the first universal surface primer lacks a cleavable moiety that can be converted to an abasic site), hi some embodiments, the first universal surface primer lacks a cleavable moiety that includes uridine, 8-oxo-7,8-dihydroguanine (e.g., 8oxoG), or deoxyinosine.

[0211] 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 cleavable moiety (e.g., the cleavable moiety in the nucleotide can be converted to an abasic site).

[0212] In some embodiments, the plurality of nucleotides used to perform the rolling circle amplification reaction includes dATP, dCTP, dGTP, dTTP, and a nucleotide having a cleavable moiety. In some embodiments, the nucleotide having a cleavable moiety includes uridine, 8-oxo-7,8-dihydroguanine (e.g., 8-oxoG), or deoxyinosine. The cleavable moiety in the nucleotide can be converted to an abasic site.

[0213] In some embodiments, a rolling circle amplification reaction is performed using nucleotides having cleavable moieties to generate a plurality of single-stranded nucleic acid concatemeric template molecules, each of which contains at least two nucleotides having a cleavable moiety distributed at random positions along each immobilized concatemeric template molecule (Figures 21-22). In some embodiments, the nucleotides having a cleavable moiety are distributed at different positions in different immobilized concatemeric template molecules.

[0214] In some embodiments, each concatemer template molecule in the plurality of concatemer template molecules comprises at least two copies of a polynucleotide unit arranged in tandem. In some embodiments, each polynucleotide unit comprises a sequence of interest. In some embodiments, each polynucleotide unit comprises at least one universal adaptor sequence. In some embodiments, each polynucleotide unit comprises a sequence of interest and at least one universal adaptor sequence. In some embodiments, each concatemer molecule comprises 2 to 100 copies of a polynucleotide unit, or 100 to 250 copies of a polynucleotide unit, or 250 to 500 copies of a polynucleotide unit, or 500 to 750 copies of a polynucleotide unit, or 750 to 1000 copies of a polynucleotide unit, or more than 1000 copies of a polynucleotide unit. In some embodiments, each concatemer molecule comprises 1000 to 2000 copies of a polynucleotide unit, or 1000 to 10,000 copies of a polynucleotide unit. In some embodiments, each concatemer comprises a plurality of tandem polynucleotide units, and the sequence of each polynucleotide unit of a given concatemer molecule is complementary to the sequence of the circular library molecule that served as the template library molecule.

[0215] In some embodiments, each concatemer molecule comprises two or more copies of a sequence of interest, and the immobilized concatemer template molecule comprises: (i) two or more copies of a universal binding sequence (or its complementary sequence) for a soluble forward sequencing primer; (ii) two or more copies of a universal binding sequence (or its complementary sequence) for a soluble reverse sequencing primer; (iii) two or more copies of a universal binding sequence (or its complementary sequence) for a first immobilized universal surface primer; (iv) two or more copies of a universal binding sequence (or its complementary sequence) for a second immobilized universal surface primer; (v) two or more copies of a universal binding sequence (or its complementary sequence) for the first soluble amplification primer; (vi) two or more copies of a universal binding sequence (or its complementary sequence) for the second soluble amplification primer; (vii) two or more copies of a universal binding sequence (or its complementary sequence) for the soluble compacting oligonucleotide; (viii) two or more copies of a sample barcode sequence; and / or (ix) two or more copies of a unique molecular index sequence.

[0216] In some embodiments, the method for generating high density nucleic acid nanostructures immobilized on a support further comprises positioning a cellular biological sample on the immobilized nanostructures.

[0217] In some embodiments, the cellular biological sample comprises a single cell, multiple cells, tissues, organs, organisms, or sections from any of these cellular biological samples. The cellular biological sample includes a fresh sample, a frozen sample, a fresh frozen sample, or an archived (e.g., formalin-fixed, paraffin-embedded, FFPE) sample. The cellular biological sample may be embedded in a matrix material. The cellular biological sample may be stained, destained, or unstained. The cellular biological sample may be permeabilized to allow nucleic acids within the cellular sample to transfer from the cell(s) to the multiple immobilized nanostructures.

[0218] On-support methods Generating high-density immobilized nanostructures containing cleavable moieties The present disclosure provides a method for generating a plurality of immobilized nucleic acid nanostructures, comprising: step (a): providing a support having a plurality of first universal surface primers immobilized thereon, the support being passivated with at least one hydrophilic polymer layer comprising a plurality of first universal surface primers, each of the first universal surface primers comprising a 3′ OH extendable end and lacking a nucleotide having a cleavable moiety, the density of the first universal surface primers being greater than or equal to 1 mm 2 Approximately 10 per 2 ~10 15 The method includes providing a first universal surface primer, wherein the first universal surface primer is a cleavable moiety that can be converted to an abasic site in a nucleic acid strand. For example, the first universal surface primer may lack uridine, 8-oxo-7,8-dihydroguanine (e.g., 8-oxoG), 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 and second universal surface primers.

[0219] In some embodiments, the immobilized first universal surface primers comprise single-stranded oligonucleotides comprising DNA, RNA, or a combination of DNA and RNA. The first universal surface primers may comprise sequences along their length that are fully or partially complementary to at least a portion of the circular nucleic acid library molecules. The first universal surface primers may comprise a terminal 3' nucleotide having a sugar 3' OH moiety that is extendible for nucleotide polymerization (e.g., polymerase-catalyzed polymerization).

[0220] In some embodiments, the immobilized first universal surface primer may be immobilized to a support or to a coating on the support. The immobilized first universal surface primer may be embedded or attached (coupled) to a coating on the support. In some embodiments, the 5' end of the immobilized first universal surface primer is immobilized to the support or to a coating on the support. Alternatively, the internal portion or 3' end of the immobilized first universal surface primer may be immobilized to the support or to a coating on the support. The support may contain multiple immobilized first universal surface primers having the same sequence. The immobilized first universal surface primer may be any length, for example, 4 to 50 nucleotides, or 50 to 100 nucleotides, or 100 to 150 nucleotides in length, or longer, or any range therebetween.

[0221] In some embodiments, the multiple immobilized first universal surface primers are located at random positions (e.g., non-predetermined positions) on the coated support. When the immobilized universal surface primers are located at random positions on the coated support, the immobilized nucleic acid nanostructures are also located at random positions. The randomly located nucleic acid nanostructures can be tightly packed and fill most of the space on the coated support.

[0222] In some embodiments, a plurality of immobilized first universal surface primers are located on the coated support at predetermined locations. For example, the immobilized first universal surface primers are arranged in an organized pattern. When the immobilized universal surface primers are located on the support or coated support at predetermined locations, the nucleic acid nanostructures are also located at predetermined locations.

[0223] In some embodiments, the plurality of immobilized first universal surface primers comprise at least one phosphorothioate diester bond at their 5' ends, which can render the first universal surface primers resistant to exonuclease degradation. In some embodiments, the plurality of immobilized first universal surface primers comprise 2 to 5 or more consecutive phosphorothioate diester bonds at their 5' ends. In some embodiments, the plurality of immobilized 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 render the first universal surface primers resistant to exonuclease degradation.

[0224] In some embodiments, the immobilized first universal surface primer comprises at least one locked nucleic acid (LNA), wherein the at least one LNA comprises a methylene bridge bond between the 2' oxygen and the 4' carbon of the pentose ring. The immobilized first universal surface primer comprising at least one LNA can be resistant to nuclease digestion and can exhibit an increased melting temperature when hybridized to a circular nucleic acid library molecule.

[0225] In some embodiments, the support further comprises a plurality of second universal surface primers immobilized thereon (Figure 29). The second universal surface primers have a different sequence from the immobilized first universal surface primers. The immobilized second immobilized primers of step (a) comprise single-stranded oligonucleotides comprising DNA, RNA, or a combination of DNA and RNA. The second universal surface primers comprise sequences along their length that are fully complementary or partially complementary to at least a portion of the immobilized single-stranded concatemer template molecules. The immobilized second universal surface primers may be immobilized to the support or to a coating on the support. The immobilized second universal surface primers may be embedded or attached (coupled) to a coating on the support. In some embodiments, the 5' end of the second universal surface primer is immobilized to the support or to a coating on the support. Alternatively, the inner portion or 3' end of the immobilized second universal surface primer may be immobilized to the support or to a coating on the support. The support may contain multiple immobilized second universal surface primers having the same sequence. The immobilized second universal surface primers can be any length, for example, 4 to 50 nucleotides, 50 to 100 nucleotides, 100 to 150 nucleotides, or longer. In some embodiments, the 3' end of the immobilized second universal surface primer contains an extendable 3'OH moiety. In some embodiments, the 3' end of the immobilized second universal surface primer contains a 3' non-extendable moiety. The 3' end of the immobilized second universal surface primer may contain a moiety that blocks primer extension, such as a phosphate group, a dideoxycytidine group, an inverted dT, or an amino group. These immobilized second universal surface primers are not extendable in a primer extension reaction. The immobilized second universal surface primer may lack a nucleotide with a cleavable moiety.

[0226] In some embodiments, the plurality of immobilized second universal surface primers comprise at least one phosphorothioate diester bond at their 5' ends, which can render the second universal surface primers resistant to exonuclease degradation. In some embodiments, the plurality of immobilized second universal surface primers comprise 2 to 5 or more consecutive phosphorothioate diester bonds at their 5' ends. In some embodiments, the plurality of immobilized 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 render the second universal surface primers resistant to exonuclease degradation.

[0227] In some embodiments, each immobilized single-stranded nucleic acid concatemer template molecule is covalently linked to an immobilized first universal surface primer, and at least a portion of each concatemer template molecule hybridizes to an immobilized second universal surface primer (Figure 29). The immobilized second universal surface primer can function to hold a portion of the immobilized concatemer template molecule to the support. In some embodiments, the immobilized concatemer template molecule has two or more copies of the universal binding sequence for the immobilized second universal surface primer. The portion of the immobilized concatemer template molecule containing the universal binding sequence for the immobilized second universal surface primer can hybridize to the immobilized second universal surface primer. In some embodiments, the second universal surface primers contain a terminal 3' blocking group that renders them non-extendible. In some embodiments, the second universal surface primers have a terminal 3' extendable end.

[0228] In some embodiments, the support is 1 mm 2 Approximately 10 per 2 ~10 15 In some embodiments, the support comprises 1 mm of immobilized first universal surface primers. 2 Approximately 10 per 2 ~10 15 In some embodiments, the support comprises 1 mm 2 Approximately 10 per 2 ~10 15 The immobilized universal surface primer comprises an immobilized first universal surface primer and an immobilized second universal surface primer.

[0229] The immobilized surface primers (e.g., first and second universal surface primers) are in fluid communication with one another, allowing various solutions, such as linear or circular nucleic acid template molecules, soluble primers, enzymes, nucleotides, divalent cations, buffers, reagents, etc., to flow over the support, thereby allowing the multiple immobilized surface primers (and primer extension products generated from the immobilized surface primers) to react with the solutions in a massively parallel manner.

[0230] In some embodiments, the method for generating a plurality of immobilized nucleic acid nanostructures further includes step (b): generating a plurality of immobilized single-stranded nucleic acid concatemeric template molecules by hybridizing the 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 with (i) a plurality of strand-displacing polymerases, (ii) a plurality of nucleotides including dATP, dCTP, dGTP, dTTP, and a nucleotide having a cleavable moiety, and (iii) a plurality of compacting oligonucleotides, whereby each concatemeric template molecule is covalently linked to the immobilized first universal surface primer, thereby generating a plurality of immobilized single-stranded nucleic acid concatemeric template molecules (e.g., Figures 22-23). ​​In some embodiments, each compacting oligonucleotide comprises a single-stranded linear oligonucleotide having a first binding region capable of hybridizing to a first portion of the concatemeric molecule and a compacting oligonucleotide having a second binding region capable of hybridizing to a second portion of the concatemeric molecule. In some embodiments, individual immobilized concatemer molecules collapse or fold into compact nucleic acid nanostructures (e.g., first strand nanostructures). In some embodiments, multiple concatemers remain immobilized on the support as they collapse or fold into nanostructures, thereby reducing the number of nanostructures immobilized on the support. 2 Approximately 10 per 2 ~about 10 15 In some embodiments, each immobilized nanostructure comprises a plurality of tandem polynucleotide units, each polynucleotide unit having a sequence that is complementary to the sequence of a covalently closed circular nucleic acid library molecule.

[0231] The multiple immobilized nucleic acid nanostructures can be in fluid communication with one another, allowing various reagents in solution, including soluble primers, enzymes, nucleotides, divalent cations, buffers, etc., to flow over the support, causing the multiple immobilized nanostructures to react with the solution in a massively parallel manner.

[0232] In some embodiments, in step (b), the first binding region of the compacted oligonucleotide can hybridize to a 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).

[0233] In some embodiments, in step (b), 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 adapter sequence according to any one of SEQ ID NOs: 157-176, or a complementary sequence thereof (see Table 2).

[0234] In some embodiments, in step (b), the compacted oligonucleotide comprises a first, second, third, fourth, fifth, sixth, or other binding region.

[0235] In some embodiments, in step (b), any binding region of the compacted oligonucleotide (e.g., the first, second, third, fourth, fifth, sixth, or other binding region) can hybridize to a portion of a concatemer molecule, where that 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).

[0236] In some embodiments, in step (b), the compacted oligonucleotide comprises two or more binding regions, all of which 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 of the binding regions have different sequences.

[0237] In some embodiments, in step (b), the first binding region of the compacted oligonucleotide may have the same sequence as the second binding region.

[0238] In some embodiments, in step (b), the first binding region of the compacted oligonucleotide may have a different sequence than the second binding region.

[0239] In some embodiments, in step (b), the first binding region of the compacting oligonucleotide comprises a sequence according to any of SEQ ID NOs: 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).

[0240] In some embodiments, in step (b), the second, third, fourth, fifth, or any subsequent binding region of the compacting oligonucleotide comprises a sequence that is the reverse sequence of the first binding region (e.g., a reverse sequence according to any one of SEQ ID NOs: 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).

[0241] In some embodiments, in step (b), the first binding region of the compacting oligonucleotide comprises a sequence according to any of SEQ ID NOs: 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).

[0242] 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, and the second, third, fourth, fifth, or any subsequent binding region is selected from the group consisting of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, , 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).

[0243] In some embodiments, in step (b), the first binding region of the compacted oligonucleotide may have a sequence that is the reverse sequence of the second binding region (e.g., a reverse sequence according to any one of SEQ ID NOs: 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).

[0244] In some embodiments, in step (b), the second binding region of the compacting oligonucleotide comprises a sequence according to any of SEQ ID NOs: 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).

[0245] In some embodiments, in step (b), the third binding region of the compacting oligonucleotide comprises a sequence according to any of SEQ ID NOs: 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).

[0246] In some embodiments, in step (b), the fourth binding region of the compacting oligonucleotide comprises a sequence according to any of SEQ ID NOs: 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).

[0247] In some embodiments, in step (b), the fifth binding region of the compacting oligonucleotide comprises a sequence according to any of SEQ ID NOs: 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).

[0248] In some embodiments, in step (b), the subsequent binding region(s) of the compacting oligonucleotide comprise a sequence according to any of SEQ ID NOs: 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).

[0249] In some embodiments, in step (b), the compacted oligonucleotide comprises a full-length sequence according to any one of SEQ ID NOs: 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).

[0250] In some embodiments, in step (b), the 3' region of any of the compacted oligonucleotides may include at least one additional base that includes one or more 2'-O-methyl RNA bases (e.g., designated mUmUmU), or the terminal 3' end lacks additional 2'-O-methyl RNA bases.

[0251] In some embodiments, in step (b), the compacted oligonucleotide comprises one or more modified bases or linkages at its 5'-end or 3'-end to confer specific functionality. In some embodiments, the compacted oligonucleotide comprises at least one phosphorothioate linkage at its 5'-end and / or 3'-end to confer exonuclease resistance. In some embodiments, at least one nucleotide at or near the 3'-end comprises a 2'-fluoro base, which confers exonuclease resistance. In some embodiments, the 3'-end of the compacted oligonucleotide comprises at least one 2'-O-methyl RNA base that blocks polymerase-catalyzed extension. For example, the 3'-end of the compacted oligonucleotide comprises at least one base that includes a 2'-O-methyl RNA base (e.g., designated mUmUmU). In some embodiments, the compacted oligonucleotide comprises a 3' inverted dT at its 3'-end to block polymerase-catalyzed extension. In some embodiments, the compacted oligonucleotide comprises a 3' phosphorylation that blocks polymerase-catalyzed extension. In some embodiments, the compacted oligonucleotide comprises at least one locked nucleic acid (LNA), which increases the thermal stability of the duplex formed by hybridizing the compacted oligonucleotide to the concatemeric molecule.

[0252] In some embodiments, in step (b), the compacted oligonucleotide may contain at least one region (e.g., hybridization / binding region) with consecutive guanines. For example, the compacted oligonucleotide may contain at least one region with 2, 3, 4, 5, or more consecutive guanines. In some embodiments, the compacted oligonucleotide contains four consecutive guanines that can form a G-quadruplex structure (see Figure 64). The G-quadruplex structure may be stabilized through Hoogsteen hydrogen bonding. The G-quadruplex structure may be stabilized by a central cation including potassium, sodium, lithium, rubidium, or cesium.

[0253] In some embodiments, in step (b), at least one compacting oligonucleotide can form a G-quadruplex (Figure 64) and hybridize to a universal binding sequence in the concatemer, which can cause the concatemer to fold and form an intramolecular G-quadruplex structure (Figure 65). The concatemer can self-collapse to form a compact nanostructure. The formation of G-quadruplexes and G-quadruplexes in the nanostructures can increase the stability of the nanostructures and retain their compact size and shape, which can withstand changes in pH, temperature, and / or repeated flow of reagents.

[0254] In some embodiments, the plurality of compacted oligonucleotides in step (b) comprises a mixture of two or more different populations of compacted oligonucleotides having different sequences, hi some embodiments, the plurality of compacted oligonucleotides in step (b) comprises a mixture of 2, 3, 4, 5, 6, 7, 8, 9, or 10 different populations of compacted oligonucleotides, wherein the compacted oligonucleotides of the different populations have different sequences. In some embodiments, in the mixture of different compacted oligonucleotides in step (b), any given population of compacted oligonucleotides comprises a sequence according to any one of SEQ ID NOs: 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.

[0255] In some embodiments, in the method for generating a plurality of immobilized nucleic acid nanostructures in step (b), the nucleic acid nanostructures may include one or more loops, or may have a spherical shape (e.g., nanoballs), elongated shape (e.g., nanorods), prototoroid shape, or toroid shape (e.g., nanotoroids). The nucleic acid nanostructures may be compact nucleic acid structures with a smaller full width at half maximum (FWHM) compared to concatemers that have not collapsed / folded into nanostructures. Inclusion of multiple compacting oligonucleotides in an on-support rolling circle amplification reaction can improve the FWHM (full width at half maximum) of nanostructure spot images. The spot images may be represented as Gaussian spots, and the size may be measured as FWHM. A smaller spot size, indicated by a smaller FWHM, typically correlates with an improved image of the spot. In some embodiments, the FWHM of the nanostructure spots may be about 10 μm or less.

[0256] In some embodiments, in the method for generating a plurality of immobilized nucleic acid nanostructures in step (b), each single-stranded covalently closed circular nucleic acid library molecule in the plurality of single-stranded covalently closed circular nucleic acid library molecules contains a sequence of interest as well as (i) a universal binding sequence (or a complementary sequence thereof) for a soluble forward sequencing primer, (ii) a universal binding sequence (or a complementary sequence thereof) for a soluble reverse sequencing primer, (iii) a universal binding sequence (or a complementary sequence thereof) for an immobilized first universal surface primer, (iv) a universal binding sequence (or a complementary sequence thereof) for an immobilized second universal surface primer, (v) a universal binding sequence (or its complementary sequence) for the first soluble amplification primer; (vi) a universal binding sequence (or its complementary sequence) for the second soluble amplification primer; (vii) a universal binding sequence (or its complementary sequence) for the soluble compacted oligonucleotide; (viii) a sample barcode sequence; and / or (ix) a unique molecular index sequence, or any combination of two or more thereof (e.g., Figures 18A and 18B, 20A and 20B).

[0257] In some embodiments, in the method for generating a plurality of immobilized nucleic acid nanostructures in step (b), the rolling circle amplification reaction generates a plurality of immobilized single-stranded nucleic acid concatemeric template molecules (e.g., first strand nanostructures), each of which contains at least two copies of a polynucleotide unit arranged in tandem. In some embodiments, each polynucleotide unit contains a sequence of interest. In some embodiments, each polynucleotide unit contains at least one universal adaptor sequence. In some embodiments, each polynucleotide unit contains a sequence of interest and at least one universal adaptor sequence (e.g., Figure 21). In some embodiments, each concatemeric molecule contains 2 to 100 copies of a polynucleotide unit, or 100 to 250 copies of a polynucleotide unit, or 250 to 500 copies of a polynucleotide unit, or 500 to 750 copies of a polynucleotide unit, or 750 to 1000 copies of a polynucleotide unit, or more than 1000 copies of a polynucleotide unit. In some embodiments, each concatemer molecule contains 1,000 to 10,000 copies of a polynucleotide unit. In some embodiments, each concatemer molecule contains multiple tandem polynucleotide units, and the sequence of each polynucleotide unit in a given concatemer molecule is complementary to the sequence of the circular library molecule that served as the template library molecule.

[0258] In some embodiments, each concatemer molecule comprises two or more copies of a sequence of interest, and the immobilized concatemer template molecule comprises: (i) two or more copies of a universal binding sequence (or its complementary sequence) for a soluble forward sequencing primer; (ii) two or more copies of a universal binding sequence (or its complementary sequence) for a soluble reverse sequencing primer; (iii) two or more copies of a universal binding sequence (or its complementary sequence) for a first immobilized universal surface primer; (iv) two or more copies of a universal binding sequence (or its complementary sequence) for a second immobilized universal surface primer; (v) two or more copies of a universal binding sequence (or its complementary sequence) for the first soluble amplification primer; (vi) two or more copies of a universal binding sequence (or its complementary sequence) for the second soluble amplification primer; (vii) two or more copies of a universal binding sequence (or its complementary sequence) for the soluble compacting oligonucleotide; (viii) two or more copies of a sample barcode sequence; and / or (ix) two or more copies of a unique molecular index sequence.

[0259] In some embodiments, in the method for generating a plurality of immobilized nucleic acid nanostructures in step (b), the rolling circle amplification reaction generates concatemer molecules containing universal binding sequences that can hybridize / bind to specific primers. In some embodiments, the universal binding sequence (or its complementary sequence) for the forward sequencing primer can hybridize to at least a portion of the forward sequencing primer. In some embodiments, the universal binding sequence (or its complementary sequence) for the reverse sequencing primer can hybridize to at least a portion of the reverse sequencing primer. In some embodiments, the universal binding sequence (or its complementary sequence) for the immobilized first universal surface primer can hybridize to at least a portion of the immobilized first universal surface primer. In some embodiments, the universal binding sequence (or its complementary sequence) for the immobilized second universal surface primer can hybridize to at least a portion of the immobilized second universal surface primer. In some embodiments, the universal binding sequence for a first soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the first soluble amplification primer. In some embodiments, the universal binding sequence for a second soluble amplification primer (or its complementary sequence) can hybridize to at least a portion of the second soluble amplification primer. In some embodiments, the universal binding sequence for a soluble compacted oligonucleotide (or its complementary sequence) can hybridize to at least a portion of the soluble compacted oligonucleotide.

[0260] In some embodiments, in the method for generating a plurality of immobilized nucleic acid nanostructures in step (b), the plurality of immobilized single-stranded nucleic acid concatemer template molecules (e.g., first-stranded nanostructures) contain two or more copies of a universal binding sequence (or its complementary sequence) for an immobilized second-sequence surface primer. In some embodiments, each immobilized single-stranded nucleic acid concatemer template molecule is linked (e.g., covalently linked) to an immobilized first universal surface primer, and at least a portion of each concatemer template molecule hybridizes to an immobilized second universal surface primer. The immobilized second universal surface primer can function to hold a portion of the immobilized concatemer template molecule to the support (see Figure 29). In some embodiments, the second universal surface primers contain a terminal 3' blocking group that renders them non-extendable.

[0261] 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 using a nucleotide mixture containing dATP, dCTP, dGTP, dTTP, and a nucleotide having a cleavable moiety to generate immobilized concatemeric template molecules (e.g., first strand nanostructures) comprising at least one nucleotide having a cleavable moiety (e.g., Figures 21-22). The cleavable moiety in the immobilized concatemeric template molecule can be converted to an abasic site. In some embodiments, the nucleotide having a cleavable moiety in the nucleotide mixture comprises uridine, 8-oxo-7,8-dihydroguanine (e.g., 8-oxoG), or deoxyinosine. In the immobilized concatemeric template molecule, uridine can be converted to an abasic site using uracil DNA glycosylase (UDG), 8-oxoG can be converted to an abasic site using FPG glycosylase, and deoxyinosine can be converted to an abasic site using 3-methyladenine DNA glycosylase II (AlkA) glycosylase.

[0262] In some embodiments, the nucleotide mixture can contain a certain amount of dUTP so 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 replaced with dUTP (e.g., 30% is the target percentage), the nucleotide mixture can 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 replaced by dUTP can be about 0.1-1%, or about 1-5%, or about 5-10%, or about 10-20%, or about 20-30%, or about 30-45%, or about 45-50%, or higher, of the dTTP in the immobilized concatemer template molecules replaced with a nucleotide having a cleavable moiety.

[0263] In some embodiments, the nucleotide mixture can contain a certain amount of deoxyinosine so that a target percentage of guanosines in the resulting concatemeric molecules are replaced with deoxyinosine. For example, when 30% of the dGTP in the concatemeric molecules are replaced with deoxyinosine (e.g., 30% is the target percentage), the nucleotide mixture can 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 replaced by deoxyinosine can be about 0.1-1%, or about 1-5%, or about 5-10%, or about 10-20%, or about 20-30%, or about 30-45%, or about 45-50%, or higher, of the dGTP in the immobilized concatemeric template molecules replaced with nucleotides having a cleavable moiety.

[0264] In some embodiments, the nucleotide mixture can include an amount of 8-oxoG such that a target percentage of guanosines in the resulting concatemeric molecules are replaced with 8-oxoG. For example, when 30% of the dGTP in the concatemeric molecules are replaced with 8-oxoG (e.g., 30% is the target percentage), the nucleotide mixture can contain 7.5% 8-oxoG (e.g., 30 / 4 = 7.5%), 17.5% dGTP, and 25% each of dATP, dCTP, and dTTP. The target percentage of dGTP replaced by 8-oxoG can be about 0.1-1%, or about 1-5%, or about 5-10%, or about 10-20%, or about 20-30%, or about 30-45%, or about 45-50%, or a higher percentage of the dGTP in the immobilized concatemeric template molecules replaced with the nucleotide having a cleavable moiety.

[0265] In some embodiments, in the method for generating a plurality of immobilized nucleic acid nanostructures in step (b), the rolling circle amplification reaction generates a plurality of single-stranded nucleic acid concatemeric template molecules (e.g., first strand nanostructures), each of which has at least two nucleotides each having a cleavable moiety distributed at random positions along each of the immobilized concatemeric template molecules. In some embodiments, the nucleotides having the cleavable moieties are distributed at different positions in different immobilized concatemeric template molecules.

[0266] In some embodiments, in the method for generating a plurality of immobilized nucleic acid nanostructures in step (b), the method may further include removing the single-stranded covalently closed circular nucleic acid library molecules from the concatemeric template molecules using at least one wash step performed under conditions suitable to retain the single-stranded nucleic acid concatemeric template molecules, with each concatemeric template molecule operably linked to the immobilized first universal surface primer.

[0267] In some embodiments, the method for generating a plurality of immobilized nucleic acid nanostructures further comprises step (c): sequencing a plurality of immobilized concatemeric template molecules (e.g., first strand nanostructures) to thereby generate a plurality of extended forward sequencing primer strands. The sequencing step (c) may further comprise contacting a plurality of immobilized concatemeric 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 concatemeric template molecules, and performing a forward sequencing reaction to generate a plurality of forward sequencing products using one or more types of sequencing polymerase, a plurality of nucleotide reagents, and the hybridized first forward sequencing primer (Figure 23). In some embodiments, the plurality of nucleotide reagents are detectably labeled. In some embodiments, the sequencing step (c) further comprises detecting and imaging the plurality of forward sequencing products.

[0268] In some embodiments, the sequencing step (c) further comprises contacting a plurality of immobilized concatemeric template molecules with a plurality of compacting oligonucleotides under conditions suitable for hybridizing the 5' end of at least one compacting oligonucleotide to a first portion of the concatemeric molecule and hybridizing a second bond of the same compacting oligonucleotide to a second portion of the same concatemeric molecule. In some embodiments, the plurality of compacting oligonucleotides in step (c) comprises any of the sequences described in step (b) above.

[0269] In some embodiments, in the sequencing 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 generate a 3' OH extendable end. In some embodiments, the soluble forward sequencing primer lacks a nucleotide with a cleavable portion. The forward sequencing reaction can generate multiple extended forward sequencing primer strands. In some embodiments, each immobilized concatemer template molecule has multiple copies of the forward sequencing primer binding site, and each forward sequencing primer binding site is capable of hybridizing to a first forward sequencing primer. Each forward sequencing primer binding site in a given immobilized concatemer template molecule can hybridize to a forward sequencing primer, and the sequencing reaction can be performed. Two or more sequencing reactions may be performed on each immobilized concatemeric template molecule, with each sequencing reaction initiated from a first forward sequencing primer hybridized to the forward sequencing primer binding site (see, e.g., Figure 23).

[0270] In some embodiments, in the sequencing step (c), the nucleotide reagents comprise a plurality of nucleotides, a plurality of nucleotide analogs, or a plurality of multivalent molecules.

[0271] In some embodiments, in sequencing step (c), each nucleotide in the plurality of nucleotides comprises an aromatic base, a 5-carbon sugar, and at least one phosphate group. In some embodiments, the plurality of nucleotides is unlabeled. In some embodiments, at least one nucleotide in the plurality of nucleotides can be labeled with a detectable reporter moiety (e.g., a fluorophore). In some embodiments, sequencing step (c) further comprises contacting the plurality of immobilized concatemeric template molecules with catalytic divalent cations or non-catalytic divalent cations. Exemplary catalytic divalent cations include magnesium and / or manganese, which promote polymerase-catalyzed nucleotide incorporation. Exemplary non-catalytic divalent cations include strontium, barium, and / or calcium, which inhibit polymerase-catalyzed nucleotide incorporation.

[0272] In some embodiments, in the sequencing step (c), each nucleotide analog in the plurality of nucleotide analogs comprises an aromatic base, a 5-carbon sugar having a 3' chain-terminating moiety that inhibits polymerase-catalyzed nucleotide incorporation, and at least one phosphate group. In some embodiments, the plurality of nucleotide analogs is unlabeled. In some embodiments, at least one nucleotide analog in the plurality of nucleotide analogs can be labeled with a detectable reporter moiety (e.g., a fluorophore). In some embodiments, the sequencing step (c) further comprises contacting the plurality of immobilized concatemeric template molecules with catalytic divalent cations or non-catalytic divalent cations. Exemplary catalytic divalent cations can include magnesium and / or manganese, which promote polymerase-catalyzed nucleotide incorporation. Exemplary non-catalytic divalent cations can include strontium, barium, and / or calcium, which inhibit polymerase-catalyzed nucleotide incorporation.

[0273] In some embodiments, in sequencing step (c), each multivalent molecule in the plurality of multivalent molecules 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, the spacer is attached to the linker, and the linker is attached to the nucleotide unit (see, e.g., Figures 55A-55C, 56-59). In some embodiments, the nucleotide unit comprises an aromatic base, a five-carbon sugar, and at least one phosphate group, and the linker is attached to the nucleotide unit via the base. In some embodiments, the plurality of multivalent molecules is unlabeled. In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules is labeled with a detectable reporter moiety (e.g., a fluorophore). In some embodiments, sequencing step (c) further comprises contacting the plurality of immobilized concatemeric template molecules with catalytic divalent cations or non-catalytic divalent cations. Exemplary catalytic divalent cations can include magnesium and / or manganese, which promote polymerase-catalyzed nucleotide incorporation. Exemplary non-catalytic divalent cations can include strontium, barium, and / or calcium, which inhibit polymerase-catalyzed nucleotide incorporation.

[0274] In some embodiments, in the sequencing step (c), the method further comprises forming at least one avidity complex, wherein forming is performed by contacting a plurality of immobilized concatemeric 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 comprising at least first and second binding complexes, wherein (i) the first binding complex comprises a first forward sequencing primer bound to a first portion of the immobilized concatemeric template molecules (e.g., first strand nanostructures). (ii) a second binding complex comprising a second forward sequencing primer, a first sequencing polymerase, and a first multivalent molecule, thereby forming a first binding complex, where the first nucleotide unit of the multivalent molecule is bound to the first polymerase; and (iii) a second binding complex comprising a second forward sequencing primer, a second sequencing polymerase, and the first multivalent molecule bound to a second portion of the same immobilized concatemeric template molecule, thereby forming a second binding complex, where the second nucleotide unit of the multivalent molecule is bound to the second polymerase. The first and second binding complexes comprising the same multivalent molecule form an avidity complex. In some embodiments, the multivalent molecule is unlabeled or labeled with a detectable reporter moiety.

[0275] 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 concatemeric template molecule in a first portion that does not prevent the soluble forward sequencing primer from hybridizing to the forward sequencing primer binding sites of the plurality of concatemeric template molecules in step (c) (e.g., there is little or no overlap).

[0276] 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 concatemeric template molecule in a second portion that does not prevent the soluble forward sequencing primer from hybridizing to the forward sequencing primer binding sites of the plurality of concatemeric template molecules in step (c) (e.g., has little or no overlap).

[0277] In some embodiments, the method for generating a plurality of immobilized nucleic acid nanostructures further comprises step (d): retaining a plurality of immobilized concatemeric template molecules (e.g., first-strand nanostructures) and replacing the plurality of extended forward sequencing primer strands with a plurality of forward extension strands hybridized to the retained immobilized nucleic acid concatemeric template molecules. The plurality of extended forward sequencing primer strands can be replaced with a plurality of forward extension strands by removing them and performing a primer extension reaction to synthesize second-strand forward extension strands (see, for example, Figures 24-25). The primer extension reaction can be performed with a plurality of compacting oligonucleotides to collapse / fold the forward extension strands and generate second-strand nanostructures. Those skilled in the art will recognize that there are several methods for performing a primer extension reaction, some of which are described below.

[0278] In some embodiments, the primer extension reaction of step (d) comprises contacting at least one extended forward sequencing primer strand with a plurality of strand-displacing polymerases and a plurality of nucleotides under conditions suitable for conducting a strand-displacing primer extension reaction in the absence of a soluble amplification primer, using the at least one extended forward sequencing primer strand to initiate a primer extension reaction, thereby generating a forward extension strand covalently linked to the extended forward sequencing primer strand, the forward extension strand hybridizing to an immobilized concatemeric template molecule (e.g., a first nanostructure) (Figures 24-25). For example, one of the extended forward sequencing primer strands can serve as a primer for the strand-displacing polymerase. The strand-displacing polymerase can extend the extended forward sequencing primer strand, synthesizing an extended strand that replaces the downstream extended forward sequencing primer strand while displacing the downstream extended forward sequencing primer strand. The newly extended strand is covalently linked to the extended forward sequencing primer strand. The immobilized concatemeric template molecule is then retained. In some embodiments, the plurality of nucleotides in the primer extension reaction of step (d) lacks a nucleotide having a cleavable moiety. The primer extension reaction can include a plurality of compacting oligonucleotides to generate forward extension strands that form nanostructures (e.g., second-strand nanostructures). Individual forward extension strands can be collapsed into nanostructures with a more compact size and / or shape compared to forward extension strands generated from a primer extension reaction performed without using compacting oligonucleotides.In some embodiments, an individual compacted oligonucleotide comprises 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 (e.g., the same forward extension strand).

[0279] In some embodiments, any of the embodiments of step (d) may be performed in the presence of a plurality of compacting oligonucleotides, in some embodiments, the plurality of compacting oligonucleotides of any of the embodiments of step (d) comprises any of the sequences described in step (b) above.

[0280] Without wishing to be bound by theory, it is believed that including compacting oligonucleotides in the primer extension reaction of step (d) can improve the FWHM (full width at half maximum) of the nanostructure spot image. The spot image can be represented as a Gaussian spot, and the size can be measured as FWHM. A smaller spot size, indicated by a smaller FWHM, typically correlates with an improved image of the spot. In some embodiments, the FWHM of the nanostructure spot can be about 10 μm or less.

[0281] Examples of strand-displacing polymerases include, but are not limited to, phi29 DNA polymerase, large fragment of Bst DNA polymerase, large fragment of Bsu DNA polymerase (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 can be a wild-type phi29 DNA polymerase (e.g., MagniPhi from Expedeon™), or a variant EquiPhi29 DNA polymerase (e.g., from Thermo Fisher Scientific™), or a chimeric QualiPhi DNA polymerase (e.g., from 4basebio™).

[0282] In some embodiments, the primer extension reaction of step (d) comprises: (i) removing a plurality of extended forward sequencing primer strands while retaining the immobilized concatemeric template molecules (e.g., the first strand nanostructure); and (ii) contacting the plurality of retained immobilized concatemeric 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 concatemeric template molecules and suitable 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 concatemeric molecules. The plurality of nucleotides in the primer extension reaction of step (d) lacks a nucleotide having a cleavable moiety. The primer extension reaction of step (d) can include multiple compacting oligonucleotides to generate forward extension strands that form nanostructures (e.g., second-strand nanostructures). Each forward extension strand can collapse into a nanostructure having a more compact size and / or shape compared to a forward extension strand generated from a primer extension reaction performed without the compacting oligonucleotide. In some embodiments, each compacting oligonucleotide comprises a single-stranded linear oligonucleotide having a 5' region capable of hybridizing to a first portion of a forward extension strand and a compacting oligonucleotide having a 3' region capable of hybridizing to a second portion of a forward extension strand (e.g., the same forward extension strand).

[0283] Without wishing to be bound by theory, it is believed that including compacting oligonucleotides in the primer extension reaction of step (d) can improve the FWHM (full width at half maximum) of the nanostructure spot image. The spot image can be represented as a Gaussian spot, and the size can be measured as FWHM. A smaller spot size, indicated by a smaller FWHM, typically correlates with an improved image of the spot. In some embodiments, the FWHM of the nanostructure spot can be about 10 μm or less.

[0284] In some embodiments, the primer extension reaction of step (d) comprises: (i) removing a plurality of extended forward sequencing primer strands while retaining the immobilized concatemeric template molecules (e.g., first strand nanostructures); and (ii) contacting the plurality of retained immobilized concatemeric 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 concatemeric template molecules and suitable for conducting a polymerase-catalyzed primer extension reaction, thereby generating a plurality of forward extension strands, wherein the soluble amplification primers hybridize to the soluble amplification primer binding sequences in the retained immobilized concatemeric molecules. The plurality of nucleotides in the primer extension reaction of step (d) may lack a nucleotide having a cleavable moiety. The primer extension reaction of step (d) can include multiple compacting oligonucleotides to generate forward extension strands that form nanostructures (e.g., second-strand nanostructures). Each forward extension strand can collapse into a nanostructure having a more compact size and / or shape compared to a forward extension strand generated from a primer extension reaction performed without the compacting oligonucleotide. In some embodiments, each compacting oligonucleotide comprises a single-stranded linear oligonucleotide having a 5' region capable of hybridizing to a first portion of a forward extension strand and a compacting oligonucleotide having a 3' region capable of hybridizing to a second portion of a forward extension strand (e.g., the same forward extension strand).

[0285] Without wishing to be bound by theory, it is believed that including compacting oligonucleotides in the primer extension reaction of step (d) can improve the FWHM (full width at half maximum) of the nanostructure spot image. The spot image can be represented as a Gaussian spot, and the size can be measured as FWHM. A smaller spot size, indicated by a smaller FWHM, typically correlates with an improved image of the spot. In some embodiments, the FWHM of the nanostructure spot can be about 10 μm or less.

[0286] In some embodiments, in any of the embodiments of step (d) above, the conditions suitable for hybridizing a plurality of soluble forward sequencing primers to a plurality of retained, immobilized, single-stranded nucleic acid concatemeric template molecules, or the conditions suitable for hybridizing a plurality of soluble amplification primers to a plurality of retained, immobilized, single-stranded nucleic acid concatemeric template molecules, comprise hybridizing the retained, immobilized concatemeric template molecules with 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, a high-efficiency hybridization buffer comprises: (i) a first polar aprotic solvent having a dielectric constant of 40 or less and a polarity index of 4-9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less 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 facilitate molecular crowding. In some embodiments, the high-efficiency hybridization buffer comprises: (i) the first polar aprotic solvent comprises acetonitrile at 25-50% by volume of the hybridization buffer; (ii) the second polar aprotic solvent comprises formamide at 5-10% by volume of the hybridization buffer; (iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0287] In some embodiments, in any of the above-described step (d), the plurality of extended forward sequencing primer strands can be removed from the immobilized concatemeric template molecule (e.g., the first strand nanostructure) using an enzyme or chemical reagent. For example, the plurality of extended forward sequencing primer strands 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). In some embodiments, the plurality of extended forward sequencing primer strands can be removed at a temperature favorable for nucleic acid denaturation.

[0288] In some embodiments, in any of the above-described step (d), a denaturing reagent can be used to remove the multiple extended forward sequencing primer strands, the denaturing reagent including any one of compounds such as formamide, acetonitrile, guanidinium chloride, and / or a buffer (e.g., Tris-HCl, MES, or HEPES, etc.), or any combination thereof.

[0289] In some embodiments, in any of the above-described step (d), the plurality of extended forward sequencing primer strands may be removed using elevated temperatures (e.g., heat) with or without a nucleic acid denaturing reagent. The plurality of extended forward sequencing primer strands may be subjected to temperatures of about 45-50°C, about 50-60°C, about 60-70°C, about 70-80°C, about 80-90°C, or about 90-95°C, or higher.

[0290] In some embodiments, in any of the above step (d), the multiple extended forward sequencing primer strands may be removed using 100% formamide at a temperature of about 65° C. for about 3 minutes and washed with about 50 mM NaCl or a reagent containing equivalent ionic strength and having a pH of about 6.5-8.5.

[0291] 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 uracil-containing template molecule (e.g., a uracil-resistant polymerase). Exemplary polymerases include, but are not limited to, Q5U Hot Start High Fidelity DNA Polymerase (e.g., catalog number M0515S from New England Biolabs™), Taq DNA polymerase, One Taq DNA polymerase (e.g., a mixture of Taq and Deep Vent DNA polymerase, catalog number M0480S from New England Biolabs™), LongAmp Taq DNA polymerase (e.g., catalog number M0323S from New England Biolabs™), Epimark Hot Start Taq DNA polymerase (e.g., catalog number M0490S from New England Biolabs™), Bst DNA polymerase (e.g., large fragment, catalog number M0275S from New England Biolabs™), Bsu DNA polymerase (e.g., large fragment, catalog number M0330S from New England Biolabs™), Phi29 DNA polymerase (e.g., catalog number M0269S from New England Biolabs™), E. coli DNA polymerase (e.g., catalog number M0209S from New England Biolabs™), Therminator DNA polymerase (e.g., catalog number M0261S from New England Biolabs), Vent DNA polymerase, and Deep Vent DNA polymerase.

[0292] In some embodiments, the method for generating a plurality of immobilized nucleic acid nanostructures further includes step (e): removing the retained immobilized concatemeric template molecules (e.g., first strand nanostructures) by creating an abasic site at the nucleotide(s) having the cleavable moiety in the immobilized single-stranded concatemeric template molecule and creating a gap at the abasic site to retain the plurality of forward-extended strands (second strand nanostructures) and generate a plurality of gap-containing single-stranded nucleic acid concatemeric template molecules while retaining the plurality of immobilized surface primers (Figures 26-27).

[0293] Abasic sites are generated on retained concatemer template strands containing nucleotides with cleavable moieties. In some embodiments, the cleavable moieties in the retained concatemer template molecules include uridine, 8-oxo-7,8-dihydroguanine (e.g., 8-oxoG), or deoxyinosine. Abasic sites can be removed to generate multiple single-stranded nucleic acid template molecules with gaps while retaining multiple forward extension strands. Abasic sites can be generated by contacting immobilized concatemer template molecules with an enzyme that removes the nucleobase in the nucleotide with the cleavable moiety. Uracil in the retained concatemer template strands can be converted to an abasic site using uracil DNA glycosylase (UDG). 8-oxoG in the retained concatemer template strands can be converted to an abasic site using FPG glycosylase. Deoxyinosine in the retained concatemer template strands can be converted to an abasic site using AlkA glycosylase.

[0294] In some embodiments, in the method for generating a plurality of immobilized nucleic acid nanostructures in step (e), gaps can be generated by contacting abasic sites in the immobilized concatemeric template molecules with an enzyme or mixture of enzymes having lyase activity that disrupts the phosphodiester backbone on the 5' and 3' sides of the abasic site, liberating abasic deoxyribose and generating gaps (Figure 26). The abasic sites can be removed using AP lyase, Endo IV endonuclease, FPG glycosylase / AP lyase, or Endo VIII glycosylase / AP lyase. In some embodiments, generating abasic sites and removing abasic sites to create gaps can be accomplished using a mixture of uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII, such as, but not limited to, USER™ (Uracil-Specific Excision Reagent Enzyme, from New England Biolabs™), or thermolabile USER™ (also from New England Biolabs™).

[0295] In some embodiments, in the method for generating a plurality of immobilized nucleic acid nanostructures in step (e), the plurality of gap-containing template molecules can be removed using enzymes, chemical compounds, and / or heat. After the gap removal procedure, the plurality of retained forward extension strands remain hybridized to the retained immobilized surface primers (Figure 27). For example, the plurality of gap-containing template molecules 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 gap-containing template molecules, the plurality of soluble amplification primers in step (e) can include at least one phosphorothioate diester bond at their 5' ends, which can render the soluble amplification primers resistant to exonuclease degradation. In some embodiments, the plurality of soluble amplification primers in step (d) comprise two to five or more consecutive phosphorothioate diester bonds at their 5' ends. In some embodiments, the plurality of 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 can render the forward sequencing primer resistant to exonuclease degradation.

[0296] In some embodiments, the plurality of gap-containing template molecules can be removed using a chemical reagent that favors nucleic acid denaturation. The denaturing reagent can include any one or any combination of compounds such as formamide, acetonitrile, guanidinium chloride, and / or a buffer (e.g., Tris-HCl, MES, or HEPES).

[0297] In some embodiments, the plurality of gap-containing template molecules may be removed using elevated temperatures (e.g., heat) with or without a nucleic acid denaturing reagent. The gap-containing template molecules may be subjected to temperatures of about 45-50°C, about 50-60°C, about 60-70°C, about 70-80°C, about 80-90°C, or about 90-95°C, or higher.

[0298] In some embodiments, the gap-containing template molecules can be removed using 100% formamide at a temperature of about 65° C. for about 3 minutes and washed with about 50 mM NaCl or a reagent containing equivalent ionic strength and having a pH of about 6.5-8.5.

[0299] 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), thereby generating a plurality of extended reverse sequencing primer strands. In some embodiments, the sequencing in 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 by performing a reverse sequencing reaction using one or more types of sequencing polymerase, a plurality of nucleotide reagents, and the hybridized reverse sequencing primers to generate reverse sequencing products (Figure 28). In some embodiments, the plurality of nucleotide reagents are detectably labeled. In some embodiments, the sequencing in step (f) further comprises detecting and imaging the plurality of reverse sequencing products. The universal adaptor sequence may be part of a concatemeric molecule having multiple copies of polynucleotide units arranged in tandem, each polynucleotide unit comprising a sequence of interest and at least one universal adaptor sequence. The first binding region of the compaction oligonucleotide can hybridize to at least a portion of any one of the universal adaptor sequences listed in Table 2. The second binding region of the compaction oligonucleotide sequence can hybridize to at least a portion of any one of the universal adaptor sequences listed in Table 2.

[0300] In some embodiments, the sequencing of step (f) further comprises contacting a plurality of the plurality of retained forward extension strands with a plurality of compaction oligonucleotides under conditions suitable for hybridizing the 5' end of at least one compaction oligonucleotide to a first portion of the plurality of retained forward extension strands and hybridizing a second binding region of the same compaction oligonucleotide to a second portion of the same plurality of retained forward extension strands.

[0301] In some embodiments, in step (f), the plurality of compacted oligonucleotides comprises any of the sequences described in step (b) above.

[0302] In some embodiments, in the sequencing step (f), the extended reverse sequencing primer strand is hybridized to the retained forward extension strand. The retained forward extension strand is hybridized to the first universal surface primer. The extended reverse sequencing primer strand is not hybridized to the first universal surface primer or covalently linked to the first universal surface primer. Therefore, the extended reverse sequencing primer strand is not immobilized on a support (see, for example, Figure 28).

[0303] In some embodiments, in the sequencing 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 generate a 3' OH extendable end. In some embodiments, the soluble reverse sequencing primer lacks a nucleotide with a cleavable portion. The reverse sequencing reaction can generate multiple extended reverse sequencing primer strands. In some embodiments, each retained forward extension strand has multiple copies of the reverse sequencing primer binding site / sequence, and each reverse sequencing primer binding site is capable of hybridizing to a reverse sequencing primer. Each reverse sequencing primer binding site in a given retained forward extension strand can then hybridize to a reverse sequencing primer, and the sequencing reaction can be performed. Each retained forward extension strand can be subjected to two or more reverse sequencing reactions, each initiated from a reverse sequencing primer hybridized to a reverse sequencing primer binding site (see, e.g., Figure 28).

[0304] In some embodiments, in the sequencing of step (f), the nucleotide reagents comprise a plurality of nucleotides, a plurality of nucleotide analogs, or a plurality of multivalent molecules.

[0305] In some embodiments, in the sequencing step (f), each nucleotide in the plurality of nucleotides comprises an aromatic base, a 5-carbon sugar, and at least one phosphate group. In some embodiments, the plurality of nucleotides is unlabeled. In some embodiments, at least one nucleotide in the plurality of nucleotides can be labeled with a detectable reporter moiety (e.g., a fluorophore). In some embodiments, the sequencing step (f) further comprises contacting the plurality of immobilized concatemeric template molecules with catalytic divalent cations or non-catalytic divalent cations. Exemplary catalytic divalent cations can include magnesium and / or manganese, which promote polymerase-catalyzed nucleotide incorporation. Exemplary non-catalytic divalent cations can include strontium, barium, and / or calcium, which inhibit polymerase-catalyzed nucleotide incorporation.

[0306] In some embodiments, in the sequencing step (f), each nucleotide analog in the plurality of nucleotide analogs comprises an aromatic base, a 5-carbon sugar having a 3' chain-terminating moiety that inhibits polymerase-catalyzed nucleotide incorporation, and at least one phosphate group. In some embodiments, the plurality of nucleotide analogs is unlabeled. In some embodiments, at least one nucleotide analog in the plurality of nucleotide analogs can be labeled with a detectable reporter moiety (e.g., a fluorophore). In some embodiments, the sequencing step (f) further comprises contacting the plurality of immobilized concatemeric template molecules with catalytic divalent cations or non-catalytic divalent cations. Exemplary catalytic divalent cations can include magnesium and / or manganese, which promote polymerase-catalyzed nucleotide incorporation. Exemplary non-catalytic divalent cations can include strontium, barium, and / or calcium, which inhibit polymerase-catalyzed nucleotide incorporation.

[0307] In some embodiments, in sequencing step (f), each multivalent molecule in the plurality of multivalent molecules 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, the spacer is attached to the linker, and the linker is attached to the nucleotide unit (see, e.g., Figures 56-59). In some embodiments, the nucleotide unit comprises an aromatic base, a five-carbon sugar, and at least one phosphate group, and the linker is attached to the nucleotide unit via the base. In some embodiments, the plurality of multivalent molecules is unlabeled. In some embodiments, at least one multivalent molecule in the plurality of multivalent molecules is labeled with a detectable reporter moiety (e.g., a fluorophore). In some embodiments, sequencing step (f) further comprises contacting the plurality of immobilized concatemeric template molecules with catalytic divalent cations or non-catalytic divalent cations. Exemplary catalytic divalent cations can include magnesium and / or manganese, which promote polymerase-catalyzed nucleotide incorporation. Exemplary non-catalytic divalent cations can include strontium, barium, and / or calcium, which inhibit polymerase-catalyzed nucleotide incorporation.

[0308] In some embodiments, in the sequencing step (f), the method further comprises forming at least one avidity complex, wherein forming is performed by contacting a plurality of immobilized concatemeric 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 comprising at least first and second binding complexes, wherein (i) the first binding complex comprises a first reverse sequencing primer bound to a first portion of the immobilized concatemeric template molecules (e.g., second strand nanostructures). (i) a first binding complex comprising a first reverse sequencing primer, a first sequencing polymerase, and a first multivalent molecule, thereby forming a first binding complex, where a 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 bound to a second portion of the same immobilized concatemeric template molecule, thereby forming a second binding complex, where a second nucleotide unit of the multivalent molecule is bound to the second polymerase, and the first and second binding complexes comprising the same multivalent molecule form an avidity complex. In some embodiments, the multivalent molecule is unlabeled or labeled with a detectable reporter moiety.

[0309] In some embodiments, in any of the primer extension reactions of step (d), the first binding region of the compacting oligonucleotide hybridizes to the forward extension strand in a first portion that does not prevent the reverse sequencing primer from hybridizing to the reverse sequencing primer binding sites of the multiple forward extension strands of step (f) (e.g., there is little or no overlap).

[0310] In some embodiments, in any of the primer extension reactions of step (d), the second binding region of the compacting oligonucleotide hybridizes to the forward extension strand in a second portion that does not prevent the reverse sequencing primer from hybridizing to the reverse sequencing primer binding sites of the multiple forward extension strands of step (f) (e.g., has little or no overlap).

[0311] 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 any length, including 20-150 nucleotides, 30-100 nucleotides, 40-80 nucleotides, or any range therebetween.

[0312] In some embodiments, the compacted oligonucleotide comprises a first binding region and a 3' region, and optionally an intervening linker between the first binding region and the second binding region. The intervening linker can be of any length, for example, about 2 to 20 nucleotides in length. The intervening linker can comprise a homopolymer having consecutive identical bases (e.g., AAA, GGG, CCC, TTT, or UUU). The intervening linker can comprise a non-homopolymer sequence.

[0313] The first binding region of the compacting oligonucleotide may be fully or partially complementary to a first portion of the concatemer molecule along its length. The second binding region of the compacting oligonucleotide may be fully or partially complementary to a second portion of the concatemer molecule along its length. The first binding region of the compacting oligonucleotide can hybridize to a first universal sequence portion of a concatemer molecule having a sequence of any one of SEQ ID NOS: 157-176 (see Table 2). The second binding region of the compacting oligonucleotide can hybridize to a second universal sequence portion of a concatemer molecule having a sequence of any one of SEQ ID NOS: 157-176 (see Table 2). The first and second binding regions of the compacting oligonucleotide can hybridize to the concatemer and pull the distal portions of the concatemer together, causing compaction of the concatemer to form a nanostructure.

[0314] In some embodiments, the method for generating a plurality of immobilized nucleic acid nanostructures further comprises positioning a cellular biological sample on the immobilized nanostructures after step (b). For example, the cellular biological sample can be placed on the immobilized nucleic acid nanostructures after step (b) and before step (c).

[0315] In some embodiments, the cellular biological sample comprises a single cell, multiple cells, tissues, organs, organisms, or sections from any of these cellular biological samples. In some embodiments, the cellular biological sample comprises a fresh sample, a frozen sample, a fresh frozen sample, or an archived (e.g., formalin-fixed paraffin-embedded, FFPE) sample. The cellular biological sample may be embedded in a matrix material. The cellular biological sample may be stained, destained, or unstained. The cellular biological sample may be permeabilized to allow nucleic acids within the cellular sample to transfer from the cell(s) to the multiple immobilized nanostructures.

[0316] In some embodiments, the conditions suitable for hybridizing the reverse sequencing primer to the reverse sequencing primer binding sequence of the retained forward extension strand in step (f) comprise 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 40 or less and a polarity index of 4 to 9; (ii) a second polar aprotic solvent having a dielectric constant of 115 or less 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 to 8; and (iv) a crowding agent in an amount sufficient to enhance or facilitate molecular crowding. In some embodiments, the high-efficiency hybridization buffer comprises: (i) the first polar aprotic solvent comprises acetonitrile at 25-50% by volume of the hybridization buffer; (ii) the second polar aprotic solvent comprises formamide at 5-10% by volume of the hybridization buffer; (iii) the pH buffer system comprises 2-(N-morpholino)ethanesulfonic acid (MES) at a pH of 5-6.5; and (iv) the crowding agent comprises polyethylene glycol (PEG) at 5-35% by volume of the hybridization buffer. In some embodiments, the high-efficiency hybridization buffer further comprises betaine.

[0317] In an alternative embodiment, the sequencing in step (f) comprises performing a sequencing reaction using the immobilized surface primers as sequencing primers to generate multiple reverse sequencing strands.

[0318] In some embodiments, at least one wash step may be performed after any of steps (a)-(f), and the wash step may be performed with a wash buffer comprising a pH buffer, a metal chelator, a salt, and a detergent.

[0319] In some embodiments, the pH buffering compound in the wash buffer comprises any one of 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), citrate compounds, citrate mixtures, NaOH, and / or KOH, or any combination of two or more thereof. In some embodiments, the pH buffering agent can be present in the wash buffer at a concentration of about 1-100 mM, about 10-50 mM, or about 10-25 mM. In some embodiments, the pH of the pH buffering agent present in any of the reagents described herein can be adjusted to a pH of about 4-9, about 5-9, or about 5-8.

[0320] 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, ammonium bicitrate, citric acid, potassium citrate, or magnesium citrate. In some embodiments, the wash buffer comprises the chelator at a concentration of about 0.01 to 50 mM, about 0.1 to 20 mM, or about 0.2 to 10 mM.

[0321] 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 can contain a monovalent salt at a concentration of about 25 to 500 mM, about 50 to 250 mM, or about 100 to 200 mM.

[0322] In some embodiments, the detergent in the wash buffer comprises a non-ionic 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 present in the wash buffer at a concentration of about 0.01-0.05%, or about 0.05-0.1%, or about 0.1-0.15%, or about 0.15-0.2%, or about 0.2-0.25%.

[0323] In some embodiments, a method for generating a plurality of immobilized nucleic acid nanostructures using on-support rolling circle amplification includes steps (a): providing a support having a plurality of first universal surface primers immobilized thereon; step (b): performing on-support rolling circle amplification; and step (c): forward sequencing. In some embodiments, the method does not include steps (d) to (f). For example, the method does not include synthesis of second-strand nanostructures (step (d)), generation of abasic sites and gaps (step (e)), and reverse sequencing (step (f)).

[0324] In some embodiments, a method for generating a plurality of immobilized nucleic acid nanostructures using on-support rolling circle amplification includes step (a): providing a support having a plurality of first universal surface primers immobilized thereon; and step (b): performing on-support rolling circle amplification. In some embodiments, the method does not include steps (c) to (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)).

[0325] On-support methods Generating densely immobilized nanostructures lacking cleavable moieties The present disclosure provides a method for generating a plurality of immobilized nucleic acid nanostructures, comprising: step (a): providing a support having a plurality of first universal surface primer...

Claims

1. 1. A method for generating high density nucleic acid nanostructures immobilized on a support, 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 greater than or equal to 1 mm ２ About 10 per ２ ~10 １５ and b) generating a plurality of immobilized single-stranded nucleic acid concatemeric template molecules, said generating comprising: 1) hybridizing a plurality of single-stranded circular nucleic acid library molecules to the plurality of immobilized first universal surface primers; 2) performing an on-support rolling circle amplification reaction with (i) a plurality of strand-displacing polymerases, (ii) a plurality of nucleotides, and (iii) a plurality of compacting oligonucleotides; thereby generating said plurality of immobilized single-stranded nucleic acid concatemeric template molecules; each compacted oligonucleotide comprises a single-stranded linear oligonucleotide having a first binding region capable of hybridizing to a first portion of a concatemeric molecule and a second binding region capable of hybridizing to a second portion of said concatemeric molecule, Multiple immobilized concatemeric molecules form compact nucleic acid nanostructures, a plurality of concatemers remain immobilized on the support as they form the compact nucleic acid nanostructure, thereby providing a nanostructure immobilized on the support in a single mm ２ About 10 per ２ ~about 10 １５ producing a support having a density of

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

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

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

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

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

7. 7. The method of any one of claims 1 to 6, wherein the plurality of nucleotides for the rolling circle amplification reaction comprises dATP, dCTP, dGTP, and dTTP, and the nucleotides lack a cleavable moiety that can be converted to an abasic site.

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

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

10. 10. The method of claim 8 or 9, wherein the rolling circle amplification reaction of step (b) generates a plurality of single-stranded nucleic acid concatemeric template molecules, each concatemeric template molecule comprising at least two nucleotides each having a cleavable moiety distributed at random locations along each immobilized concatemeric template molecule.

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

12. 12. The method of claim 11, wherein the sequence is set forth in any one of SEQ ID NOs: 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. 13. The method of 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, and the compacted oligonucleotides of the different populations have different sequences.

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

15. 15. The method of claim 13 or 14, wherein each population of compacted oligonucleotides in the mixture comprises a sequence set forth in any of SEQ ID NOs: 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. 16. The method of any one of claims 1 to 15, wherein the compact nucleic acid nanostructure comprises one or more loops, or comprises a spherical, elongated, prototoroid, or toroid shape.

17. The method of claim 16 , wherein the spherical shapes are nanoballs.

18. The method of claim 16 , wherein the elongated shapes are nanorods.

19. The method of claim 16 , wherein the toroidal shape is a nanotoroid.

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

21. The method of any one of claims 1 to 20, further comprising imaging the high-density nucleic acid nanostructures immobilized on the support.

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

23. connecting 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; hybridizing the plurality of soluble sequencing primers to each of the immobilized nanostructures to generate a plurality of nucleic acid duplexes along each of the nanostructures; 22. The method of any one of claims 1 to 21, further comprising contacting at least one nucleic acid duplex with a sequencing polymerase and a nucleotide reagent under conditions suitable for binding.

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

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

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

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

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

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

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

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

32. 32. The method of any one of claims 23-31, wherein the plurality of nucleotide reagents comprises a plurality of multivalent molecules, each multivalent molecule comprising (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, and the linker is attached to the nucleotide unit, and the nucleotide unit comprises an aromatic base, a five-carbon sugar, and at least one phosphate group.

33. forming a plurality of binding complexes, said forming comprising: c) binding a first sequencing primer, a first sequencing polymerase, and a first multivalent molecule to a first portion of each immobilized nanostructure, thereby forming a first binding complex, wherein a first nucleotide unit of the first multivalent molecule binds to the first sequencing polymerase; d) binding a second sequencing primer, a second sequencing polymerase, and the first multivalent molecule to a second portion of the same individual immobilized nanostructure, thereby forming a second binding complex, wherein a second nucleotide unit of the first multivalent molecule binds to the second sequencing polymerase, and the first and second binding complexes comprising the same multivalent molecule form an avidity complex.

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

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

36. c) contacting the plurality of immobilized nucleic acid nanostructures with a labeled multivalent molecule; d) imaging the high-density nucleic acid nanostructures immobilized on the support.

37. 21. The method of any one of claims 1 to 20, further comprising contacting the plurality of immobilized nanostructures with a cellular biological sample.

38. 37. 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. 39. The method of any one of claims 1 to 38, wherein the plurality of immobilized nucleic acid nanostructures are in fluid communication with one another, allowing a solution of reagents to flow over the support such that the plurality of immobilized nucleic acid nanostructures on the support react with the solution of reagents in a massively parallel manner.

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