Reversibly crosslinked hydrogels and methods for using them for cluster amplification

Abstract

Some examples herein provide a hydrogel on a substrate. The hydrogel includes a three-dimensional network of polymer chains, first functional groups coupled to the polymer chains, amplification primers coupled to the polymer chains via the first functional groups, and second functional groups coupled to the polymer chains and reversibly cross-linking the polymer chains to each other. Some examples herein provide a method of using the hydrogel. The method includes hybridizing a target polynucleotide to the amplification primer coupled to the hydrogel, cleaving a bridge in the hydrogel where the target polynucleotide is hybridized to the amplification primer, and amplifying the target polynucleotide using an additional amplification primer in the hydrogel whose cross-link has been cleaved.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 427,018, filed November 21, 2022, entitled "Reversibly Cross-Linked Hydrogels, and Methods of Using the Same for Cluster Amplification," the entire disclosure of which is incorporated herein by reference.

[0002] This application relates to hydrogels such as can be used in cluster amplification. [Background technology]

[0003] Cluster amplification is an approach for amplifying polynucleotides, for example, intended for use in gene sequencing. Target polynucleotides are captured by primers (e.g., P5 and P7 primers) coupled to the surface of a substrate within a flow cell, forming "seeds" at random locations on the surface. Amplification cycles are performed to form clusters on the surface around each seed. The clusters contain copies of the seed polynucleotide and complementary copies. In some situations, the substrate is patterned to define regions bound to different clusters, such as wells that can be filled with each cluster. Summary of the Invention

[0004] The examples provided herein relate to reversibly crosslinked hydrogels and methods of using same for cluster amplification.

[0005] Some examples herein provide a hydrogel on a substrate. The hydrogel may comprise a three-dimensional network of polymer chains. The hydrogel may comprise a first functional group coupled to the polymer chain. The hydrogel may comprise an amplification primer coupled to the polymer chain via the first functional group. The hydrogel may comprise a second functional group coupled to the polymer chain, which reversibly crosslinks the polymer chains to each other.

[0006] In some examples, the first and second functional groups are of different types from each other. In other examples, the first and second functional groups are of the same type from each other. In some examples, the first and second functional groups are independently selected from the group consisting of azide, amine, thiol, diol, aldehyde, alkyne, strained cyclooctyne, and inverse electron-demand (IED) Diels-Alder groups.

[0007] In some examples, the second functional group reversibly crosslinks the polymer chains via a cleavable molecule. In some examples, the cleavable molecule can be cleaved using a chemical agent, an enzyme, light, or heat. In some examples, the chemical agent can include an acid. In some examples, the cleavable molecule can include an acetal, a ketal, an imine, a hydrazone, or a t-butyl ester, which can be cleaved by an acid. In other examples, the chemical agent can include a reducing agent. In some examples, the cleavable molecule can include a disulfide bond or an azidoalkyl ether, which can be cleaved using a reducing agent, or an allyl ether, which can be cleaved using a palladium complex of a reducing agent. In other examples, the enzyme can include a DNAase, an RNAase, a protease, or a restriction enzyme, and the cleavable molecule can include an oligonucleotide, which can be cleaved using a DNAase, an RNAase, a protease, or a restriction enzyme. In other examples, the enzyme can include a protease enzyme or a lysosomal enzyme, and the cleavable molecule can include a peptide, which can be cleaved using a protease enzyme or a lysosomal enzyme. In other examples, the cleavable molecule comprises a Diels-Alder conjugation that is cleavable using heat, hi other examples, the cleavable molecule comprises a coumarin group or a nitrobenzene group that is cleavable using light.

[0008] In some examples, the second functional group comprises a host molecule that reversibly crosslinks the backbone via a guest molecule. In some examples, the guest molecule is removable via salt, heat, or pH. Additionally or alternatively, in some examples, the guest molecule is removable via substitution of a binding partner for the guest molecule. Additionally or alternatively, in some examples, the host molecule comprises a crown ether and the guest molecule comprises an ammonium moiety. Additionally or alternatively, the host molecule comprises beta-cyclodextrin and the guest molecule comprises adamantane, ferrocene, or bipyridine. In some examples, the second functional group comprises a ligand molecule that reversibly crosslinks the backbone via a multivalent binding protein. In some examples, the multivalent binding protein is removable using a denaturing agent.

[0009] Some examples herein provide methods of using hydrogels. The methods may include depositing a hydrogel on a substrate. The hydrogel may include a three-dimensional network of polymer chains and at least first and second types of functional groups coupled to the polymer chains. The methods may include coupling amplification primers to the first functional groups of the deposited hydrogel. The methods may include reversibly stabilizing the deposited hydrogel by reversibly crosslinking the second functional groups of the deposited hydrogel to which the amplification primers are coupled.

[0010] Some examples herein provide another method of using a hydrogel. The method can include depositing a hydrogel on a substrate. The hydrogel can include a three-dimensional network of polymer chains, amplification primers coupled to the polymer chains, and functional groups coupled to the polymer chains. The method can include reversibly stabilizing the hydrogel by reversibly crosslinking the functional groups of the deposited hydrogel to which the amplification primers are coupled.

[0011] Some examples herein provide another method of using a hydrogel. The method may include depositing a hydrogel on a substrate, the hydrogel comprising a three-dimensional network of polymer chains and first functional groups coupled to the polymer chains. The method may include coupling amplification primers to a first subset of the first functional groups of the deposited hydrogel. The method may include converting a second subset of the first functional groups to second functional groups. The method may include reversibly stabilizing the hydrogel by reversibly crosslinking the second functional groups.

[0012] Some examples herein provide another method of using a hydrogel. The method may include hybridizing a target polynucleotide to an amplification primer coupled to the hydrogel. The method may include cleaving a crosslink within the hydrogel where the target polynucleotide is hybridized to the amplification primer. The method may include amplifying the target polynucleotide using an additional amplification primer within the hydrogel where the crosslink has been cleaved. Optionally, the method may further include swelling the hydrogel after cleavage and before amplification.

[0013] It should be understood that any respective feature / example of each of the aspects of the present disclosure described herein may be implemented together in any suitable combination, and that any feature / example from any one or more of these aspects may be implemented together in any suitable combination with any of the features of the other aspects described herein, to achieve the benefits described herein. [Brief explanation of the drawings]

[0014] [Figure 1A] 1A-1C schematically illustrate exemplary compositions and operations in a process flow for using reversibly crosslinked hydrogels for cluster amplification. [Figure 1B] 1A-1C schematically illustrate exemplary compositions and operations in a process flow for using reversibly crosslinked hydrogels for cluster amplification. [Figure 1C] 1A-1C schematically illustrate exemplary compositions and operations in a process flow for using reversibly crosslinked hydrogels for cluster amplification. [Figure 1D] 1A-1C schematically illustrate exemplary compositions and operations in a process flow for using reversibly crosslinked hydrogels for cluster amplification. [Figure 1E] 1A-1C schematically illustrate exemplary compositions and operations in a process flow for using reversibly crosslinked hydrogels for cluster amplification. [Figure 1F]1A-1C schematically illustrate exemplary compositions and operations in a process flow for using reversibly crosslinked hydrogels for cluster amplification. [Figure 2A] 10A-10C schematically illustrate alternative exemplary compositions and operations in a process flow for using reversibly crosslinked hydrogels for cluster amplification. [Figure 2B] 10A-10C schematically illustrate alternative exemplary compositions and operations in a process flow for using reversibly crosslinked hydrogels for cluster amplification. [Figure 2C] 10A-10C schematically illustrate alternative exemplary compositions and operations in a process flow for using reversibly crosslinked hydrogels for cluster amplification. [Figure 3] 10A-10C schematically illustrate another alternative exemplary composition and operation in a process flow for using a reversibly crosslinked hydrogel for cluster amplification. [Figure 4-1] 1A and 1B illustrate schematic diagrams of exemplary hydrogels and exemplary crosslinkers that may be used with such hydrogels. [Figure 4-2] 1A and 1B illustrate schematic diagrams of exemplary hydrogels and exemplary crosslinkers that may be used with such hydrogels. [Figure 5A] 10A-10C schematically illustrate additional exemplary hydrogels and exemplary crosslinkers that may be used with such hydrogels. [Figure 5B] 10A-10C schematically illustrate additional exemplary hydrogels and exemplary crosslinkers that may be used with such hydrogels. [Figure 6] 1A-1C show schematic diagrams of exemplary cleavable molecules that can be used to reversibly crosslink hydrogels in the manner described herein. [Figure 7] 1A-1C are schematic diagrams illustrating additional exemplary cleavable molecules that can be used to reversibly crosslink hydrogels in the manner described herein. [Figure 8A] 10A-10C schematically illustrate additional exemplary compositions and operations in a process flow for using reversibly crosslinked hydrogels for cluster amplification. [Figure 8B] 10A-10C schematically illustrate additional exemplary compositions and operations in a process flow for using reversibly crosslinked hydrogels for cluster amplification. [Figure 9A] 10A-10C schematically illustrate additional exemplary compositions and operations in a process flow for using reversibly crosslinked hydrogels for cluster amplification. [Figure 9B] 10A-10C schematically illustrate additional exemplary compositions and operations in a process flow for using reversibly crosslinked hydrogels for cluster amplification. DETAILED DESCRIPTION OF THE INVENTION

[0015] The examples provided herein relate to reversibly crosslinked hydrogels and methods of using same for cluster amplification.

[0016] To determine the sequence of the target polynucleotides in each cluster, it may be desirable to perform sequencing-by-synthesis (SBS) using a functionalized hydrogel in a flow cell. For example, the hydrogel may contain functional groups to which amplification primers can be coupled. The hydrogel can then be seeded by flowing a target polynucleotide coupled with an adapter that is complementary to the amplification primers and hybridizes to one of the primers through the flow cell. The seeds can then be amplified using the amplification primers in the hydrogel to produce clusters containing multiple amplicons of the target polynucleotide, which can then be sequenced using SBS.

[0017] However, certain previously known hydrogels may degrade during transport or storage, thereby causing degradation of the hydrogel's performance. For example, during transport or storage, the hydrogel may irreversibly disintegrate. Therefore, amplification primers within the hydrogel may become inaccessible during amplification, resulting in the generation of clusters containing fewer amplicons, and therefore generating fewer signals during SBS than clusters formed using hydrogels that have not been so degraded. This may reduce the accuracy of sequencing reads.

[0018] The reversibly crosslinked hydrogels provided herein are expected to be significantly more stable during transport and storage than previously known hydrogels, such as those described above. More specifically, the hydrogels can be crosslinked before transport and storage. The crosslinks can reduce or prevent irreversible collapse of the hydrogel during transport and storage, for example, by restricting the mobility of the polymer chains to form strong inter-chain and / or intra-chain physical bonds, in other words, by restricting the mobility of the polymer chains to rearrange into a lower entropy state that would be energetically unfavorable to reverse upon rehydration. The crosslinks can be controllably reversed, for example, by applying an appropriate stimulus, and can then be broken or removed, after which the hydrogel can be used as desired.

[0019] Additionally, in some instances, the seeding process can be performed before reversing the crosslinks. Thus, amplification primers within the hydrogel can be relatively inaccessible during the seeding process, while amplification primers at the exposed surface of the hydrogel are available for seeding. In some instances, the crosslinks can then be reversed to make the amplification primers within the hydrogel accessible for amplification. Using the present reversible crosslinks to reduce the number of amplification primers available for seeding can reduce the number (or density) of seeding events that occur and thus improve the monoclonality of subsequently formed clusters compared to hydrogels that were not crosslinked during seeding. Additionally, using the present reversible crosslinks to subsequently increase the number of amplification primers available for amplification can generate more amplicons than hydrogels that were irreversibly collapsed due to not being crosslinked. Thus, it can be seen that the present reversibly crosslinked hydrogels can be expected to provide clusters with enhanced monoclonality and enhanced amplification compared to previously known hydrogels. However, it will be understood that cross-linking can be used at any suitable time, for example, to improve the stability of the hydrogel during shipping and storage, can be reversed prior to seeding, and need not necessarily be used to regulate the accessibility of amplification primers during seeding and / or amplification.

[0020] First, a brief explanation of some terms used herein will be provided, followed by a description of some exemplary methods for reversibly crosslinking hydrogels, the resulting compositions, and methods of using the reversibly crosslinked hydrogels.

[0021] term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The use of the term "including" and other forms such as "include," "includes," and "included" is not limiting. The use of the term "having" and other forms such as "have," "has," and "had" is not limiting. As used herein, whether in a transitional phrase or in the body of a claim, the terms "comprise" and "comprising" should be interpreted as having an open-ended meaning. That is, the above terms should be interpreted as synonymous with the phrase "having at least" or "including at least." For example, when used in the context of a process, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components.

[0022] As used throughout this specification, the terms "substantially," "approximately," and "about" are used to describe and account for small variations due to processing variations, etc. For example, they can refer to ±10% or less, such as ±5% or less, such as ±2% or less, such as ±1% or less, such as ±0.5% or less, such as ±0.2% or less, such as ±0.1% or less, such as ±0.05% or less.

[0023] As used herein, "hybridize" refers to the non-covalent binding of a first polynucleotide to a second polynucleotide along the length of the polynucleotides to form a double-stranded "duplex." For example, two DNA polynucleotide strands can associate through complementary base pairing. The strength of association between a first and second polynucleotide increases with the complementarity between the nucleotide sequences within the polynucleotides. The strength of hybridization between polynucleotides can be characterized by the melting temperature (Tm), at which 50% of the polynucleotide strands of the duplex separate.

[0024] As used herein, the term "nucleotide" is intended to mean a molecule comprising a sugar and at least one phosphate group, and in some instances, also comprising a nucleobase. A nucleotide lacking a nucleobase may be referred to as "abasic." Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate sugar backbone nucleotides, and mixtures thereof. Examples of nucleotides include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine monophosphate (UMP), uridine diphosphate (UDP), and uridine triphosphate (GTP). deoxyadenosine monophosphate (UTP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (deoxythymidineThese include deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), and deoxyuridine triphosphate (dUTP).

[0025] As used herein, the term "nucleotide" is also intended to encompass any nucleotide analog, which is a type of nucleotide that contains a modified nucleobase, sugar, and / or phosphate moiety compared to naturally occurring nucleotides. Exemplary modified nucleobases include inosine, xanthate, hypoxanthate, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azothio ... Examples of nucleotide analogs include cytosine, 6-azothymine, 5-uracil, 4-thiouracil, 8-halo adenine or guanine, 8-amino adenine or guanine, 8-thiol adenine or guanine, 8-thioalkyl adenine or guanine, 8-hydroxyl adenine or guanine, 5-halo substituted uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, and the like. As is known in the art, certain nucleotide analogs cannot be incorporated into polynucleotides, such as adenosine 5'-phosphosulfate. A nucleotide can contain any suitable number of phosphates, for example, 3, 4, 5, 6, or more than 6 phosphates.

[0026] As used herein, the term "polynucleotide" refers to a molecule comprising a sequence of nucleotides linked together. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and their analogs. A polynucleotide can be a single-stranded sequence of nucleotides, such as RNA or single-stranded DNA, a double-stranded sequence of nucleotides, such as double-stranded DNA, or a mixture of single- and double-stranded sequences of nucleotides. Double-stranded DNA (dsDNA) includes genomic DNA and PCR and amplification products. Single-stranded DNA (ssDNA) can be converted to dsDNA, and vice versa. Polynucleotides can include non-naturally occurring DNA, such as enantiomeric DNA. The exact sequence of nucleotides in a polynucleotide may or may not be known. The following are examples of polynucleotides: a gene or gene fragment (e.g., a probe, primer, expressed sequence tag (EST), or serial analysis of gene expression (SAGE) tag), genomic DNA, a genomic DNA fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, synthetic polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, primers, or amplified copies of any of the foregoing.

[0027] As used herein, "polymerase" is intended to mean an enzyme having an active site that assembles polynucleotides by polymerizing nucleotides into polynucleotides. A polymerase can bind to a primed single-stranded target polynucleotide and grow it by sequentially adding nucleotides to the primer to form a "complementary copy" polynucleotide having a sequence complementary to that of the target polynucleotide. Another polymerase, or the same polymerase, can then create a copy of the target polynucleotide by forming a complementary copy of the complementary copy polynucleotide. Any such copy may be referred to herein as an "amplicon." A DNA polymerase can bind to a target polynucleotide and then move downstream along the target polynucleotide while sequentially adding nucleotides to the free hydroxyl group at the 3' end of the polynucleotide chain to grow it (amplicon growth). DNA polymerases can synthesize complementary DNA molecules from DNA templates, and RNA polymerases can synthesize RNA molecules from DNA templates (transcription). Polymerases can use short RNA or DNA strands (primers) to initiate strand growth. Some polymerases can displace the strand upstream of the site where they add a base to the strand. Such polymerases can also be referred to as strand displacing, which means they have the activity of removing a complementary strand from the template strand being read by the polymerase. Exemplary polymerases with strand displacement activity include, but are not limited to, Bst (Bacillus stearothermophilus) polymerase, exo-Klenow polymerase, or large fragments of sequencing-grade T7 exo-polymerase. Some polymerases cleave the strand in front of them and effectively replace it with the growing strand (5' exonuclease activity). Some polymerases have the activity of degrading the strand behind them (3' exonuclease activity). Some useful polymerases have been mutated or otherwise modified to reduce or eliminate 3' and / or 5' exonuclease activity.

[0028] As used herein, the term "primer" is defined as a polynucleotide to which a nucleotide can be added via a free 3'OH group. A primer may contain a 3' block that prevents polymerization until the block is removed. A primer may contain a 5'-end modification to enable a coupling reaction or to allow the primer to be coupled to another moiety. A primer may contain one or more moieties that can be cleaved under suitable conditions, such as UV light, chemicals, enzymes, etc. A primer may be any suitable number of bases long and may contain a suitable combination of natural and / or non-natural nucleotides. A target polynucleotide may contain an "adapter" (having a sequence complementary to the primer) that hybridizes to the primer and can be amplified to generate a complementary copy polynucleotide by adding a nucleotide to the free 3'OH group of the primer. "Amplification primer" is intended to mean a primer that can be coupled to a substrate and hybridize to a second adapter of a target polynucleotide, and "orthogonal amplification primer" is intended to mean a primer that can be coupled to a substrate and hybridize to a first adapter of that target polynucleotide. The first adapter can have a sequence complementary to that of an orthogonal amplification primer, and the second adapter can have a sequence complementary to that of an amplification primer. The amplification primer and the orthogonal amplification primer can have different and independent sequences from each other. In one non-limiting example, the amplification primer comprises a P5 primer and the orthogonal amplification primer comprises a P7 primer.

[0029] As used herein, the term "substrate" refers to a material used as a support for the compositions described herein. Exemplary substrate materials can include glass, silica, plastic, quartz, metal, metal oxide, organo-silicates (e.g., polyhedral organic silsesquioxanes (POSS)), polyacrylate, tantalum oxide, complementary metal oxide semiconductor (CMOS), or a combination thereof. An example of a POSS can be that described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776-778, which is incorporated herein by reference in its entirety. In some examples, the substrate used in this application includes a silica-based substrate, such as glass, fused silica, or other silica-containing material. In some examples, the substrate can include silicon, silicon nitride, or silicon hydride. In some examples, the substrate used in this application includes plastic materials or components such as polyethylene, polystyrene, poly(vinyl chloride), polypropylene, nylon, polyester, polycarbonate, and poly(methyl methacrylate). Examples of plastic materials include poly(methyl methacrylate), polystyrene, and cyclic olefin polymer substrates. In some examples, the substrate is or includes a silica-based material or a plastic material, or a combination thereof. In certain examples, the substrate has at least one surface including glass or a silicon-based polymer. In some examples, the substrate can include a metal. In some such examples, the metal is gold. In some examples, the substrate has at least one surface including a metal oxide. In one example, the surface includes tantalum oxide or tin oxide. Acrylamides, enones, or acrylates can also be utilized as substrate materials or components. Other substrate materials may include, but are not limited to, gallium arsenide, indium phosphide, aluminum, ceramic, polyimide, quartz, resin, polymer, quartz, resin, polymer, and copolymer.In some examples, the substrate and / or substrate surface may be or may include quartz. In some other examples, the substrate and / or substrate surface may be or may include a semiconductor, such as GaAs or ITO. The above list is intended to illustrate, but not limit, the present application. The substrate may include a single material or multiple different materials. The substrate may be a composite or laminate. In some examples, the substrate includes an organosilicate material. The substrate may be flat, round, spherical, rod-shaped, or any other suitable shape. The substrate may be rigid or flexible. In some examples, the substrate is a bead or a flow cell.

[0030] In some examples, the surface is a patterned surface. A "patterned surface" refers to an arrangement of distinct regions within or on an exposed layer of a substrate. For example, one or more of the regions can be features in which one or more amplification primers are present, e.g., in a hydrogel located within the feature. The features can be separated by interstitial regions where amplification primers and hydrogel are absent. In some examples, the pattern can be an xy format of features in rows and columns. In some examples, the pattern can be a repeating arrangement of features and / or interstitial regions. In some examples, the pattern can be a random arrangement of features and / or interstitial regions. In some examples, the substrate includes an array of wells (depressions) on the surface. The wells can be provided with substantially vertical sidewalls. The wells can be fabricated using a variety of techniques, including, but not limited to, photolithography, stamping techniques, molding techniques, and microetching techniques, as commonly known in the art. As understood in the art, the technique used will depend on the composition and shape of the array substrate.

[0031] The features on the patterned surface of the substrate can include an array of wells (e.g., microwells or nanowells) on glass, silicon, plastic, or other suitable material onto which a hydrogel as provided herein is patterned. The patterning can provide a hydrogel pad that can be used for sequencing and can be stable, for example, over multiple cycles of sequencing runs. In some examples, covalently binding the hydrogel to the wells can help maintain the hydrogel in the structured features (e.g., wells) throughout the life of the structured substrate during various uses. However, in some examples, the hydrogel does not need to be covalently bound to the wells.

[0032] In certain examples, structured substrates can be created by patterning a substrate formed of a suitable material with wells (e.g., microwells or nanowells), coating the substrate material with a hydrogel, and polishing the surface of the hydrogel-coated material, e.g., via chemical or mechanical polishing, thereby retaining the hydrogel within the wells but removing or inactivating substantially all of the hydrogel from the interstitial regions on the surface of the structured substrate between the wells. Amplification primers can then be attached to the hydrogel, e.g., in a manner as provided herein. A solution containing multiple target polynucleotides (e.g., a fragmented human genome or portion thereof) can then be contacted with the polished substrate such that individual target polynucleotides are seeded into individual wells through interaction with the amplification primers attached to the hydrogel; however, the target polynucleotides will not occupy the interstitial regions due to the absence or inactivity of the hydrogel. The absence or inactivity of the hydrogel in the interstitial regions can inhibit the outward migration of growing clusters, thereby confining amplification of the target polynucleotides to the wells. The process is easily manufacturable, scalable, and utilizes conventional micro- or nano-fabrication methods.

[0033] Patterned substrates can include, for example, wells etched into a slide or chip. The etching and geometric pattern of the wells can be of a variety of different shapes and sizes, and such features can be physically or functionally separable from one another. Particularly useful substrates with such structural features include patterned substrates that allow for the selection of sizes of solid particles, such as microspheres. An example of a patterned substrate with these properties is the etched substrate used in connection with BEAD ARRAY technology (Illumina, Inc., San Diego, Calif.).

[0034] In some examples, the substrates described herein form at least a portion of, are located within, or are coupled to a flow cell. A flow cell may include a flow chamber divided into multiple lanes or multiple sectors. Examples of flow cells and substrates for the manufacture of flow cells that can be used in the methods and compositions described herein include, but are not limited to, those commercially available from Illumina, Inc. (San Diego, CA).

[0035] As used herein, the term "directly," when used in reference to a layer covering the surface of a substrate, is intended to mean that the layer covers the surface of the substrate without any significant intermediate layer, such as, for example, an adhesive layer or a polymer layer. A layer that directly covers a surface can be attached to this surface through any chemical or physical interaction, such as a covalent or non-covalent bond.

[0036] As used herein, the term "immobilized," when used with respect to polynucleotides, is intended to mean direct or indirect attachment to a substrate via covalent or non-covalent bonds. In certain instances, covalent bonds may be used, or any other suitable bond that allows the polynucleotide to remain stationary or attached to the substrate under the conditions for which the substrate is intended to be used, such as for amplifying or sequencing the polynucleotide. Polynucleotides used as amplification primers or target polynucleotides may be immobilized so that the 3' end is available for enzymatic extension and at least a portion of the sequence is capable of hybridizing to a complementary sequence. Immobilization may occur via hybridization to a surface-attached oligonucleotide, in which case the immobilized oligonucleotide or polynucleotide may be in a 3' to 5' orientation. Alternatively, immobilization may occur by means other than base-pairing hybridization, such as covalent bonding.

[0037] As used herein, the term "array" refers to a collection of substrate regions that can be distinguished from one another according to their relative positions. Different molecules (such as polynucleotides) in different regions of an array can be distinguished from one another according to the position of the region within the array. An individual region of an array can contain one or more molecules of a particular type. For example, a substrate region can contain a single target polynucleotide having a particular sequence, or a substrate region can contain several polynucleotides having the same sequence (or its complementary sequence). Regions of an array can each contain different features on the same substrate. Example features include, but are not limited to, wells within a substrate, beads (or other particles) within or on a substrate, protrusions from a substrate, ridges on a substrate, or channels within a substrate. Regions of an array can each comprise different regions on different substrates. Different molecules attached to separate substrates can be identified according to the position of the substrate on a surface to which the substrates are associated, or according to the position of the substrate within a liquid or gel. An example array in which separate substrates are located on a surface includes, but is not limited to, beads within wells.

[0038] As used herein, the term "plurality" is intended to mean a population of two or more distinct members. Pluralities can range in size from small, medium, large, to very large. A small-sized plurality can range, for example, from a few members to tens of members. A medium-sized plurality can range, for example, from tens of members to about 100 or hundreds of members. A large plurality can range, for example, from about hundreds of members to about 1,000 members, thousands of members, and tens of thousands of members. A very large plurality can range, for example, from tens of thousands of members to about hundreds of thousands, millions, tens of millions, or hundreds of millions or more members. Thus, pluralities can range in size from 2 to well over 100 million members, as well as all sizes measured by number of members and larger than the exemplary ranges listed above. Exemplary pluralities of polynucleotides include, for example, about 1 x 10 5 That's it, 5 x 10 5 or more or 1 x 10 6 The term "multiple" includes a population of more than two different polynucleotides. Therefore, the definition of this term is intended to include all integer values ​​greater than 2. The upper limit of the multiple value can be set, for example, by the theoretical diversity of polynucleotide sequences in a sample.

[0039] As used herein, the term "double-stranded," when used in reference to a polynucleotide, is intended to mean that all or substantially all of the nucleotides in the polynucleotide are hydrogen bonded to each nucleotide in a complementary polynucleotide.

[0040] As used herein, the term "single-stranded," when used in reference to a polynucleotide, means that none of the nucleotides in the polynucleotide are hydrogen bonded to each nucleotide in a complementary polynucleotide.

[0041] As used herein, the term "target polynucleotide" is intended to mean a polynucleotide that is the subject of an analysis or operation. The analysis or operation may include subjecting the polynucleotide to amplification, sequencing, and / or other procedures. A target polynucleotide may contain additional nucleotide sequences to the target sequence being analyzed. For example, a target polynucleotide may contain one or more adapters, including adapters that function as primer binding sites, that flank the target polynucleotide sequence being analyzed. A target polynucleotide hybridized to an amplification primer may contain nucleotides that extend beyond the 5' or 3' end of the amplification primer in such a way that a portion of the target polynucleotide is inappropriate for extension. In certain examples, multiple target polynucleotides may have first and second adapters that are identical to each other but have different sequences. Two adapters that may flank a particular target polynucleotide sequence may have the same sequence as each other or complementary sequences to each other, or the two adapters may have different sequences. Thus, a species in a plurality of target polynucleotides can include, for example, a region of known sequence flanked by regions of unknown sequence that are evaluated by sequencing (e.g., SBS). In some instances, the target polynucleotide carries an adapter at a single end, and such adapter can be located at either the 3' or 5' end of the target polynucleotide. The target polynucleotide can be used without an adapter, in which case the primer binding sequence can directly use a sequence present in the target polynucleotide.

[0042] The terms "polynucleotide" and "oligonucleotide" are used interchangeably herein. The difference in terminology is not intended to indicate any particular difference in size, sequence, or other properties unless otherwise specified. For clarity of explanation, when describing a particular method or composition that includes several polynucleotide species, different terms may be used to distinguish one species of polynucleotide from another.

[0043] As used herein, the term "amplicon," when used in reference to a polynucleotide, refers to the product of replication of the polynucleotide, which product has a nucleotide sequence that is substantially the same as or substantially complementary to at least a portion of the nucleotide sequence of the polynucleotide. "Amplification" and "amplifying" refer to the process of producing an amplicon of a polynucleotide. A first amplicon of a target polynucleotide can typically be a complementary copy. Additional amplicons are copies made from the target polynucleotide or the first amplicon after the generation of the first amplicon. Subsequent amplicons can have a sequence that is substantially complementary to or substantially identical to the target polynucleotide. It will be understood that minor variations of a polynucleotide (e.g., due to amplification artifacts) may occur when generating an amplicon of that polynucleotide.

[0044] As used herein, "hydrogel" refers to a three-dimensional polymer network structure comprising polymer chains, which is at least partially hydrophilic and contains water within the interchain spaces. Hydrogels can include any suitable combination of hydrophilic, hydrophobic, and / or amphiphilic polymers, so long as the entire polymer network is hydrophilic and contains water within the interchain spaces. Hydrogels include chemical hydrogels, in which both the bonds forming the polymer chains and any crosslinks between the polymer chains are covalent; such crosslinks during hydrogel formation can be irreversible, as distinguished from the reversible crosslinks performed after the hydrogel is formed. In some cases, chemical hydrogels can comprise or consist essentially of polymer chains in a brush-like structure attached to a surface with substantially no physical or covalent crosslinks between the polymer chains, or alternatively, polymer chains with multiple attachment points to a surface, resulting in loops but lacking interchain crosslinks. Hydrogels also include physical hydrogels, in which the bonds forming the polymer chains and any crosslinks within the polymer chains are not covalent. Non-limiting examples of physical hydrogels include agarose and alginate.

[0045] As used herein, the "polymer chains" of a hydrogel are intended to mean those portions of the hydrogel that are polymerized together during the polymerization process. The polymer chains can be crosslinked to form the hydrogel. For example, a crosslinking agent can be added during or after the polymerization process to form the polymer chains. Additionally or alternatively, in some instances, the polymer chains can be deposited on a substrate surface that includes functional groups to which the functional groups of the polymer chains can be coupled. The polymer chains can be coupled to the surface, for example, via a reaction between the functional groups of the polymer chains and the functional groups of the surface, and such coupling can crosslink the polymer chains to form the hydrogel. Such crosslinking can result in covalent or non-covalent attachment of the polymer chains to each other or can occur as a result of chain entanglement during polymerization and / or attachment to the surface. As provided herein, the polymer chains can also or alternatively be reversibly crosslinked after the hydrogel is formed, for example, using functional groups in a manner described in more detail below.

[0046] As used herein, the term "functional group" is intended to mean a chemical entity that is reactive with another chemical entity of the same or different type to form a covalent or non-covalent bond. As provided herein, when a hydrogel is described as having a functional group, it should be understood that the functional group is chemically distinct from the polymer chain. Thus, the functional group does not necessarily participate in the formation of a three-dimensional network structure of polymer chains during the formation of the hydrogel or during the attachment of the polymer chains to a surface; therefore, as provided herein, the functional group is available to perform additional crosslinking of the hydrogel at a later time after the hydrogel itself has been formed and placed on a substrate.

[0047] Methods of forming and using reversibly crosslinked hydrogels As provided herein, the stability of a hydrogel can be enhanced, for example, by reversibly crosslinking the hydrogel after hydrogel formation (after the formation and crosslinking of polymer chains to form the hydrogel is complete) and before hydrogel transport and / or storage. Additionally or alternatively, as provided herein, the hydrogel can optionally be reversibly crosslinked at any suitable time before seeding the hydrogel with template polynucleotides, and the crosslinks can optionally be reversed at any suitable time before or during cluster amplification using the seeded template polynucleotides. Alternatively, the crosslinks can optionally be reversed before seeding.

[0048] For example, FIGS. 1A-1F schematically illustrate exemplary compositions and operations in a process flow for using reversibly crosslinked hydrogels for cluster amplification. The composition shown in FIG. 1A includes a hydrogel 100 on a substrate 140. Optionally, the substrate 140 may include vertical sidewalls 150 that laterally constrain the hydrogel 100. The hydrogel 100 includes a three-dimensional network of polymer chains 110, intended to be represented by elongated lines that intersect with each other. The hydrogel 100 also includes first functional groups 120 coupled to the polymer chains 110 and second functional groups 130 coupled to the polymer chains 110. In the non-limiting example shown in FIG. 1A, the first functional groups 120 and the second functional groups 130 are different types of chemical entities, and are different types of chemical entities from the monomers that form the polymer chains 110 of the hydrogel 100. The first functional groups 120 can be identical to each other or different from each other. The second functional groups 130 can be identical to one another or can be different from one another. The hydrogel 100 can be formed in any suitable manner to form a three-dimensional crosslinked network of polymer chains 110 comprising the first functional groups 120 and the second functional groups 130. Non-limiting examples of monomers that can be used to form the hydrogel 100 are described in more detail below. In some examples, the hydrogel 100 is a chemical hydrogel in which both the bonds and crosslinks within the polymer chains 110 of the hydrogel are covalent and therefore irreversible. The first functional groups 120 and the second functional groups 130 do not substantially participate in the formation of the polymer chains 110 of the hydrogel 100 or in the crosslinking of such polymer chains during the formation of the hydrogel 100. Alternatively, in a manner described in more detail below, first functional group 120 may be used to couple amplification primers to polymer chains 110 after formation of hydrogel 100, and second functional group 130 may be used to crosslink polymer chains 110 to each other after the amplification primers have been coupled to the polymer chains. Polymer chains 110 and / or substrate 140 may include other functional groups (not specifically shown) through which polymer chains may be crosslinked to each other and / or to substrate 140 during formation of hydrogel 100.Such functional groups, and such cross-linking during hydrogel formation, should be distinguished from functional groups 130 that are used to reversibly cross-link the hydrogel after it has been formed, e.g., minutes, hours, days, or longer after the hydrogel has been formed.

[0049] The hydrogel 100 shown in FIG. 1A may be contacted with a fluid containing amplification primers that include functional groups (not specifically shown) configured to react with the first functional groups 120 to covalently bond the amplification primers to the polymer chains 110. During such contact between the fluid and the hydrogel 100, the amplification primers 121, 122 can diffuse through the open spaces within the three-dimensional crosslinked network of the hydrogel and thus become covalently coupled to the polymer chains 110 at various different lateral positions and depths, as intended to be shown in FIG. 1B. For example, the hydrogel 100 may have a sufficient amount of open space to facilitate diffusion of the amplification primers 121, 122 through the open spaces. Such contact between the fluid and the hydrogel 100, as described with reference to FIG. 1A, forms the modified hydrogel 101 shown in FIG. 1B, which includes the amplification primers 121, 122 coupled to the polymer chains 110 via the first functional groups 120. In Figure IB, first functional group 120 is represented by a dark fill, and reference numeral 120' indicates that the functional group has reacted with the functional groups of amplification primers 121, 122. Additionally, in Figure IB, amplification primers 121, 122 are shown with different fills to represent a non-limiting example in which different types of amplification primers are coupled to a hydrogel. For example, amplification primer 121 can include a P5 primer, and amplification primer 122 can include an orthogonal amplification primer, such as a P7 primer.

[0050] The hydrogel 101 shown in FIG. 1B may be contacted with a fluid containing at least one chemical or enzymatic reagent configured to react with the second functional groups 130, causing such functional groups to covalently or non-covalently crosslink the polymer chains 110 of the hydrogel 101 to one another. During such contact between the fluid and the hydrogel 101, the at least one chemical or enzymatic reagent can diffuse through the open spaces within the three-dimensional crosslinked network of the hydrogel 101 and crosslink the polymer chains 110 at various different lateral positions and depths, as intended to be shown in FIG. 1C. The open spaces within the hydrogel 101 may facilitate the diffusion of the at least one chemical or enzymatic reagent through the open spaces. Such contact forms the modified hydrogel 102 shown in FIG. 1C, in which the second functional groups 130 coupled to the polymer chains 110 reversibly crosslink the polymer chains to one another. In Figure 1C, the second functional groups 130 are represented by a dark fill, and reference numeral 130' indicates that the functional groups have reacted to crosslink the polymer chains. Additionally, in Figure 1C, the crosslinks 131 between the polymer chains 110 are shown using dashed lines to indicate that such crosslinks are reversible, for example, in a manner described in more detail below.

[0051] 1C , hydrogel 102 may have a significantly smaller percent open space than hydrogel 100 and significantly smaller than hydrogel 101, as intended by the closer proximity of polymer chains 110 in FIG. 1C . The reduced open space of hydrogel 102 may reduce or inhibit diffusion of any reagents through the open space compared to the open spaces of hydrogels 100 and 101, for example, in a manner as described with reference to FIG. 1D . Additionally, crosslinks 131 within hydrogel 102 may be expected to significantly stabilize hydrogel 102 compared to hydrogels 100 and 101, for example, during storage or transport. Illustratively, if hydrogel 100 or 101 is transported or stored under conditions that cause the hydrogel to completely or partially dry out (lose water), the resulting reduction in the distance between polymer chains 110 may cause the polymer chains to become irreversibly hydrogen bonded to each other in such a manner as to irreversibly collapse the hydrogel and close off a significant portion of the open space in the hydrogel. Thus, even if water is added to a collapsed hydrogel, the water may not significantly penetrate the collapsed hydrogel, and the hydrogel may remain in a collapsed state that, at best, is of limited utility for further use. In comparison, the crosslinks 131 in the hydrogel 102 can hold the polymer chains 110 at a distance sufficiently spaced apart to inhibit such irreversible hydrogen bonding and, therefore, inhibit irreversible collapse of the hydrogel. Thus, reversing the crosslinks in the manner described below allows the hydrogel to swell back to an open state in which water and reagents can again diffuse into the hydrogel.

[0052] Additionally, the reduced open space of the hydrogel 102 can optionally be used to improve the monoclonality of clusters generated using the hydrogel 102 compared to clusters generated using the hydrogel 101. For example, the hydrogel 102 shown in FIG. 1C may be contacted with a fluid containing a target polynucleotide 160, which optionally includes first and second adaptors (not specifically shown) complementary to amplification primers 121 and 122, respectively. During such contact between the fluid and the hydrogel 102, the reduced open space within the three-dimensional crosslinked network of the hydrogel 102 may reduce or inhibit diffusion of the target polynucleotide 160 into the hydrogel 102 compared to diffusion within the hydrogel 101. As a result of the reduced open space within the hydrogel 102, the number of amplification primers 121 and 122 available for hybridization of the target polynucleotide 160 may be significantly less than the total number of amplification primers 121 and 122 within the hydrogel 102. Indeed, in some instances, the crosslinks 131 within the hydrogel 102 reduce the open space within the hydrogel 102 to the extent that the target polynucleotide 160 cannot substantially diffuse into the hydrogel and is therefore substantially limited to hybridizing to the amplification primers 121, 122 at the surface of the hydrogel. In comparison, if the hydrogel 101 were contacted with the same fluid containing the target polynucleotide 160, the target polynucleotide would be expected to readily diffuse into the hydrogel and hybridize to the amplification primers 121, 122 at a variety of different lateral locations and depths. Because the number of amplification primers available to hybridize with the target polynucleotide 160 is significantly reduced in the hydrogel 102 compared to the hydrogel 101, the likelihood of having only a single hybridization event (a single seeding event) is significantly higher in the hydrogel 102 than in the hydrogel 101. Therefore, it can be expected that clusters generated using the hydrogel 102 are significantly more likely to be monoclonal than clusters generated using the hydrogel 101.Illustratively, in the example shown in FIG. 1D , the crosslinks 131 sufficiently reduce the availability of amplification primers at greater depths within the hydrogel for such hybridization, so that the hydrogel 103 contains only a single target polynucleotide 160 hybridized to an amplification primer 121 at the surface of the hydrogel.

[0053] In the non-limiting example shown in FIG. 1D , after the target polynucleotide 160 hybridizes to the amplification primer 121, the crosslinks 131 between the second functional groups 130′ can be reversed. For example, the hydrogel 103 illustrated in FIG. 1D can be contacted with a fluid containing at least one chemical or enzymatic reagent configured to react with the crosslinks 131 and reverse the covalent or non-covalent crosslinks between the polymer chains 110. During such contact between the fluid and the hydrogel 103, the at least one chemical or enzymatic reagent can diffuse through the open spaces within the three-dimensional crosslink network of the hydrogel 103 and reverse the crosslinks 131 between the polymer chains 110 at various different lateral positions and various different depths, as intended to be shown in FIG. 1E . Note that upon initiation of such contact with the fluid, the hydrogel 103 can be substantially closed by the crosslinks 131 in a manner as described with reference to FIG. 1D . However, fluid may contact the surface of the hydrogel 103 and reverse crosslinks at that surface, thereby causing the hydrogel to open at its surface. Such opening may allow the fluid to diffuse slightly into the hydrogel, where it may reverse additional crosslinks and cause a slight opening of the hydrogel below the surface. In this manner, as the hydrogel gradually and directionally opens from its surface, fluid may diffuse even further into the hydrogel, eventually causing the entire hydrogel to reopen. The resulting open space may facilitate the diffusion of at least one chemical or enzymatic reagent through the open space. Such contact forms the modified hydrogel 104 shown in FIG. 1E, where the crosslinks using the second functional group 130′ are broken as intended, indicated using a shortened dashed line and reference number 131′.

[0054] In some examples, the target polynucleotide 160 hybridized to the amplification primer 121 can then be amplified using, for example, bridge amplification or other cluster-generating techniques described elsewhere herein or known in the art. For example, the target polynucleotide 160 can be amplified using processes such as those known in the art that use a strand invasion process, which may be referred to as surface-bound polymerase chain reaction (PCR), bridge amplification, or ExAmp, to form the clusters of amplicons 160′ shown in FIG. 1F. Such an amplification process can use chemical and / or enzymatic reagents that diffuse through the open spaces within the three-dimensional crosslinked network of the hydrogel 104, resulting in amplicons 160′ coupled to the polymer chains 110 at various different lateral positions and depths, as intended to be shown in FIG. 1F. As shown in FIG. 1F, the amplicons 160′ can be directly coupled to the polymer chains 110, for example, via the first functional groups 120 that originally coupled the primers 121, 122 to the polymer chains.

[0055] In some examples, the amount of open space in hydrogel 101 may be similar to the amount of open space in hydrogel 100, e.g., within about 20%, or within about 10%, or within about 5% of the open space in hydrogel 100. Additionally or alternatively, in some examples, the crosslink density of hydrogel 101 may be similar to the crosslink density of hydrogel 100, e.g., within about 20%, or within about 10%, or within about 5% of the crosslink density in hydrogel 100. Additionally or alternatively, in some examples, the amount of open space in hydrogel 103 may be similar to the amount of open space in hydrogel 102, e.g., within about 20%, or within about 10%, or within about 5% of the open space in hydrogel 102. Additionally or alternatively, in some examples, the crosslink density of hydrogel 103 may be similar to the crosslink density of hydrogel 102, e.g., within about 20%, or within about 10%, or within about 5% of the crosslink density within hydrogel 102. Additionally or alternatively, in some examples, the amount of open space within hydrogel 104 may be similar to the amount of open space within hydrogel 101, e.g., within about 20%, or within about 10%, or within about 5% of the open space within hydrogel 101. Additionally or alternatively, in some examples, the crosslink density of hydrogel 104 may be similar to the crosslink density of hydrogel 101, e.g., within about 20%, or within about 10%, or within about 5% of the crosslink density within hydrogel 101. Additionally or alternatively, in some examples, the amount of open space within hydrogel 105 may be similar to the amount of open space within hydrogel 101, e.g., within about 20%, or within about 10%, or within about 5% of the open space within hydrogel 101. Additionally or alternatively, in some examples, the crosslink density of hydrogel 105 may be similar to the crosslink density of hydrogel 101, e.g., within about 20%, or within about 10%, or within about 5% of the crosslink density within hydrogel 101.Additionally or alternatively, in some examples, the open space of hydrogel 102 or 103 may be significantly smaller than the open space of hydrogel 100, hydrogel 101, hydrogel 104, and / or hydrogel 105, e.g., less than about 80%, or less than about 60%, or less than about 40%, or less than about 20% of the open space in hydrogel 100, hydrogel 101, hydrogel 104, and / or hydrogel 105. Additionally or alternatively, in some examples, the crosslink density of hydrogel 102 or 103 may be significantly higher than the crosslink density of hydrogel 100, hydrogel 101, hydrogel 104, and / or hydrogel 105, e.g., about 20% higher, or about 40% higher, or about 60% higher, or about 80% higher than the crosslink density in hydrogel 100, hydrogel 101, hydrogel 104, and / or hydrogel 105. Additionally or alternatively, in some examples, hydrogels 100, 101, 104, and / or 105 may have a thickness that is at least 10 times greater than the thickness of hydrogels 102 and / or 103. For example, hydrogels 100, 101, 104, and / or 105 may have a thickness of about 80-120 nm, while hydrogels 102 and / or 103 may have a thickness of about 8-12 nm.

[0056] The amount of open space and / or crosslink density within a hydrogel can be characterized using mechanical measurements such as the elastic modulus determined from the force-distance curve in a wet atomic force microscopy (AFM). Additionally or alternatively, the amount of open space and / or crosslink density within a hydrogel can be measured by mesh size, for example, using small-angle x-ray scattering (SAXS) or small-angle neutron scattering (SANS). Additionally or alternatively, the amount of open space and / or crosslink density within a hydrogel can be characterized using the swelling capacity (mass ratio between dry and wet hydrogels), for example, using a quartz crystal microbalance (QCM) or ellipsometry in the wet state compared to the dry state. Additionally or alternatively, the amount of open space and / or crosslink density within a hydrogel can be characterized using the elastic modulus (E' from nanoindentation), which correlates with the hardness / softness of the hydrogel due to crosslink density. For example, nanoindentation can be used to differentiate between highly crosslinked and lightly crosslinked hydrogels or polymers. Illustratively, one exemplary strategy for assessing the openness of a hydrogel in its true state on the flow cell surface (e.g., approximately 80-120 nm thick in the wet state and approximately 8-12 nm thick in the dry state) is the use of fluorescent probes via hybridization or base incorporation. Alternatively, ellipsometry may be used to detect swelling. QCM is also compatible with thin hydrogel layers for assessing swelling, but in some instances, the hydrogel may be deposited on a QCM sensor instead of directly on the flow cell substrate. Nanoindentation can be used for relatively thin film hydrogels (e.g., <100 nm thick). QCM can be used to measure mass changes (open vs. closed) through water adsorption or to measure primers tethered inside the hydrogel.

[0057] Additionally or alternatively, the amount of open space and / or crosslink density within a hydrogel (e.g., hydrogel 101, 102, 103, or 104) containing amplification primers available for hybridization can be characterized using surface charge. For example, crosslinks can obscure the primers (which are negatively charged), thus preventing them from contributing to the surface charge. In some examples, surface charge can be characterized using atomic force microscopy (AFM), for example, by measuring the interaction between the surface and an AFM tip. Additionally or alternatively, Kelvin probe force microscopy can be used to characterize the surface charge of the hydrogel. Additionally or alternatively, electrokinetic analysis can be used to characterize the surface charge of the hydrogel at the solid-liquid interface. Additionally or alternatively, the amount of open space and / or crosslink density within a hydrogel (e.g., hydrogels 101, 102, 103, or 104) containing amplification primers available for hybridization can be characterized using complementary fluorescent oligonucleotide probes that hybridize to the amplification primers within the hydrogel, and the fluorescence intensity of the hybridized probes can be used to quantify the primers on the surface; for bulky probes such as hairpins, the fluorescence can be an indicator of how accessible the surface primers are to the template during a seeding event. Additionally or alternatively, the amount of open space and / or crosslink density within a hydrogel can be measured by quantifying broken bonds using infrared (IR) spectroscopy, e.g., by comparing the intensity of the crosslinker signal in the open or broken state with the intensity of the crosslinker signal in the crosslinked state. Additionally or alternatively, the amount of open space and / or crosslink density within a hydrogel can be measured by viscosity, e.g., by comparing the viscosity of a broken or open polymer to the viscosity of a fully crosslinked polymer. Additionally or alternatively, the amount of open space and / or crosslink density within a hydrogel can be measured by comparing the molecular weight of the hydrogel in an open or cut state to the molecular weight in a crosslinked state.

[0058] While FIGS. 1A-1F describe the use of first functional groups 120 and second functional groups 130 that are different from one another, it should be understood that the first and second functional groups may alternatively be the same type. For example, FIGS. 2A-2C schematically illustrate alternative exemplary compositions and operations in a process flow for using a reversibly crosslinked hydrogel for cluster amplification. As shown in FIG. 2A, a hydrogel 200 can be deposited on a substrate 140 in a manner similar to that described with reference to FIG. 1A. The hydrogel 200 can include a three-dimensional network of polymer chains 110 and first functional groups 120 coupled to the polymer chains. Amplification primers 121, 122 can be coupled to the first functional groups 120 of the deposited hydrogel in a manner similar to that described with reference to FIG. 1B. However, as shown in FIG. 2B, amplification primers 121, 122 may be coupled to a first subset of the deposited hydrogel's first functional groups 120 (such functional groups are represented by a darker shade and reference number 120′ to indicate their reaction). A second subset of the first functional groups 120 may remain unreacted, resulting in the modified hydrogel 210 shown in FIG. 2B. The second subset of the first functional groups 120 of the hydrogel 201 may then be converted to second functional groups 130, for example, in a manner as shown in FIG. 2C. For example, one or more suitable chemical or enzymatic reagents may be diffused into the hydrogel 201 to convert the second subset of the first functional groups 120 to second functional groups 130, resulting in the hydrogel 202 shown in FIG. 2C, which may be configured similarly to the hydrogel 101 described with reference to FIG. 1B. The hydrogel 202 can then be stabilized by reversibly crosslinking the second functional groups 130, for example, in a manner as described with reference to Figure 1C. The resulting reversibly crosslinked hydrogel can then be used in a manner as described with reference to Figures 1D-1F.

[0059] In another example, the hydrogel can be formed to include amplification primers. For example, FIG. 3 schematically illustrates another alternative exemplary composition and operation in a process flow for using a reversibly crosslinked hydrogel for cluster amplification. In the example shown in FIG. 3, a hydrogel 300 can be deposited on a substrate 140 in a manner similar to that described with reference to FIGS. 1A and 2B. The hydrogel 300 can include a three-dimensional network of polymer chains 110, similar to hydrogels 100 and 200. The hydrogel 300 can also include amplification primers 121, 122 coupled to the polymer chains 110. Illustratively, the amplification primers can be coupled to the polymer chains 110 during preparation of the hydrogel 300, e.g., before the hydrogel is deposited on the substrate 140. The hydrogel 300 can also include functional groups 230 coupled to the polymer chains 110 and configured similarly to the second functional groups 130 as described with reference to FIGS. 1A-1F and 2C. Hydrogel 300 can be stabilized by reversibly crosslinking functional groups 230, for example, in a manner as described with reference to Figure 1C. The resulting reversibly crosslinked hydrogel can then be used in a manner as described with reference to Figures 1D-1F.

[0060] It should be noted that the specific configurations and specific operational sequences described with reference to Figures 1A-1F, 2A-2C, and 3 can be suitably modified in a variety of different ways. For example, a reversibly cross-linked hydrogel (e.g., hydrogel 102 described with reference to Figure 1C) containing amplification primers 121, 122 can be prepared by a manufacturer and can be stable for storage and transportation to another entity, such as a customer. The entity can seed the polymer with target polynucleotides (e.g., to form hydrogel 103 described with reference to Figure 1D). The entity can then reverse cross-links 131 to open the hydrogel (e.g., to form hydrogel 104 described with reference to Figure 1E). The entity can then amplify the seeded target polynucleotides, within which cross-links have been reversed, to form clusters (e.g., to form hydrogel 105 described with reference to Figure 1F). The entity can optionally swell the hydrogel after reversing the cross-links and before amplification.

[0061] As further noted above, seeding does not necessarily have to be performed before reversing the hydrogel crosslinks. Illustratively, the hydrogel can be crosslinked after coupling the amplification primers to provide a crosslinked hydrogel 102 in a manner similar to that described with reference to FIGS. 1A-1C. Rather than seeding the crosslinked hydrogel 102 in the manner described with reference to FIG. 1D, in some instances, the crosslinks within the hydrogel 102 can instead be reversed before seeding. For example, FIGS. 8A-8B schematically illustrate additional exemplary compositions and operations in a process flow for using a reversibly crosslinked hydrogel for cluster amplification. The hydrogel 801 shown in FIG. 8A can be produced by cleaving the crosslinks within the hydrogel 102 described with reference to FIG. 1C in the manner described with reference to FIG. 1E, omitting the intervention of seeding the hydrogel as described with reference to FIG. 1D. After cleaving the crosslinks to form hydrogel 801 containing amplification primer 121 and broken crosslinks 131', the hydrogel can be seeded with target polynucleotide 160 in the manner shown in Figure 8B to form hydrogel 802, which is similar to hydrogel 104 described above but obtained through a different procedure.

[0062] Additionally or alternatively, as further described above, reversing the crosslinks need not necessarily be performed in whole or in its entirety prior to amplification. Instead, amplification can be performed partially or completely before reversing the crosslinks. For example, FIGS. 9A-9B schematically illustrate additional exemplary compositions and operations in a process flow for using a reversibly crosslinked hydrogel for cluster amplification. The hydrogel 901 shown in FIG. 9A can be produced by performing cluster amplification in a manner similar to that described with reference to FIG. 1F, but using the seeded crosslinked hydrogel 103 described with reference to FIG. 1D. Note that due to the crosslinking of the hydrogel 901, significantly fewer amplification primers 121 are accessible to the amplification reagents, and therefore the hydrogel 901 can contain significantly fewer amplicons 160′ than the hydrogel 105 described with reference to FIG. 1F. For example, amplicons 160' may be generated using amplification primers 121 close enough to the outer surface of hydrogel 901 to be available for cluster amplification, while other amplification primers 121 deeper within hydrogel 901 may remain inaccessible for cluster amplification due to crosslinks. The crosslinks within hydrogel 901 may then be cleaved in a manner as described with reference to FIG. 1E to provide hydrogel 902 (FIG. 9B) containing amplification primers 121 and broken crosslinks 131'. Optionally, amplicons 160' may be further amplified using newly accessible amplification primers 121 in a manner similar to that described with reference to FIG. 1F to form hydrogel 105 through a series of operations different from those described with reference to FIGS. 1E-1F.

[0063] Some non-limiting examples of functional groups and their uses for forming reversible crosslinks within hydrogels are provided herein.

[0064] Any suitable functional group can be included in the hydrogel and can be used to couple amplification primers 121, 122 to the hydrogel and / or to reversibly crosslink the polymer chains 110 of the hydrogel to one another. In some examples, the functional groups (e.g., 120, 130, and / or 230) can be independently selected from the group consisting of azide, amine, thiol, diol, aldehyde, alkyne, strained cyclooctyne, and inverse electron demand (IED) Diels-Alder groups. The functional groups can be included in the polymer chains 110 of the hydrogel 100, 200, or 300 during synthesis of the hydrogel. The crosslink density can be controlled by adjusting the content of functional moieties 120, 130, and / or 230 in the polymer chains. Additionally, as described above with reference to Figures 2A-2C, one type of functional group can be converted to another type of functional group within the hydrogel. In one non-limiting example where the functional groups 120 include azides, a first set of azides is used to couple the amplification primers 121, 122 to the polymer chains 110, and a second, unreacted set of azides is reduced to amines that are used as functional groups 130 to crosslink the polymer chains 110 to each other via a crosslinker molecule 131 that includes an amine-reactive functional group. Optionally, any suitable number of azides can be coupled to a heterobifunctional molecule that includes one or more different types of azide-reactive functional groups (such as alkynes or DBCO) and also has orthogonal functional groups (such as amines, thiols, diols, aldehydes, or inverse electron-demanding (IED) Diels-Alder groups). These functional groups can be used to crosslink the hydrogel using cleavable molecules that react with them.

[0065] 1A-1B, amplification primers 121, 122 can be coupled to a functional group that reacts with functional group 120 in such a manner as to couple amplification primers 121, 122 to polymer chain 110. In examples where functional group 120 is an amine, non-limiting examples of functional groups that can be coupled to amplification primers 121, 122 and react with the amine to couple the amplification primer to a polymer chain include N-hydroxysuccinimide (NHS) ester, imidoester, pentofluorophenyl ester, hydroxymethylphosphine, and carboxylcarbodiimide. In examples where functional group 120 is a thiol, non-limiting examples of functional groups that can be coupled to amplification primers 121, 122 and react with the thiol to couple the amplification primer to a polymer chain include maleimide, haloacetyl, pyridyl disulfide, thiosulfonate, and vinyl sulfone. In an example where the functional group 120 is an aldehyde, non-limiting examples of functional groups that can be coupled to the amplification primers 121, 122 and react with the aldehyde to couple the amplification primer to a polymer chain include hydrazides, alkoxyamines, and isocyanates. In an example where the functional group 120 is an azide, non-limiting examples of functional groups that can be coupled to the amplification primers 121, 122 and react with the azide to couple the amplification primer to a polymer chain include alkynes, cyclooctynes, phosphines, and norbornenes. In an example where the functional group is a diol, non-limiting examples of functional groups that can be coupled to the amplification primers 121, 122 and react with the diol to couple the amplification primer to a polymer chain include sulfonyl fluoride, where unreacted diol can be converted to a dialdehyde that can be used to crosslink polymer chains.In instances where functional group 120 is an IED Diels-Alder group, non-limiting examples of IED Diels-Alder groups that can be coupled to amplification primers 121, 122 and react with functional group 120 to couple the amplification primer to a polymer chain include transcyclooctene or norbornene (which can react with functional group 120, which is tetrazine); tetrazine (which can react with functional group 120, which is transcyclooctene or norbornene). It will be understood that any combination of the foregoing functional groups can be used; for example, the functional group coupled to the amplification primer can include an amine, thiol, diol, aldehyde, or IED Diels-Alder group, and that functional group 120 can be selected to react with such functional groups in a manner similar to that described above.

[0066] The functional groups 130, 230 can be used to reversibly crosslink polymer chains in any suitable manner, as described with reference to FIGS. 1C-1E, 2C, and 3. In some examples, the functional groups 130, 230 reversibly crosslink polymer chains via cleavable molecules. For example, the cleavable molecules can form covalent bonds with functional groups 130, 230 coupled to different polymer chains 110, thereby covalently crosslinking these polymer chains to each other, as shown in FIG. 1C. The cleavable molecules can include, for example, aliphatic, aromatic, hydrophilic, or amphiphilic moieties. The cleavable molecules can be cleavable in any suitable manner, for example, using chemical agents, enzymes, light, or heat. For example, the cleavable molecules can include at least one labile bond that can be cleaved upon application of a stimulus, such as a chemical agent, enzyme, light, or heat.

[0067] 6 schematically illustrates an exemplary cleavable molecule 600 that can be used to reversibly crosslink hydrogels in the manner described herein. Molecule 600 includes a first functional group 601, a second functional group 601′, an optional first linker 602, an optional second linker 602′, a first cleavable component 603, and a second cleavable component 603′. First linker 602 can couple first functional group 601 to first cleavable component 603; alternatively, if first linker 602 is omitted, first functional group 601 may be directly coupled to first cleavable component 603. The second linker 602 can couple the second functional group 601′ to the second cleavable component 603′; alternatively, if the second linker 602′ is omitted, the second functional group 601′ can be coupled directly to the second cleavable component 603′. The first cleavable component 603 can be coupled to the second cleavable component 603′ in the first relatively closed state of the hydrogel and can be detached from the second cleavable component in the second relatively open state of the hydrogel. The first functional group 601 can be coupled to a first one of the functional groups 130, and the second functional group 601′ can be coupled to a second one of the functional groups 130, in a manner as described with reference to FIG. 1C . Thus, molecule 600 can couple a first polymer chain 110 of hydrogel 101 (to which a first functional group of functional groups 130 is coupled) to a second polymer chain 110 (to which a second functional group of functional groups 130 is coupled), causing the hydrogel to adopt a relatively closed, more highly crosslinked configuration 102 as described with reference to FIG. 1C.

[0068] The optional linkers 602, 602′ can have the same configuration as each other or can have different configurations from each other. In some examples, one linker 602, 602′ is used and the other linker is omitted. The optional linkers 602, 602′ can independently comprise an alkyl chain, polyethylene glycol (PEG), peptide, or polyphosphate, such as those shown below, where * indicates a connection to the first or second functional group 601 or 601′ or the first or second cleavable component 603, 603′, R1 and R2 represent residues of natural or unnatural amino acids, and X represents a natural nucleoside or a spacer such as PEG or alkyl.

[0069] [ka] In non-limiting examples, n can range from about 1 to about 100, or from about 2 to about 80, or from about 5 to about 50, or from about 2 to about 16.

[0070] 6 , the first cleavable component 603 and the second cleavable component 603′ may correspond to any components of molecules that can be coupled to one another at a first time and cleaved from one another at a second, different time in response to exposure to a suitable stimulus. For example, the first cleavable component 603 and the second cleavable component 603′ may be covalently bonded to one another at a first time, e.g., while the hydrogel is in a relatively closed state as described with reference to FIGS. 1C and 1D , and the covalent bond may be dissociated at a second time to reopen the hydrogel, e.g., in a manner as described with reference to FIG. 1E . Table 1 below lists different types of chemical bonds that can be used to couple the first cleavable component to the second cleavable component 603′, as well as different categories of stimuli (e.g., chemical, enzymatic, or photolabile) that can be used to dissociate such bonds, and specific examples of such stimuli.

[0071] [Table 1]

[0072] Non-limiting examples of cleavable entities having a covalent bond that can be released using a chemical stimulus as listed in Table 1 are shown below:

[0073] [ka] wherein A and Q (if shown) correspond to the first or second functional group 601 or 601′, X and M (if shown) correspond to the first or second linker 602, 602′, the wavy line (if shown) corresponds to the first or second functional group 601 or 601′ or the first or second cleavable component 603, 603′, and R corresponds to a functional group containing one or more carbon atoms and optionally one or more heteroatoms, such as alkyl, allyl, or aryl. For further details regarding chemically cleavable linkers, see Leriche et al., “Cleavable linkers in chemical biology,” Bioorg. Med. Chem. 20(2):571-782 (2012), the entire contents of which are incorporated herein by reference.

[0074] Non-limiting examples of cleavable entities having covalent bonds that can be reversibly associated and dissociated using a thermal stimulus are shown below:

[0075] [ka] wherein R and R′ may be the same or different from each other and comprise functional groups that are reactive with functional groups on the polymer in a manner as described elsewhere herein.

[0076] In some instances where the cleavable molecule is cleavable by a chemical agent, the chemical agent may comprise an acid. Non-limiting examples of labile moieties include acetals, ketals, imines, hydrazones, or t-butyl esters that contain a bond that is cleavable by acid, for example, by use of a buffer having a pH of about 5 or less. An exemplary cleavable molecule is shown below, however, the ketal moiety may be replaced by any of the alternative labile moieties shown below in the molecule:

[0077] [ka]

[0078] For further details regarding pH-sensitive linkers containing imine, hydrazine, or acetal moieties, see Myrgorodska et al., "A novel acid-degradable PEG cross-linker for the fabrication of pH-responsive soft materials," Macro-Molecular Rapid Communications 42(12):2100102 (2021), the entire contents of which are incorporated herein by reference.

[0079] In one specific, non-limiting example, the acid may include periodic acid, sodium periodate, or lead tetraacetate. The cleavable molecule may include a vicinal diol that is cleavable by periodic acid. Non-limiting examples of cleavable molecules that include a vicinal diol that is cleavable by periodic acid include:

[0080] [ka] where A and Q correspond to the first or second functional group 601 or 601′, and X and M correspond to the first or second linker 602, 602′.

[0081] In other examples where the cleavable molecule is cleavable by a chemical agent, the chemical agent may include a reducing agent. A non-limiting example of a labile bond is a disulfide bond, which is cleavable using a reducing agent. Another non-limiting example of a labile bond is an azidoalkyl ether, which is cleavable using a reducing agent. Another non-limiting example of a labile bond is an allyl ether, which is cleavable using a palladium complex of a reducing agent. Illustratively, reducing agents may include glutathione, dithiothreitol (DTT), beta-mercaptoethanol (BME), cystamine, or tris(2-carboxyethyl)phosphine (TCEP). Non-limiting examples of cleavable molecules having a disulfide bond that is cleavable using a reducing agent include:

[0082] [ka] where A and Q correspond to the first or second functional group 601 or 601', and X and M correspond to the first or second linker 602, 602'. Non-limiting examples of cleavable molecules comprising azidoalkyl ethers that are cleavable using a reducing agent include:

[0083] [ka] where A and Q correspond to the first or second functional group 601 or 601′, and X and M correspond to the first or second linker 602, 602′.

[0084] 7 schematically illustrates additional exemplary cleavable molecules 701, 711, 721 that can be used to reversibly crosslink hydrogels in the manner described herein. Molecule 700 includes a first functional group 701, a second functional group 701′, a first linker 702, a second linker 702′, and a cleavable component 703, which can include a first cleavable component and a second cleavable component in a manner similar to that described with reference to FIG. 6. First linker 702 can couple first functional group 701 to cleavable component 703; alternatively, if first linker 702 is omitted, first functional group 701 can be directly coupled to cleavable component 703. A second linker 702′ can couple the second functional group 701′ to the cleavable component 703; alternatively, if the second linker 702′ is omitted, the second functional group 701′ may be coupled directly to the cleavable component 703. The cleavable components may be coupled to one another in a first, relatively closed state of the hydrogel and may be cleaved from one another in a second, relatively open state of the hydrogel, and / or may be completely or partially removed from the molecule 701. In a manner as described with reference to FIG. 1C , the first functional group 701 may be coupled to a first one of the functional groups 130, and the second functional group 701′ may be coupled to a second one of the functional groups 130. Thus, molecule 700 can couple a first polymer chain 110 (to which a first one of functional groups 130 is coupled) of hydrogel 101 to a second polymer chain 110 (to which a second one of functional groups 130 is coupled), causing the hydrogel to adopt a relatively closed, more highly crosslinked configuration 102 as described with reference to FIG. 1C. In the non-limiting example shown in FIG. 7, cleavable component 703 comprises a polypeptide that can be fully or partially degraded using a protease to generate gaps 704 that cause the hydrogel to reopen, in a manner as described with reference to FIG. 1E. Note that such proteases can be non-specific for the polypeptide.Alternatively, the cleavable component 703 may comprise a specific peptide sequence that is recognized and specifically cleaved by a lysosomal enzyme.

[0085] 7 includes a first functional group 711, a second functional group 711′, a first linker 712, a second linker 712′, and a cleavable component 713, which may include a first cleavable component and a second cleavable component in a manner similar to that described with reference to FIG. 6. The first linker 712 can couple the first functional group 711 to the cleavable component 713; alternatively, if the first linker 712 is omitted, the first functional group 711 may be directly coupled to the cleavable component 713. The second linker 712′ can couple the second functional group 711′ to the cleavable component 713; alternatively, if the second linker 712′ is omitted, the second functional group 711′ may be directly coupled to the cleavable component 713. The cleavable components can be coupled to one another in the first, relatively closed state of the hydrogel, can be cleaved from one another in the second, relatively open state of the hydrogel, and / or can be completely or partially removed from the molecule 711. In a manner as described with reference to FIG. 1C , the first functional group 711 can be coupled to a first functional group of the functional groups 130, and the second functional group 711′ can be coupled to a second functional group of the functional groups 130. Thus, the molecule 710 can couple the first polymer chain 110 (to which the first functional group of the functional groups 130 is coupled) of the hydrogel 101 to the second polymer chain 110 (to which the second functional group of the functional groups 130 is coupled), causing the hydrogel to adopt a relatively closed, more highly crosslinked configuration 102 as described with reference to FIG. 1C . In the non-limiting example shown in FIG. 7, cleavable component 713 comprises an oligonucleotide that can be fully or partially degraded using a nuclease (such as a DNAase in the example where the oligonucleotide comprises DNA, or an RNAase in the example where the oligonucleotide comprises RNA, or a protease in the example where the oligonucleotide comprises peptide nucleic acid (PNA)) to generate a gap 714 that causes the hydrogel to reopen in a manner as described with reference to FIG. 1E.Note that a DNAase may be non-specific for DNA oligonucleotides, an RNAase may be non-specific for RNA oligonucleotides, or a protease may be non-specific for PNA oligonucleotides. Alternatively, the cleavable component 703 may comprise a specific oligonucleotide sequence that is recognized and specifically cleaved by a restriction enzyme.

[0086] 7 includes a first functional group 721, a second functional group 721′, a first linker 722, a second linker 722′, and a cleavable component 723, which may include a first cleavable component and a second cleavable component in a manner similar to that described with reference to FIG. 6. The first linker 722 can couple the first functional group 721 to the cleavable component 723; alternatively, if the first linker 722 is omitted, the first functional group 721 may be coupled directly to the cleavable component 723. The second linker 722′ can couple the second functional group 721′ to the cleavable component 723; alternatively, if the second linker 722′ is omitted, the second functional group 721′ may be coupled directly to the cleavable component 723′. The cleavable components can be coupled to one another in the first, relatively closed state of the hydrogel, can be cleaved from one another in the second, relatively open state of the hydrogel, and / or can be completely or partially removed from the molecule 721. In a manner as described with reference to FIG. 1C , the first functional group 721 can be coupled to a first functional group of the functional groups 130, and the second functional group 721′ can be coupled to a second functional group of the functional groups 130. Thus, the molecule 720 can couple the first polymer chain 110 (to which the first functional group of the functional groups 130 is coupled) of the hydrogel 101 to the second polymer chain 110 (to which the second functional group of the functional groups 130 is coupled), causing the hydrogel to adopt a relatively closed, more highly crosslinked configuration 102 as described with reference to FIG. 1C . 7, the cleavable component 723 comprises a polymer that can be fully or partially degraded to generate a gap 724 that causes the hydrogel to reopen, in a manner as described with reference to FIG. 1E. In some examples, the polymer can comprise a (poly)ester that can be fully or partially degraded using a lipase to generate the gap 724.In other examples, the polymer may comprise an ester containing a crosslinker that can be cleaved in a manner as described herein, or an ester-containing oligomer / polymer that can be cleaved in a manner as described herein.

[0087] Functional groups 701, 701′, 711, 711′, and 721, 721′ may have the same configuration as one another or may have different configurations from one another, and in some examples may be selected from the options provided above for functional groups 601, 601′. Linkers 702, 702′, 712, 712′, and 722, 722′ may have the same configuration as one another or may have different configurations from one another, and in some examples may be selected from the options provided above for linkers 602, 602′.

[0088] In some examples where the cleavable molecule is enzymatically cleavable, the enzyme may comprise a restriction enzyme, and the cleavable molecule may comprise an oligonucleotide that is cleavable using the restriction enzyme in a manner as described with reference to molecule 710 in FIG. 7 . The oligonucleotide may comprise a functional group at each end that can form a covalent bond with functional groups 130, 230 to reversibly crosslink the polymer chains 110 to one another. In other examples where the cleavable molecule is enzymatically cleavable, the enzyme may comprise a protease enzyme, and the cleavable molecule may comprise a peptide that is cleavable using the protease enzyme in a manner as described with reference to molecule 700 in FIG. 7 . The peptide may comprise a functional group at each end that can form a covalent bond with functional groups 130, 230 to reversibly crosslink the polymer chains 110 to one another. Non-limiting examples of commercially available peptides that can be used as cleavable building blocks 703 and that are cleavable using proteases are shown below:

[0089] [ka]

[0090] In some examples, Val-Cit-PABS (valine-citrulline-p-aminobenzyl alcohol) can be coupled to functional groups such as maleimide and / or p-nitrophenyl carbonate for cross-linking. Another example of an enzyme-cleavable component 703 is matrix metalloproteinase-2 cleavable peptide (MMP2), which can also be functionalized with reactive groups for cross-linking. As provided elsewhere herein, the functional groups of the present cross-linkers can be identical to each other or different from each other.

[0091] In examples where the cleavable molecule is cleavable by light, the cleavable molecule may contain a coumarin group or a nitrobenzene group that is cleavable using light. A non-limiting example of a molecule containing a nitrobenzene group that is cleavable using light at about 360 nm is:

[0092] [ka]

[0093] For further details regarding molecules that can be cleaved using light (optionally in combination with chemical reagents), see Leriche et al., cited above, and Hansen et al., "Wavelength-selected cleavage of photoprotecting groups: strategies and applications in dynamic systems," Chemical Society Reviews 44:3358-3377 (2015), the entire contents of which are incorporated herein by reference.

[0094] FIG. 4 schematically illustrates an exemplary hydrogel and exemplary crosslinkers that may be used with such hydrogels. The exemplary hydrogel 400 shown in FIG. 4 comprises poly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM), as described in International Patent Publication No. 2013 / 184796, the entire contents of which are incorporated herein by reference. In the non-limiting example shown in FIG. 4, a first subset of functional groups 420 (e.g., azide groups) of hydrogel 400 are coupled to oligonucleotides 411, e.g., amplification primers, in a manner as described with reference to FIG. 1B via "primer grafting" as described with reference to FIG. 1B. A second subset of functional groups 420 (e.g., azide groups) are not coupled to amplification primers and thus may be considered "residual." Such residual functional groups are available for use in reversibly crosslinking the hydrogel. Optionally, the residual functional groups can be modified to form other types of functional groups. For example, in the non-limiting example shown in FIG. 4, a second subset of the azide groups can be contacted with a reducing agent, such as a phosphine, which reduces the azide groups to amines. The grafted hydrogel with the modified functional groups is then contacted with a reversibly cleavable molecule 430 (as described in more detail above, some non-limiting examples of which are shown in FIG. 4). In the non-limiting example shown in FIG. 4, the functional group (e.g., an NHS group) of the cleavable molecule 430 reacts with the amine resulting from the reaction of the azide with the reducing agent, thus forming a reversibly crosslinked hydrogel 400′ comprising polymer chains 110 that are crosslinked using the reacted cleavable molecule 431. Later (e.g., before or after seeding and / or amplification), the hydrogel 400' may be contacted with a stimulus suitable for cleaving the cleavable molecule 431, allowing the hydrogel to reopen in a manner as described with reference to Figure 1E.

[0095] Alternatively, non-covalent interactions can be used to crosslink the polymer chains 110 within the hydrogel, as described elsewhere herein. Illustratively, the functional groups 130, 230 can include host molecules that reversibly crosslink the backbone via guest molecules. The guest molecules can be removable by salt, heat, or pH. Alternatively, the guest molecules can be removable via displacement by a binding partner for the guest molecules. For example, FIGS. 5A-5B schematically illustrate additional exemplary hydrogels and exemplary crosslinkers that can be used with such hydrogels. The hydrogel 500 shown in FIG. 5A can be prepared using a procedure that includes reacting the functional groups of the hydrogel 400 from FIG. 4 with a host molecule 530. As shown in FIG. 5A, the functionalized hydrogel can include a functional group X that is reactive with the functional group X′ of the host molecule 530. X and X′ can include any suitable reactive pair of functional groups, non-limiting examples of which are provided elsewhere herein. As shown in FIG. 5A, the hydrogel is contacted with a guest molecule 532. During the complexation process, guest molecules 532 non-covalently bond to reacted host molecule moieties 531, which are coupled to different polymer chains 110, thus providing a hydrogel 500' comprising crosslinked polymer chains. During the degradation process, the non-covalent bonds between guest molecules 532 and host molecule moieties 531 are broken (e.g., using salt, heat, or pH), causing reversal of the crosslinks formed using the complexation process. In some examples, host molecule moieties 531 include macrocyclic pendant groups 533 or 534, and guest molecules 531 are suitably selected for use with such macrocyclic pendant groups. Various host-guest partners and their binding affinities have been reported in the literature. Non-limiting examples of hydrogels 510 and 510' are shown in FIG. 5B, which may be formed using pending groups 533 or 534, respectively, as shown in FIG. 5A. The hydrogel 510 includes a crown ether as a host molecule portion 531, which can be used with a guest molecule 532 that includes a terminal ammonium moiety coupled to a linker as described elsewhere herein.The hydrogel 510' includes beta-cyclodextrin as a host molecule portion 531, which may be used with a guest molecule 532, such as adamantane, ferrocene, or bipyridine.

[0096] In yet another example, the functional groups of the hydrogel can be coupled to ligand molecules that reversibly crosslink the backbone via a multivalent binding protein. The multivalent binding protein can be removed using a denaturing agent. For example, protein-ligand complexes held together by strong noncovalent interactions can be used. In a manner similar to that described with reference to macrocycle-guest binding, hydrogels 500 can include ligand molecules 535 in their backbone, as shown in FIG. 5A. Incubation with an appropriate multivalent binding protein can induce crosslinks between chains, which can be reversed by displacing the protein with excess free ligand or a strong chaotropic agent. One example of this is biotin-streptavidin binding, in which streptavidin binds up to four biotin molecules 535. Another example is binding between concanavalin A protein and four specific sugars, such as D-glucose and D-mannose. The hydrogel 510″ shown in FIG. 5B includes ligands 535, a non-limiting example of which is shown in FIG. 5A, which can be crosslinked using multivalent proteins. For example, strong but reversible interactions between certain proteins and their small molecule binding partners can be used to crosslink the hydrogel 510″. The small molecule ligands 535 bind to specific pockets in the protein through non-covalent interactions such as H-bonds, salt bridges, etc. When a protein is introduced into a polymer network with ligand side chains 535, the protein can bind to the ligands from two or more separate polymer chains, resulting in a crosslinked hydrogel. Crosslinking can be reversed by introducing excess free-floating ligands to outcompete the binding of the polymer side chains.

[0097] Further comments It will be understood that the present compositions and methods can be used in any suitable application, such as amplifying a template polynucleotide. For example, the present compositions can be used to perform "bridge amplification" or "surface-bound polymerase chain reaction," although it will be understood that the present compositions and methods can be easily adapted for use with other amplification modalities. One such amplification modality is "exclusion amplification," or ExAmp. Exclusion amplification methods can enable the amplification of a single target polynucleotide per hydrogel region and the production of a substantially monoclonal population of amplicons in the region. For example, the amplification rate of a first seeded target polynucleotide within a hydrogel region can be rapid compared to the very slow transport rate and seeding of the target polynucleotide in the region. Thus, a first target polynucleotide seeded into a hydrogel region can be rapidly amplified, filling the entire hydrogel region and thus inhibiting the seeding of additional target polynucleotides in the same hydrogel region. Alternatively, if a second target polynucleotide is attached to the same hydrogel region after the first polynucleotide, relatively rapid amplification of the first target polynucleotide may sufficiently fill the region (e.g., the region may be at least functionally monoclonal) to produce a signal strong enough to perform sequencing biosynthesis. The use of exclusion amplification may also result in a super-Poisson distribution of monoclonal regions, i.e., the fraction of regions within the array that are functionally monoclonal may exceed the fraction predicted by a Poisson distribution.

[0098] Increasing the super-Poisson distribution of useful clusters is useful because more functionally monoclonal regions can result in higher-quality signals and therefore improved SBS. However, seeding of target polynucleotides into hydrogel regions can follow a spatial Poisson distribution, and the tradeoff for increasing the number of occupied regions is increasing the number of polyclonal regions. One way to achieve a higher super-Poisson distribution is for seeding to occur rapidly, followed by a delay between seeded target polynucleotides. This delay, called "kinetic delay," is thought to occur through biochemical reaction kinetics, allowing some seeded target polynucleotides to initiate earlier than other seeded targets. Exclusion amplification works by using a recombinase to promote infiltration of primers (e.g., primers attached to substrate regions) into double-stranded DNA (e.g., target polynucleotides) when the recombinase mediates a sequence match. The present compositions and methods can be adapted for use with recombinases to promote infiltration of the present amplification primers and orthogonal amplification primers into the present target polynucleotides when the recombinase mediates a sequence match. Indeed, the compositions and methods of the present invention can be adapted for use with any surface-based polynucleotide amplification method, such as thermal PCR, chemically denaturing PCR, and enzyme-mediated methods (which may be referred to as recombinase polymerase amplification (RPA) or ExAmp).

[0099] While various illustrative examples have been described above, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the invention. It is intended that the appended claims cover all such changes and modifications that fall within the true spirit and scope of the invention.

[0100] It should be understood that any respective feature / example of each of the aspects of the present disclosure described herein may be implemented together in any suitable combination, and that any feature / example from any one or more of these aspects may be implemented together in any suitable combination with any of the features of the other aspects described herein, to achieve the benefits described herein.

Claims

1. A hydrogel on a substrate, comprising: a three-dimensional network of polymer chains; a first functional group coupled to the polymer chain; an amplification primer coupled to the polymer strand via the first functional group; and second functional groups coupled to said polymer chains that reversibly crosslink said polymer chains to one another.

2. The hydrogel of claim 1 , wherein the first and second functional groups are of different types.

3. The hydrogel of claim 1 , wherein the first and second functional groups are of the same type.

4. 4. The hydrogel of claim 1, wherein the first and second functional groups are independently selected from the group consisting of azide, amine, thiol, diol, aldehyde, alkyne, strained cyclooctyne, and inverse electron demand (IED) Diels-Alder groups.

5. The hydrogel of any one of claims 1 to 4, wherein the second functional groups reversibly crosslink the polymer chains via cleavable molecules.

6. 6. The hydrogel of claim 5, wherein the cleavable molecule is cleavable using a chemical agent, an enzyme, light, or heat.

7. The hydrogel of claim 6 , wherein the chemical agent comprises an acid.

8. 8. The hydrogel of claim 7, wherein the cleavable molecule comprises an acetal, ketal, imine, hydrazone, or t-butyl ester that is cleavable by the acid.

9. The hydrogel of claim 6 , wherein the chemical agent comprises a reducing agent.

10. 9. The hydrogel of claim 8, wherein the cleavable molecule comprises a disulfide bond or an azidoalkyl ether that is cleavable using the reducing agent, or an allyl ether that is cleavable using a palladium complex of the reducing agent.

11. 7. The hydrogel of claim 6, wherein the enzyme comprises a DNAase, RNAse, protease, or restriction enzyme, and the cleavable molecule comprises an oligonucleotide that is cleavable using the DNAase, RNAase, protease, or restriction enzyme.

12. 7. The hydrogel of claim 6, wherein the enzyme comprises a protease enzyme or a lysosomal enzyme, and the cleavable molecule comprises a peptide that is cleavable using the protease enzyme or the lysosomal enzyme.

13. 7. The hydrogel of claim 6, wherein the cleavable molecule comprises a Diels-Alder conjugation that is cleavable using heat.

14. 7. The hydrogel of claim 6, wherein the cleavable molecule comprises a coumarin group or a nitrobenzene group that is cleavable using light.

15. 10. The hydrogel of claim 1, wherein the second functional group comprises a host molecule that reversibly crosslinks the backbone via a guest molecule.

16. 16. The hydrogel of claim 15, wherein the guest molecule is removable via salt, heat, or pH.

17. 17. The hydrogel of claim 15 or 16, wherein the guest molecule is removable via displacement with a binding partner for the guest molecule.

18. 18. The hydrogel of any one of claims 15 to 17, wherein the host molecule comprises a crown ether and the guest molecule comprises an ammonium moiety.

19. 19. The hydrogel of any one of claims 15 to 18, wherein the host molecule comprises beta-cyclodextrin and the guest molecule comprises adamantane, ferrocene, or bipyridine.

20. 10. The hydrogel of claim 1, wherein the second functional groups comprise ligand molecules that reversibly crosslink the backbone via multivalent binding proteins.

21. 21. The hydrogel of claim 20, wherein the multivalent binding protein is removable using a denaturing agent.

22. 1. A method of using a hydrogel, comprising: depositing a hydrogel onto a substrate, the hydrogel comprising a three-dimensional network of polymer chains and at least first and second types of functional groups coupled to the polymer chains; coupling an amplification primer to the first functional group of the deposited hydrogel; reversibly stabilizing the deposited hydrogel by reversibly crosslinking the second functional groups of the deposited hydrogel to which the amplification primers are coupled.

23. 1. A method of using a hydrogel, comprising: depositing a hydrogel onto a substrate, the hydrogel comprising a three-dimensional network of polymer chains, amplification primers coupled to the polymer chains, and functional groups coupled to the polymer chains; reversibly stabilizing the hydrogel by reversibly crosslinking the functional groups of the deposited hydrogel to which the amplification primers are coupled.

24. 1. A method of using a hydrogel, comprising: depositing a hydrogel onto a substrate, the hydrogel comprising a three-dimensional network of polymer chains and first functional groups coupled to the polymer chains; coupling amplification primers to a first subset of the first functional groups of the deposited hydrogel; converting a second subset of the first functional groups to second functional groups; reversibly stabilizing the hydrogel by reversibly crosslinking the second functional groups.

25. 1. A method of using a hydrogel, comprising: hybridizing a target polynucleotide to an amplification primer coupled to a hydrogel; cleaving crosslinks within the hydrogel where the target polynucleotide is hybridized to the amplification primer; amplifying the target polynucleotide using additional amplification primers within the hydrogel where the crosslinks have been cleaved.

26. 26. The method of claim 25, further comprising swelling the hydrogel after said cutting and before said amplifying.

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