Patent Application
20240182959
This application relates to hydrogels, such as may be used in cluster amplification.
Cluster amplification is an approach to amplifying polynucleotides, for example for use in genetic sequencing. Target polynucleotides are captured by primers (e.g., P5 and P7 primers) coupled to a substrate surface in a flowcell, and form “seeds” at random locations on the surface. Cycles of amplification are performed to form clusters on the surface around each seed. The clusters include copies, and complementary copies, of the seed polynucleotides. In some circumstances, the substrate is patterned so as to define regions that bound different clusters, such as wells that may be filled with respective clusters.
Examples provided herein are related to reversibly cross-linked hydrogels, and methods of using the same for cluster amplification.
Some examples herein provide a hydrogel on a substrate. The hydrogel may include a three-dimensional network of polymer chains. The hydrogel may include first functional groups coupled to the polymer chains. The hydrogel may include amplification primers coupled to the polymer chains via the first functional groups. The hydrogel may include second functional groups coupled to the polymer chains and reversibly cross-linking the polymer chains to one another.
In some examples, the first and second functional groups are of different types than one another. In other examples, the first and second functional groups are of the same type as one another. In some examples, the first and second functional groups independently are selected from the group consisting of: azide, amine, thiol, diol, aldehyde, alkyne, strained cyclooctyne, and an inverse electron-demand (IED) Diels-Alder group.
In some examples, the second functional groups reversibly cross-link the polymer chains via cleavable molecules. In some examples, the cleavable molecules are cleavable using a chemical agent, an enzyme, light, or heat. In some examples, the chemical agent includes an acid. In some examples, the cleavable molecules include an acetal, ketal, imine, hydrazone, or t-butyl ester that is cleavable by the acid. In other examples, the chemical agent includes a reducing agent. In some examples, the cleavable molecules include a disulfide bond or azidoalkyl ether that is cleavable using the reducing agent, or allyl ether that is cleavable using a palladium complex of the reducing agent. In other examples, the enzyme includes a DNAase, RNAase, protease, or restriction enzyme, and wherein the cleavable molecules include an oligonucleotide that is cleavable using the DNAase, RNAase, protease, or restriction enzyme. In other examples, the enzyme includes a protease enzyme or lysosomal enzyme, and wherein the cleavable molecules include a peptide that is cleavable using the protease enzyme or lysosomal enzyme. In other examples, the cleavable molecules include a Diels-Alder conjugation that is cleavable using heat. In other examples, the cleavable molecules include a coumarin or nitrobenzene group that is cleavable using light.
In some examples, the second functional groups include host molecules that reversibly cross-link the backbone via guest molecules. In some examples, the guest molecules are removable via salt, heat, or pH. Additionally, or alternatively, in some examples, the guest molecules are removable via displacement with a binding partner to the guest molecules. Additionally, or alternatively, in some examples the host molecules include crown ethers and the guest molecules include ammonium moieties. Additionally, or alternatively, host molecules include beta-cyclodextrins and the guest molecules include adamantanes, ferrocenes, or bipyridines. In some examples, the second functional groups include ligand molecules that reversibly cross-link the backbone via multivalent binding proteins. In some examples, the multivalent binding proteins are removable using a denaturing agent.
Some examples herein provide a method of using a hydrogel. The method 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 method may include coupling amplification primers to the first functional groups of the deposited hydrogel. The method may include reversibly stabilizing the deposited hydrogel by reversibly cross-linking the second functional groups of the deposited hydrogel to which the amplification primers are coupled.
Some examples herein provide another method of using a hydrogel. The method may include depositing a hydrogel on a substrate. The hydrogel may 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 may include reversibly stabilizing the hydrogel by reversibly cross-linking the functional groups of the deposited hydrogel to which the amplification primers are coupled.
Some examples herein provide another method of using a hydrogel. The method may include depositing a hydrogel on a substrate, the hydrogel including 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 cross-linking the second functional groups.
Some examples herein provide another method of using a hydrogel. The method may include hybridizing a target polynucleotide to an amplification primer coupled to a hydrogel. The method may include cleaving cross-linkages within the hydrogel within which the target polynucleotide is hybridized to the amplification primer. The method may include amplifying the target polynucleotide using additional amplification primers within the hydrogel within which the cross-linkages have been cleaved. Optionally, the method further may include swelling the hydrogel after the cleaving and before the amplifying.
It is to be understood that any respective features/examples of each of the aspects of the disclosure as described herein may be implemented together in any appropriate combination, and that any features/examples from any one or more of these aspects may be implemented together with any of the features of the other aspect(s) as described herein in any appropriate combination to achieve the benefits as described herein.
Examples provided herein are related to reversibly cross-linked hydrogels, and methods of using the same for cluster amplification.
It may be desirable to perform sequencing-by-synthesis (SBS) using functionalized hydrogels within flowcells, to determine the sequence of target polynucleotides in respective clusters. For example, a hydrogel may include functional groups to which amplification primers may be coupled. The hydrogel then may be seeded by flowing through the flowcell a target polynucleotide to which has been coupled an adapter that is complementary to the amplification primers, and that hybridizes to one of the primers. The seed then may be amplified using amplification primers that are within the hydrogel, resulting in a cluster including a plurality of amplicons of the target polynucleotide that then may be sequenced using SBS.
However, certain previously known hydrogels may degrade during shipping or storage, which may cause their performance to deteriorate. For example, during shipping or storage, the hydrogel may irreversibly collapse. As such, amplification primers within the hydrogel may be inaccessible during amplification, resulting in generation of clusters that include fewer amplicons and therefore generate less signal during SBS than would clusters formed using a hydrogel that was not so degraded. This can reduce accuracy of the sequencing read.
The reversibly cross-linked hydrogels provided herein are expected to be significantly more stable during shipping and storage than previously known hydrogels such as described above. More specifically, the present hydrogels may be cross-linked prior to shipping and storage. The cross-linkages may reduce or inhibit irreversible collapse of the hydrogel during shipping and storage, for example by limiting polymer chain mobility to form strong inter-chain physical bonds and/or intra-chain physical bonds, in other words, by limiting polymer chain mobility to rearrange into a lower entropy state that would be energetically unfavorable to reverse upon rehydration. The cross-linkages may be controllably reversed, e.g., cleaved or removed by applying an appropriate stimulus, following which the hydrogel may be used as desired.
Additionally, in some examples, the seeding process may be performed before reversing the cross-linkages. As such, amplification primers within the hydrogel may be relatively inaccessible during the seeding process, while amplification primers at the exposed surface of the hydrogel may be used for seeding. In some examples, the cross-linking then may be reversed, making the amplification primers within the hydrogel accessible for amplification. Using the present reversible cross-linking to reduce the number of amplification primers that are available for seeding may reduce the number (or density) of seeding events that occur and thus may improve monoclonality of the clusters that are later formed than would a hydrogel that was not cross-linked during seeding. Additionally, using the present reversible cross-linking to subsequently increase the number of amplification primers that are available for amplification may generate more amplicons than would a hydrogel that had irreversibly collapsed because it had not been cross-linked. As such, it may be understood that the present reversibly cross-linked hydrogels may be expected to provide clusters with enhanced monoclonality and enhanced amplification as compared to previously known hydrogels. However, it will be appreciated that the cross-linking may be used at any suitable time, e.g., may be used to improve hydrogel stability during shipping and storage and may be reversed prior to seeding, and need not necessarily be used to modulate accessibility of amplification primers during seeding and/or amplification.
First, some terms used herein will be briefly explained. Then, some example methods for reversibly cross-linking hydrogels, resulting compositions, and methods of using reversibly cross-linked hydrogels will be described.
Terms
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. The use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting. The use of the term “having” as well as other forms, such as “have,” “has,” and “had,” is not limiting. As used in this specification, whether in a transitional phrase or in the body of the claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the above terms are to be interpreted synonymously with the phrases “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.
The terms “substantially,” “approximately,” and “about” used throughout this specification are used to describe and account for small fluctuations, such as due to variations in processing. For example, they may refer to less than or equal to ±10%, such as less than or equal to ±5%, such as less than or equal to ±2%, such as less than or equal to ±1%, such as less than or equal to ±0.5%, such as less than or equal to ±0.2%, such as less than or equal to ±0.1%, such as less than or equal to ±0.05%.
As used herein, “hybridize” is intended to mean noncovalently associating a first polynucleotide to a second polynucleotide along the lengths of those polynucleotides to form a double-stranded “duplex.” For instance, two DNA polynucleotide strands may associate through complementary base pairing. The strength of the association between the first and second polynucleotides increases with the complementarity between the sequences of nucleotides within those polynucleotides. The strength of hybridization between polynucleotides may be characterized by a temperature of melting (Tm) at which 50% of the duplexes have polynucleotide strands that disassociate from one another.
As used herein, the term “nucleotide” is intended to mean a molecule that includes a sugar and at least one phosphate group, and in some examples also includes a nucleobase. A nucleotide that lacks 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), uridine triphosphate (UTP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), 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).
As used herein, the term “nucleotide” also is intended to encompass any nucleotide analogue which is a type of nucleotide that includes a modified nucleobase, sugar and/or phosphate moiety compared to naturally occurring nucleotides. Example modified nucleobases include inosine, xathanine, hypoxathanine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethyl cytosine, 2-aminoadenine, 6-methyl adenine, 6-methyl guanine, 2-propyl guanine, 2-propyl adenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 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 or the like. As is known in the art, certain nucleotide analogues cannot become incorporated into a polynucleotide, for example, nucleotide analogues such as adenosine 5′-phosphosulfate. Nucleotides may include any suitable number of phosphates, e.g., three, four, five, six, or more than six phosphates.
As used herein, the term “polynucleotide” refers to a molecule that includes a sequence of nucleotides that are bonded to one another. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and analogues thereof. A polynucleotide may 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 may include a mixture of a single stranded 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 may include non-naturally occurring DNA, such as enantiomeric DNA. The precise sequence of nucleotides in a polynucleotide may be known or unknown. The following are examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, expressed sequence tag (EST) or serial analysis of gene expression (SAGE) tag), genomic DNA, genomic DNA fragment, exon, intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, recombinant polynucleotide, synthetic polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, primer or amplified copy of any of the foregoing.
As used herein, a “polymerase” is intended to mean an enzyme having an active site that assembles polynucleotides by polymerizing nucleotides into polynucleotides. A polymerase can bind a primed single stranded target polynucleotide, and can sequentially add nucleotides to the growing primer to form a “complementary copy” polynucleotide having a sequence that is complementary to that of the target polynucleotide. Another polymerase, or the same polymerase, then can form a copy of the target nucleotide by forming a complementary copy of that complementary copy polynucleotide. Any of such copies may be referred to herein as “amplicons.” DNA polymerases may bind to the target polynucleotide and then move down the target polynucleotide sequentially adding nucleotides to the free hydroxyl group at the 3′ end of a growing polynucleotide strand (growing amplicon). DNA polymerases may synthesize complementary DNA molecules from DNA templates and RNA polymerases may synthesize RNA molecules from DNA templates (transcription). Polymerases may use a short RNA or DNA strand (primer), to begin strand growth. Some polymerases may displace the strand upstream of the site where they are adding bases to a chain. Such polymerases may be said to be strand displacing, meaning they have an activity that removes a complementary strand from a template strand being read by the polymerase. Example polymerases having strand displacing activity include, without limitation, the large fragment of Bst (Bacillus stearothermophilus) polymerase, exo-Klenow polymerase or sequencing grade T7 exo-polymerase. Some polymerases degrade the strand in front of them, effectively replacing it with the growing chain behind (5′ exonuclease activity). Some polymerases have an activity that degrades the strand behind them (3′ exonuclease activity). Some useful polymerases have been modified, either by mutation or otherwise, to reduce or eliminate 3′ and/or 5′ exonuclease activity.
As used herein, the term “primer” is defined as a polynucleotide to which nucleotides may be added via a free 3′ OH group. A primer may include a 3′ block preventing polymerization until the block is removed. A primer may include a modification at the 5′ terminus to allow a coupling reaction or to couple the primer to another moiety. A primer may include one or more moieties which may be cleaved under suitable conditions, such as UV light, chemistry, enzyme, or the like. The primer length may be any suitable number of bases long and may include any suitable combination of natural and non-natural nucleotides. A target polynucleotide may include an “adapter” that hybridizes to (has a sequence that is complementary to) a primer, and may be amplified so as to generate a complementary copy polynucleotide by adding nucleotides to the free 3′ OH group of the primer. An “amplification primer” is intended to mean a primer that is coupled to the substrate and may hybridize to a second adapter of the target polynucleotide, while an “orthogonal amplification primer” is intended to mean a primer that is coupled to the substrate and may hybridize to a first adapter of that target polynucleotide. The first adapter may have a sequence that is complementary to that of the orthogonal amplification primer, and the second adapter may have a sequence that is complementary to that of the amplification primer. An amplification primer and an orthogonal amplification primer may have different and independent sequences than one another. In one nonlimiting example, the amplification primer includes a P5 primer, and the orthogonal amplification primer includes a P7 primer.
As used herein, the term “substrate” refers to a material used as a support for compositions described herein. Example substrate materials may include glass, silica, plastic, quartz, metal, metal oxide, organo-silicate (e.g., polyhedral organic silsesquioxanes (POSS)), polyacrylates, tantalum oxide, complementary metal oxide semiconductor (CMOS), or combinations thereof. An example of POSS can be that described in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776-778, which is incorporated by reference in its entirety. In some examples, substrates used in the present application include silica-based substrates, such as glass, fused silica, or other silica-containing material. In some examples, substrates may include silicon, silicon nitride, or silicone hydride. In some examples, substrates used in the present application include plastic materials or components such as polyethylene, polystyrene, poly(vinyl chloride), polypropylene, nylons, polyesters, polycarbonates, and poly(methyl methacrylate). Example plastics materials include poly(methyl methacrylate), polystyrene, and cyclic olefin polymer substrates. In some examples, the substrate is or includes a silica-based material or plastic material or a combination thereof. In particular examples, the substrate has at least one surface comprising glass or a silicon-based polymer. In some examples, the substrates may include a metal. In some such examples, the metal is gold. In some examples, the substrate has at least one surface comprising a metal oxide. In one example, the surface comprises a tantalum oxide or tin oxide. Acrylamides, enones, or acrylates may also be utilized as a substrate material or component. Other substrate materials may include, but are not limited to gallium arsenide, indium phosphide, aluminum, ceramics, polyimide, quartz, resins, polymers and copolymers. In some examples, the substrate and/or the substrate surface may be, or include, quartz. In some other examples, the substrate and/or the substrate surface may be, or include, semiconductor, such as GaAs or ITO. The foregoing lists are intended to be illustrative of, but not limiting to the present application. Substrates may comprise a single material or a plurality of different materials. Substrates may be composites or laminates. In some examples, the substrate comprises an organo-silicate material. Substrates may be flat, round, spherical, rod-shaped, or any other suitable shape. Substrates may be rigid or flexible. In some examples, a substrate is a bead or a flow cell.
In some examples, a substrate includes a patterned surface. A “patterned surface” refers to an arrangement of different regions in or on an exposed layer of a substrate. For example, one or more of the regions may be features where one or more amplification primers are present, for example in a hydrogel located within that feature. The features can be separated by interstitial regions where amplification primers and hydrogel are not present. In some examples, the pattern may be an x-y format of features that are in rows and columns. In some examples, the pattern may be a repeating arrangement of features and/or interstitial regions. In some examples, the pattern may be a random arrangement of features and/or interstitial regions. In some examples, a substrate includes an array of wells (depressions) in a surface. The wells may be provided by substantially vertical sidewalls. Wells may be fabricated as is generally known in the art using a variety of techniques, including, but not limited to, photolithography, stamping techniques, molding techniques and microetching techniques. As will be appreciated by those in the art, the technique used will depend on the composition and shape of the array substrate.
The features in a patterned surface of a substrate may include wells in an array of wells (e.g., microwells or nanowells) on glass, silicon, plastic or other suitable material(s) with patterned hydrogel such as provided herein. The patterning may provide hydrogel pads that may be used for sequencing, e.g., may be stable over sequencing runs with a large number of cycles. In some examples, covalent linking of the hydrogel to the wells may be helpful for maintaining the hydrogel in the structured features (e.g., wells) throughout the lifetime of the structured substrate during a variety of uses. However in some examples, the hydrogel need not be covalently linked to the wells.
In particular examples, a structured substrate may be made by patterning a substrate formed of 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, for example via chemical or mechanical polishing, thereby retaining hydrogel in 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 may be attached to the hydrogel, e.g., in a manner such as provided herein. A solution including a plurality of target polynucleotides (e.g., a fragmented human genome or portion thereof) may then be contacted with the polished substrate such that individual target polynucleotides will seed individual wells via interactions with amplification primers attached to the hydrogel; however, the target polynucleotides will not occupy the interstitial regions due to absence or inactivity there of the hydrogel. Amplification of the target polynucleotides may be confined to the wells because absence or inactivity of hydrogel in the interstitial regions may inhibit outward migration of the growing cluster. The process is conveniently manufacturable, being scalable and utilizing conventional micro- or nano-fabrication methods.
A patterned substrate may include, for example, wells etched into a slide or chip. The pattern of the etchings and geometry of the wells may take on a variety of different shapes and sizes, and such features may be physically or functionally separable from each other. Particularly useful substrates having such structural features include patterned substrates that may select the size of solid particles such as microspheres. An example patterned substrate having these characteristics is the etched substrate used in connection with BEAD ARRAY technology (Illumina, Inc., San Diego, Calif.).
In some examples, a substrate described herein forms at least part of a flow cell or is located in or coupled to a flow cell. Flow cells may include a flow chamber that is divided into a plurality of lanes or a plurality of sectors. Example flow cells and substrates for manufacture of flow cells that may be used in methods and compositions set forth herein include, but are not limited to, those commercially available from Illumina, Inc. (San Diego, CA).
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 substrate's surface without a significant intermediate layer, such as, e.g., an adhesive layer or a polymer layer. Layers directly covering a surface may be attached to this surface through any chemical or physical interaction, including covalent bonds or non-covalent adhesion.
As used herein, the term “immobilized” when used in reference to a polynucleotide is intended to mean direct or indirect attachment to a substrate via covalent or non-covalent bond(s). In certain examples, covalent attachment may be used, or any other suitable attachment in which the polynucleotides remain stationary or attached to a substrate under conditions in which it is intended to use the substrate, for example, in polynucleotide amplification or sequencing. Polynucleotides to be used as amplification primers or as target polynucleotides may be immobilized such that a 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 the 3′-5′ orientation. Alternatively, immobilization may occur by means other than base-pairing hybridization, such as covalent attachment.
As used herein, the term “array” refers to a population of substrate regions that may be differentiated from each other according to relative location. Different molecules (such as polynucleotides) that are at different regions of an array may be differentiated from each other according to the locations of the regions in the array. An individual region of an array may include one or more molecules of a particular type. For example, a substrate region may include a single target polynucleotide having a particular sequence, or a substrate region may include several polynucleotides having the same sequence (or complementary sequences thereof). The regions of an array respectively may include different features than one another on the same substrate. Example features include without limitation, wells in a substrate, beads (or other particles) in or on a substrate, projections from a substrate, ridges on a substrate or channels in a substrate. The regions of an array respectively may include different regions on different substrates than each other. Different molecules attached to separate substrates may be identified according to the locations of the substrates on a surface to which the substrates are associated or according to the locations of the substrates in a liquid or gel. Example arrays in which separate substrates are located on a surface include, without limitation, those having beads in wells.
As used herein, the term “plurality” is intended to mean a population of two or more different members. Pluralities may range in size from small, medium, large, to very large. The size of small plurality may range, for example, from a few members to tens of members. Medium sized pluralities may range, for example, from tens of members to about 100 members or hundreds of members. Large pluralities may range, for example, from about hundreds of members to about 1000 members, to thousands of members and up to tens of thousands of members. Very large pluralities may range, for example, from tens of thousands of members to about hundreds of thousands, a million, millions, tens of millions and up to or greater than hundreds of millions of members. Therefore, a plurality may range in size from two to well over one hundred million members as well as all sizes, as measured by the number of members, in between and greater than the above example ranges. Example polynucleotide pluralities include, for example, populations of about 1×105 or more, 5×105 or more, or 1×106 or more different polynucleotides. Accordingly, the definition of the term is intended to include all integer values greater than two. An upper limit of a plurality may be set, for example, by the theoretical diversity of polynucleotide sequences in a sample.
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 respective nucleotides in a complementary polynucleotide.
As used herein, the term “single-stranded,” when used in reference to a polynucleotide, means that essentially none of the nucleotides in the polynucleotide are hydrogen bonded to a respective nucleotide in a complementary polynucleotide.
As used herein, the term “target polynucleotide” is intended to mean a polynucleotide that is the object of an analysis or action. The analysis or action includes subjecting the polynucleotide to amplification, sequencing and/or other procedure. A target polynucleotide may include nucleotide sequences additional to a target sequence to be analyzed. For example, a target polynucleotide may include one or more adapters, including an adapter that functions as a primer binding site, that flank(s) a target polynucleotide sequence that is to be analyzed. A target polynucleotide hybridized to an amplification primer may include nucleotides that extend beyond the 5′ or 3′ end of the amplification primer in such a way that not all of the target polynucleotide is amenable to extension. In particular examples, target polynucleotides may have different sequences than one another but may have first and second adapters that are the same as one another. The two adapters that may flank a particular target polynucleotide sequence may have the same sequence as one another, or complementary sequences to one another, or the two adapters may have different sequences. Thus, species in a plurality of target polynucleotides may include regions of known sequence that flank regions of unknown sequence that are to be evaluated by, for example, sequencing (e.g., SBS). In some examples, target polynucleotides carry an adapter at a single end, and such adapter may be located at either the 3′ end or the 5′ end the target polynucleotide. Target polynucleotides may be used without any adapter, in which case a primer binding sequence may come directly from a sequence found in the target polynucleotide.
The terms “polynucleotide” and “oligonucleotide” are used interchangeably herein. The different terms are not intended to denote any particular difference in size, sequence, or other property unless specifically indicated otherwise. For clarity of description the terms may be used to distinguish one species of polynucleotide from another when describing a particular method or composition that includes several polynucleotide species.
As used herein, the term “amplicon,” when used in reference to a polynucleotide, is intended to means a product of copying the polynucleotide, wherein the product has a nucleotide sequence that is substantially the same as, or is substantially complementary to, at least a portion of the nucleotide sequence of the polynucleotide. “Amplification” and “amplifying” refer to the process of making an amplicon of a polynucleotide. A first amplicon of a target polynucleotide may be a complementary copy. Additional amplicons are copies that are created, after generation of the first amplicon, from the target polynucleotide or from the first amplicon. A subsequent amplicon may have a sequence that is substantially complementary to the target polynucleotide or is substantially identical to the target polynucleotide. It will be understood that a small number of mutations (e.g., due to amplification artifacts) of a polynucleotide may occur when generating an amplicon of that polynucleotide.
As used herein, a “hydrogel” refers to a three-dimensional polymer network structure that includes polymer chains and is at least partially hydrophilic and contains water within spaces between the polymer chains. A hydrogel may include any suitable combination of hydrophilic, hydrophobic, and/or amphiphilic polymer(s), so long as the overall polymer network is hydrophilic and contains water within spaces between the polymer chains. Hydrogels include chemical hydrogels in which both the bonding to form the polymer chains, and any cross-linking between the polymer chains, is covalent; such cross-linking during hydrogel formation may be irreversible, as distinguished from the present reversible cross-linking which is performed after the hydrogel is formed. In some cases, the chemical hydrogel may include, or may consist essentially of, brush-like structures of polymer chains attached to a surface, substantially without physical or covalent crosslinks between polymer chains, or alternatively polymer chains with multiple attachment points to a surface, resulting in loops, but also lacking interchain crosslinks. Hydrogels also include physical hydrogels in which the bonding to form the polymer chains, and any cross-linking within the polymer chains, is not covalent. Nonlimiting examples of physical hydrogels include agarose and alginate.
As used herein, the “polymer chain” of a hydrogel is intended to mean those portions of the hydrogel that are polymerized with one another during the polymerization process. Polymer chains may be cross-linked to form the hydrogel. For example, cross-linkers may be added during or after the polymerization process that forms the polymer chains. Additionally, or alternatively, in some examples the polymer chains may be deposited on a substrate surface that includes functional groups to which functional groups of the polymer chains become coupled. The polymer chains may be coupled to the surface, e.g., via reactions between the functional groups of the polymer chains and the functional groups at the surface, and such coupling may cross-link the polymer chains to form the hydrogel. Such cross-linking may cause the polymer chains to covalently or non-covalently attach to one another, or may occur as a result of chain entanglement during polymerization and/or attachment to a surface. As provided herein, polymer chains also or alternatively may be reversibly cross-linked after the hydrogel is formed, e.g., using functional groups in a manner such as described in greater detail below.
As used herein, the term “functional group” is intended to mean a chemical entity that is reactive with another chemical entity of the same type, or of a different type, to form a covalent bond or a non-covalent bond. As provided herein, when a hydrogel is described as having a functional group, it is to be understood that the functional group is chemically different from the polymer chains. Accordingly, the functional groups may not necessarily participate in forming the three-dimensional network of polymer chains during formation of the hydrogel or attachment of the polymer chains to a surface; as such, and as provided herein, the functional groups are available to perform additional cross-linking of the hydrogel at a time after the hydrogel itself is formed and is disposed on a substrate.
Methods of forming and using reversibly cross-linked hydrogels
As provided herein, stability of a hydrogel may be enhanced by reversibly cross-linking the hydrogel, e.g., after hydrogel formation (after formation and cross-linking of the polymer chains to form the hydrogel is complete) and before hydrogel shipping and/or storage. Additionally, or alternatively, as provided herein, the hydrogel optionally may be reversibly cross-linked at any suitable time before seeding the hydrogel with a template polynucleotide, and the cross-linkages optionally may be reversed at any suitable time before or during conducting cluster amplification using the seeded template polynucleotide. Alternatively, the cross-linkages optionally may be reversed before seeding.
For example,
Hydrogel 100 illustrated in
Hydrogel 101 illustrated in
As a result of cross-linking such as illustrated in
Additionally, the reduced open space of hydrogel 102 optionally may be used to improve monoclonality of a cluster that is generated using hydrogel 102, as compared to a cluster generated using hydrogel 101. For example, hydrogel 102 illustrated in
In the nonlimiting example shown in
In some examples, the target polynucleotide 160 hybridized to amplification primer 121 then may be amplified, e.g., using bridge amplification or other cluster generation technique described elsewhere herein or otherwise known in the art. For example, target polynucleotide 160 may be amplified using processes such as known in the art, e.g., using surface-bound polymerase chain reaction (PCR), bridge amplification, or a strand invasion process which may be referred to as ExAmp, forming a cluster of amplicons 160′ illustrated in
In some examples, the amount of open space within hydrogel 101 may be similar to the amount of open space within hydrogel 100, e.g., may be within about 20%, or within about 10%, or within about 5%, of the open space within hydrogel 100. Additionally, or alternatively, in some examples, the cross-link density of hydrogel 101 may be similar to the cross-link density of hydrogel 100, e.g., may be within about 20%, or within about 10%, or within about 5%, of the cross-link density within hydrogel 100. Additionally, or alternatively, in some examples, the amount of open space within hydrogel 103 may be similar to the amount of open space within hydrogel 102, e.g., may be within about 20%, or within about 10%, or within about 5%, of the open space within hydrogel 102. Additionally, or alternatively, in some examples, the cross-link density of hydrogel 103 may be similar to the cross-link density of hydrogel 102, e.g., may be within about 20%, or within about 10%, or within about 5%, of the cross-link 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., may be 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 cross-link density of hydrogel 104 may be similar to the cross-link density of hydrogel 101, e.g., may be within about 20%, or within about 10%, or within about 5%, of the cross-link 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., may be 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 cross-link density of hydrogel 105 may be similar to the cross-link density of hydrogel 101, e.g., may be within about 20%, or within about 10%, or within about 5%, of the cross-link density within hydrogel 101. Additionally, or alternatively, in some examples, the open space of hydrogel 102 or 103 may be significantly lower than the open space of hydrogel 100, hydrogel 101, hydrogel 104, and/or hydrogel 105, e.g., may be less than about 80%, or less than about 60%, or less than about 40%, or less than about 20% of the open space within hydrogel 100, hydrogel 101, hydrogel 104, and/or hydrogel 105. Additionally, or alternatively, in some examples, the cross-link density of hydrogel 102 or 103 may be significantly higher than the cross-link density of hydrogel 100, hydrogel 101, hydrogel 104, and/or hydrogel 105, e.g., may be about 20% greater, or about 40% greater, or about 60% greater, or about 80% greater, than the cross-link density within hydrogel 100, hydrogel 101, hydrogel 104, and/or hydrogel 105. Additionally, or alternatively, in some examples, hydrogel 100, 101, 104, and/or 105 may have a thickness that is at least ten times greater than the thickness of hydrogel 102 and/or 103. For example, hydrogel 100, 101, 104, and/or 105 may have a thickness of about 80-120 nm, while hydrogel 102 and/or 103 may have a thickness of about 8-12 nm.
The amount of open space and/or cross-link density within a hydrogel may be characterized using mechanical measurements, such as modulus determined from force distance curves in wet atomic force microscopy (AFM). Additionally, or alternatively, the amount of open space and/or cross-link density within a hydrogel can be measured by the mesh size, e.g., 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 may be characterized using swelling capacity (mass ratio between dry and wet hydrogel), e.g., using a quartz crystal microbalance (QCM) or ellipsometry in the wet state as compared the dry state. Additionally, or alternatively, the amount of open space and/or crosslink density within a hydrogel may be characterized using modulus (E′ from nano-indentation), which correlate with the hardness/softness of the hydrogel due to crosslinking density. For example, using nano-indentation there will be a difference between highly and less cross-linked hydrogel or polymers. Illustratively, one example strategy to assess openness of the hydrogel in its real condition on flowcell surface (e.g., which may be about 80-120 nm thick in the wet state and about 8-12 nm in the dry state), is using a fluorescence probe by hybridization or base incorporation. Ellipsometry also or alternatively may be used to detect swelling. QCM is compatible with thin hydrogel layers for assessing swelling too, but in some examples the hydrogel may be deposited on the QCM sensor instead of directly on the flowcell substrate. For relatively thin film hydrogel (e.g., <100 nm thickness) nanoindentation may be used. QCM may be used to measure the mass change through water adsorption (open vs close) or measuring primers tethered inside the hydrogel.
Additionally, or alternatively, the amount of open space and/or crosslink density within a hydrogel that includes amplification primers that are available to be hybridized to (e.g., hydrogel 101, 102, 103, or 104) may be characterized using the surface charge. For example, the cross-linking may obscure the primers (which are negatively charged), thus inhibiting the primers from contributing to the surface charge. In some examples, the surface charge may be characterized using atomic force microscopy (AFM), e.g., by measuring the interaction between the surface and the AFM tip. Additionally, or alternatively, Kelvin probe force microscopy may be used to characterize the surface charge of the hydrogel. Additionally, or alternatively, electrokinetic analysis may be used to characterize the surface charge of the hydrogel at a solid-liquid interface. Additionally, or alternatively, the amount of open space and/or crosslink density within a hydrogel that includes amplification primers that are available to be hybridized to (e.g., hydrogel 101, 102, 103, or 104) may be characterized using complementary fluorescent oligonucleotide probes to hybridize to the amplification primers within the hydrogel; the fluorescent intensity of hybridized probes can be used to quantify primers on the surface, and in case of bulky probes such as hairpins, fluorescence can be an indication of how accessible surface primers are to templates in a seeding event. Additionally, or alternatively, the amount of open space and/or cross-link density within a hydrogel can be measured by quantifying cleaved bonds using infrared (IR) spectroscopy, for example by comparing the intensity of a crosslinker signal in the open or cleaved state to the intensity of the crosslinker signal in crosslinked state. Additionally, or alternatively, the amount of open space and/or cross-link density within a hydrogel can be measured by viscosity, for example by comparing the cleaved or open polymer viscosity to fully crosslinked polymer viscosity. Additionally, or alternatively, the amount of open space and/or cross-link density within a hydrogel can be measured by comparing the hydrogel's molecular weight in the open or cleaved state to the molecular weight in the crosslinked state.
Although
In another example, the hydrogel may be formed so as to include the amplification primers. For example,
Note that the particular compositions, and the particular order of operations described with reference to
As noted further above, seeding need not necessarily be performed before reversing the hydrogel cross-linking. Illustratively, the hydrogel may be cross-linked after coupling the amplification primers, to provide cross-linked hydrogel 102 in a manner similar to that described with reference to
Additionally, or alternatively, as noted further above, reversing the cross-linking need not necessarily be performed at all, or completely, before amplification. Instead, amplification may be partially or fully performed before reversing the cross-linking. For example,
Some nonlimiting examples of functional groups and their uses to form reversible cross-linkages within hydrogels now will be provided.
Any suitable functional groups may be included in a hydrogel and used to couple amplification primers 121, 122 to the hydrogel and/or to reversibly cross-link polymer chains 110 of the hydrogel to one another. In some examples, the functional groups (e.g., 120, 130, and/or 230) independently may be selected from the group consisting of: azide, amine, thiol, diol, aldehyde, alkyne, strained cyclooctyne, and an inverse electron-demand (IED) Diels-Alder group. The functional groups may be included in polymer chains 110 of the hydrogel 100, 200, or 300 during synthesis of the hydrogel. The cross-linking density may be controlled by tuning the content of the functional moieties 120, 130, and/or 230 in the polymer chains. Additionally, as noted above with reference to
As noted above with reference to
In a manner such as described with reference to
Optional linkers 602, 602′ may have the same configuration as one another, or may have different configurations than one another. In some examples, one linker 602, 602′ is used, and
the other linker is omitted. Optional linkers 602, 602′ independently may include alkyl chains, polyethylene glycol (PEG), peptides, or polyphosphates, such as illustrated below: in which * denotes connection to the first or second functional group 601 or 601′ or to the first or second cleavable component 603, 603′, R1 and R2 denote residues of natural or non-natural amino acids, and X denotes a natural nucleoside or a spacer such as PEG or alkyl. In nonlimiting examples, n may be in the range of about 1 to 100, or about 2 to about 80, or about 5 to about 50, or about 2 to about 16.
Referring still to
Nonlimiting examples of cleavable components with covalent bonds that may be dissociated using chemical stimuli such as listed in Table 1 are shown below:
in which A and Q (if shown) correspond to first or second functional group 601 or 601′, X and M (if shown) correspond to first or second linker 602, 602′, where the squiggly lines (if shown) correspond to the first or second functional group 601 or 601′ or to the first or second cleavable component 603, 603′, and where R corresponds to a functional group that includes one or more carbon atoms and optionally includes 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 by reference herein.
Nonlimiting examples of cleavable components with a covalent bond that may be reversibly associated and disassociated using thermal stimuli are shown below:
in which R and R′ are the same as one another or may differ from one another, and include functional groups that are reactive with functional groups on the polymer in a manner such as described elsewhere herein.
In some examples in which the cleavable molecules are cleavable by a chemical agent, the chemical agent may include an acid. Nonlimiting examples of labile moieties include an acetal, ketal, imine, hydrazone, or t-butyl ester including a bond that is cleavable by the acid, e.g., by use of a buffer with a pH that is about 5 or less. An example cleavable molecule is illustrated below, in which the ketal moiety may be replaced by any of the alternative labile moieties illustrated below the molecule:
For further details regarding pH sensitive linkers including 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 by reference herein.
In one specific, nonlimiting example, the acid may include periodic acid, sodium periodate, or lead tetraacetate. The cleavable molecules may include a vicinal diol that is cleavable by the periodic acid. A nonlimiting example of a cleavable molecule that includes a vicinal diol cleavable by periodic acid is:
in which A and Q correspond to first or second functional group 601 or 601′, and X and M correspond to first or second linker 602, 602′.
In other examples in which the cleavable molecules are cleavable by a chemical agent, the chemical agent may include a reducing agent. A nonlimiting example of a labile bond is a disulfide bond that is cleavable using the reducing agent. Another nonlimiting example of a labile bond is an azidoalkyl ether that is cleavable using the reducing reagent. Another nonlimiting example of a labile bond is an allyl ether that is cleavable using a palladium complex of the reducing agent. Illustratively, the reducing agent may include glutathione, dithiothreitol (DTT), beta-mercaptoethanol (BME), cystamine, or tris(2-carboxyethyl)phosphine (TCEP). A nonlimiting example of a cleavable molecule with a disulfide bond that is cleavable using a reducing agent is:
in which A and Q correspond to first or second functional group 601 or 601′, and X and M correspond to first or second linker 602, 602′. A nonlimiting example of a cleavable molecule with an azidoalkyl ether that is cleavable using a reducing agent is:
in which A and Q correspond to first or second functional group 601 or 601′, and X and M correspond to first or second linker 602, 602′.
Molecule 710 illustrated in
Molecule 720 illustrated in
Functional groups 701, 701′, 711, 711′, and 721, 721′ may have the same configuration as one another, or may have different configurations than 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 than one another, and in some examples may be selected from the options provided above for linkers 602, 602′.
In some examples in which the cleavable molecules are cleavable by an enzyme, the enzyme may include a restriction enzyme, and the cleavable molecules may include an oligonucleotide that is cleavable using the restriction enzyme in a manner such as described with reference to molecule 710 of
In some examples, Val-Cit-PABS (valine-citrulline-p-aminobenzyl alcohol) may be coupled to functional groups, such as maleimide and/or p-nitrophenyl carbonate, for cross-linking. Another example of enzyme-cleavable component(s) 703 is matrix metalloproteinase-2 cleavable peptide (MMP2), which may be functionalized by reactive groups for cross-linking as well. As provided elsewhere herein, the functional groups of the present cross-linkers may be identical to one another, or may be different than one another.
In examples in which the cleavable molecules are cleavable by light, the cleavable molecules may include a coumarin or nitrobenzene group that is cleavable using light. A nonlimiting example of a molecule that includes a nitrobenzene group that is cleavable using light of about 360 nm is:
For further details regarding molecules that may be cleaved using light (optionally in combination with a chemical reagent), see Leriche et al., cited above, as well as 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 by reference herein.
As noted elsewhere herein, noncovalent interactions alternatively may be used to cross-link polymer chains 110 within the hydrogel. Illustratively, functional groups 130, 230 may include host molecules that reversibly cross-link the backbone via guest molecules. The guest molecules may be removable via salt, heat, or pH. Alternatively, the guest molecules may be removable via displacement with a binding partner to the guest molecules. For example,
In still other examples, the functional groups of the hydrogel may be coupled to ligand molecules that reversibly cross-link the backbone via multivalent binding proteins. The multivalent binding proteins are removable using a denaturing agent. For example, protein-ligand complexes held by strong non-covalent interactions may be employed. In a manner similar to that described with reference to macrocycle-guest binding, hydrogel 500 may include ligand molecules 535 in their backbone as illustrated in
Additional comments
It will be appreciated that the present compositions and methods may be used in any suitable application, such as amplifying a template polynucleotide. For example, although the present compositions may be used to perform “bridge amplification” or “surface-bound polymerase chain reaction,” it will be appreciated that the present compositions and methods readily may be adapted for use with other amplification modalities. One such amplification modality is “exclusion amplification,” or ExAmp. Exclusion amplification methods may allow for the amplification of a single target polynucleotide per region of the hydrogel and the production of a substantially monoclonal population of amplicons in that region. For example, the rate of amplification of the first seeded target polynucleotide within a region of the hydrogel may be more rapid relative to much slower rates of transport and seeding of target polynucleotides in that region. As such, the first target polynucleotide that seeds in a hydrogel region may be amplified rapidly and fill the entire hydrogel region, thus inhibiting the seeding of additional target polynucleotide(s) in the same hydrogel region. Alternatively, if a second target polynucleotide attaches to same region of the hydrogel after the first polynucleotide, the relatively rapid amplification of the first target polynucleotide may fill enough of the region to result in a signal that is sufficiently strong to perform sequencing by synthesis (e.g., the region may be at least functionally monoclonal). The use of exclusion amplification may also result in super-Poisson distributions of monoclonal regions; that is, the fraction of regions in an array that are functionally monoclonal may exceed the fraction predicted by the Poisson distribution.
Increasing super-Poisson distributions of useful clusters is useful because more functionally monoclonal regions may result in higher quality signal, and thus improved SBS; however, the seeding of target polynucleotides into regions of the hydrogel may follow a spatial Poisson distribution, where the trade-off for increasing the number of occupied regions is increasing the number of polyclonal regions. One method of obtaining higher super-Poisson distributions is to have seeding occur quickly, followed by a delay among the seeded target polynucleotide. The delay, termed “kinetic delay” because it is thought to arise through the biochemical reaction kinetics, gives one seeded target polynucleotide an earlier start over the other seeded targets. Exclusion amplification works by using recombinase to facilitate the invasion of primers (e.g., primers attached to a substrate region) into double-stranded DNA (e.g., a target polynucleotide) when the recombinase mediates a sequence match. The present compositions and methods may be adapted for use with recombinase to facilitate the invasion of the present amplification primers and orthogonal amplification primers into the present target polynucleotides when the recombinase mediates a sequence match. Indeed, the present compositions and methods may be adapted for use with any surface-based polynucleotide amplification methods such as thermal PCR, chemically denatured PCR, and enzymatically mediated methods (which may also be referred to as recombinase polymerase amplification (RPA) or ExAmp).
While various illustrative examples are described above, it will be apparent to one skilled in the art that various changes and modifications may be made therein without departing from the invention. The appended claims are intended to cover all such changes and modifications that fall within the true spirit and scope of the invention.
It is to be understood that any respective features/examples of each of the aspects of the disclosure as described herein may be implemented together in any appropriate combination, and that any features/examples from any one or more of these aspects may be implemented together with any of the features of the other aspect(s) as described herein in any appropriate combination to achieve the benefits as described herein.
This application claims the benefit of U.S. Provisional Patent Application No. 63/427,018 filed Nov. 21, 2022, and entitled “Reversibly Cross-Linked Hydrogels, and Methods of Using the Same for Cluster Amplification,” the disclosure of which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63427018 | Nov 2022 | US |
