Enzymatic selection of nucleic acids

Abstract

The present invention provides methods for generating a population of captured target nucleic acids and compositions for use in the methods. An exemplary method can include contacting a plurality of oligonucleotides individually linked to a solid support with a sample comprising a target nucleic acid having a 3'end and a 5 'end, where the target nucleic acid is annealed to some free 3' ends of the oligonucleotides, and where there is an excess of oligonucleotides such that at least some of the oligonucleotides still have free 3 'ends; then extending the 3'end of the target nucleic acid with a polymerase using an oligonucleotide as a template to form a double-stranded portion comprising a restriction enzyme recognition sequence; then cleaving the restriction enzyme recognition sequence with a restriction enzyme to form a released target nucleic acid having a single-stranded 5'end and a double-stranded 3 'end, and leaving an oligonucleotide not cleaved from the solid support and still having a free 3' end; the solid support and the uncleaved oligonucleotide are then optionally separated from the released target nucleic acid to form a target nucleic acid solution.

Description

[0001] Cross-referencing of related patent applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 544,372, filed October 16, 2023, which is incorporated by reference for all purposes. Background Technology

[0003] Single-cell genomic analysis, including single-cell sequencing, is performed in partitions (e.g., droplets). Sequencing is performed by attaching oligonucleotides containing partition-specific barcodes to target nucleic acids from cells, allowing the differentiation of target nucleic acids from different cells based on the properties of the partition-specific barcode sequences. In some methods, a large number of copies of oligonucleotides are delivered to partitions using solid carriers such as beads. The delivery of beads, as well as individual cells, to partitions follows a Poisson distribution. Because one goal is to avoid the presence of multiple cells in a partition, users adjust the number of partitions to be higher than the number of cells, resulting in many partitions lacking cells and therefore lacking target nucleic acids, but often containing beads and their oligonucleotides. Oligonucleotides can be released from the beads before or after annealing to the target nucleic acid, but generally, the release mechanism of the beads is independent of whether the partition contains the target nucleic acid. An example of a release mechanism is, for example, containing one or more uracils that link the oligonucleotides to the beads, and using uracil DNA glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII to release the oligonucleotides at the uracil. In other cases, UV-cleavable chemical fractions, disulfides, or other chemical components that can be released by the user are used. However, generally speaking, these methods do not distinguish between oligonucleotides annealed to the target nucleic acid and those that are not annealed, resulting in excess release of oligonucleotides in both sample-containing and sample-deficient regions, such as regions without single cells (and therefore without target nucleic acids). The presence of released oligonucleotides that are not annealed to the target nucleic acid can cause background interference in subsequent sequencing reactions. Summary of the Invention

[0004] In some embodiments, a method is provided for generating a captured population of target nucleic acids. In some embodiments, the method includes providing a plurality of oligonucleotides individually ligated to a solid carrier, wherein the oligonucleotides have a free 3' end; contacting the plurality of oligonucleotides with a sample containing a target nucleic acid having a 3' end and a 5' end, wherein the target nucleic acid annealed to some of the free 3' ends of the oligonucleotides, and wherein an excess of oligonucleotides is present such that at least some oligonucleotides still have a free 3' end; then using the oligonucleotides as a template, extending the 3' end of the target nucleic acid with a polymerase to form a double-stranded portion containing a restriction enzyme recognition sequence; then cleaving the restriction enzyme recognition sequence with a restriction enzyme to form (i) a released target nucleic acid having a single-stranded 5' end and a double-stranded 3' end and (ii) cleaved oligonucleotide ends ligated to the solid carrier, an affinity agent, or the link-blocking portion, leaving (iii) some uncleaved oligonucleotides that are not cleaved from the solid carrier, the affinity agent, or the link-blocking portion and still have a free 3' end; then separating the uncleaved oligonucleotides from the released target nucleic acid to form a target nucleic acid solution. In some embodiments, the double-stranded portion, rather than the restriction enzyme recognition sequence, can be a sequence recognized by different enzymes that selectively cleave the double-stranded sequence, as described elsewhere herein.

[0005] In some embodiments, multiple oligonucleotides are individually ligated to a solid support. In some embodiments, the oligonucleotides include a 5' end, and the 5' end is ligated to the solid support.

[0006] In some embodiments, the oligonucleotide from 5' to 3' comprises: one strand of a double-stranded restriction enzyme recognition sequence, a solid-carrier-specific barcode sequence, and a 3' target-specific sequence.

[0007] In some embodiments, the double-stranded restriction enzyme recognition sequence is selected from the group consisting of ApaLI, PmeI, AflII, AflIII, and PacI.

[0008] In some embodiments, the oligonucleotide includes a 5' end and the 5' end is linked to the bead, and the oligonucleotide also includes 2-10 nucleotides at the 5' end of one strand of a double-stranded restriction enzyme recognition sequence.

[0009] In some embodiments, the double-stranded portion contains two or more distinct restriction enzyme recognition sequences.

[0010] In some embodiments, the oligonucleotide is covalently linked to a solid carrier. In some embodiments, the oligonucleotide is non-covalently linked to a solid carrier. In some embodiments, the oligonucleotide is biotinylated, and the solid carrier contains streptavidin. In some embodiments, after cleavage, biotinylated streptavidin affinity separates the biotinylated nucleic acid at the cleaved oligonucleotide terminus from the released target nucleic acid.

[0011] In some embodiments, the target nucleic acid is an RNA molecule, and the polymerase is a reverse transcription polymerase. In some embodiments, the reverse transcription polymerase is HIV reverse transcription polymerase, M-MLV reverse transcription polymerase, or AMV reverse transcription polymerase.

[0012] In some embodiments, the target nucleic acid is a DNA molecule, and the polymerase is a DNA polymerase.

[0013] In some embodiments, the solid carrier is one or more beads, wherein copies of oligonucleotides attached to the beads contain bead-specific barcode sequences, and wherein oligonucleotides attached to different beads have different bead-specific barcode sequences.

[0014] In some embodiments, the method includes providing a solid carrier within the partitions. In some embodiments, the partitions are droplets or micropores in an oil-based emulsion. In some embodiments, at least some partitions also contain single cells, and wherein the target nucleic acid is a nucleic acid derived from a cell. In some embodiments, the method further includes lysing or permeabilizing the cells within the partitions.

[0015] In some embodiments, the cutting occurs within a partition.

[0016] In some embodiments, the oligonucleotide comprises a solid-vector specific barcode sequence and an optional PCR handle sequence.

[0017] In some embodiments, multiple oligonucleotides are individually linked to an affinity agent, and separation includes contacting the affinity agents on the cleaved oligonucleotide ends and uncleaved oligonucleotides with a reagent that binds the affinity agent; and separating the reagent bound to the affinity agent from the released target nucleic acid. In some embodiments, the affinity agent is biotin, and the reagent that binds the affinity agent is avidin or streptavidin.

[0018] In some embodiments, multiple oligonucleotides are individually linked to a link-blocking moiety. In some embodiments, the link-blocking moiety is selected from 3′ dideoxycytidine (ddC), 3′ reverse dT, 3′ C3 spacer, 3′ amino, and 3′ phosphorylation. In some embodiments, separation includes linking the 3′ end of the released nucleic acid to a polynucleotide, wherein uncut oligonucleotides or cleaved oligonucleotide ends are not linked to the polynucleotide; and separating the nucleic acid linked to the polynucleotide from the oligonucleotide containing the link-blocking moiety and the cleaved oligonucleotide ends. In some embodiments, the polynucleotide is linked to a solid carrier or affinity agent, thereby allowing selective separation of the polynucleotide from other nucleic acids in the mixture.

[0019] In some embodiments, the oligonucleotide is a completely DNA oligonucleotide. In some embodiments, the oligonucleotide contains DNA and uracil bases.

[0020] In some embodiments, the single cell is a mammalian cell.

[0021] In some embodiments, a plurality of beads are provided, each bead being linked to a plurality of oligonucleotides, the plurality of oligonucleotides comprising, from 5' to 3': one strand of a double-stranded restriction enzyme recognition sequence, a bead-specific barcode sequence, and a 3' target-specific sequence. In some embodiments, the double-stranded restriction enzyme recognition sequence is selected from the group consisting of ApaLI, PmeI, AflII, AflIII, and PacI. In some embodiments, the oligonucleotide comprises one strand of two or more double-stranded restriction enzyme recognition sequences.

[0022] In some embodiments, the oligonucleotide includes a 5' end and the 5' end is linked to the bead, and the oligonucleotide also includes 2-10 nucleotides at the 5' end of one strand of a double-stranded restriction enzyme recognition sequence.

[0023] In some embodiments, the oligonucleotide is a completely DNA oligonucleotide. In some embodiments, the oligonucleotide contains DNA and uracil bases.

[0024] In some embodiments, a kit is provided. In some embodiments, the kit includes a plurality of beads as described above or elsewhere herein; and a restriction enzyme that cleaves at a double-stranded restriction enzyme recognition sequence. In some embodiments, the kit also includes a reverse transcriptase.

[0025] In some embodiments, multiple partitions are provided. In some embodiments, a partition comprises multiple oligonucleotides individually linked to a solid carrier, wherein the oligonucleotides have a free 3' end and the oligonucleotides from 5' to 3' comprise: one strand of a double-stranded restriction enzyme recognition sequence, a solid carrier-specific barcode sequence, and a 3' target-specific sequence. In some embodiments, the partition is a droplet or micropore in an oil-based emulsion. In some embodiments, the solid carrier is a bead. In some embodiments, the double-stranded restriction enzyme recognition sequence is selected from the group consisting of ApaLI, PmeI, AflII, AflIII, and PacI. In some embodiments, the oligonucleotide comprises one strand of two or more double-stranded restriction enzyme recognition sequences. In some embodiments, the oligonucleotide includes a 5' end and the 5' end is linked to the bead, and the oligonucleotide further comprises 2-10 nucleotides at the 5' end of one strand of the double-stranded restriction enzyme recognition sequence. In some embodiments, the oligonucleotide is a complete DNA oligonucleotide. In some embodiments, the oligonucleotide comprises DNA and uracil bases. In some embodiments, some, but not all, partitions contain a target nucleic acid that is annealed to some free 3' ends of an oligonucleotide to form a double-stranded portion, and there is an excess of oligonucleotides such that at least some oligonucleotides still have free 3' ends. Attached Figure Description

[0026] Figure 1 Background options for oligonucleotide-based target nucleic acid capture are described. Hybridization-based target nucleic acid capture requires an excess of capture molecules for each target molecule. Capture molecules may also include tags or primer sites necessary for downstream applications, such as NGS library creation, cloning constructs, etc.

[0027] Figure 2 The problems experienced in previous methods are described: the removal of excess capture oligonucleotides from the reaction mixture. For example, in the past, methods for removing excess capture oligonucleotides included exonuclease digestion or biotinylation and streptavidin enrichment.

[0028] Figure 3 Some aspects of the method described herein are depicted. The target nucleic acid is annealed to a single-stranded oligonucleotide containing a restriction enzyme recognition sequence and ligated to a solid vector. After contact with a polymerase and extension of the target nucleic acid using the oligonucleotide (including the single-stranded restriction enzyme recognition sequence) as a template (not depicted), a double-stranded restriction enzyme recognition sequence is generated. (Also not depicted: extension of the 3' end of the oligonucleotide using the target nucleic acid as a template to generate a sequence to be sequenced later.) The restriction enzyme cleaves at or near the double-stranded restriction enzyme recognition sequence, releasing those oligonucleotides that have been extended by the polymerase to contain the inverse complementary sequence of the target nucleic acid. The uncut oligonucleotides remain ligated to the solid vector and can be separated from the released, extended oligonucleotides, which can then be used for any desired downstream application, including but not limited to nucleotide sequencing.

[0029] Figure 4An exemplary simplified workflow of the method described herein is depicted. Droplet partitions are formed to contain (i) a single cell and (ii) at least one bead linked to multiple copies of a capturing oligonucleotide (only one oligonucleotide is depicted for simplicity). The cell and bead can be separated within a droplet surrounded by oil, micropores, or other partitions. After separation, the cell can be lysed to release the cellular component of interest, such as mRNA. The released mRNA can be captured with an oligonucleotide containing a 3' Poly T terminus, which also has a diverse sequence for barcoding the bead. Following mRNA capture, a reverse transcriptase (RT) reaction can generate a hybrid DNA molecule containing a first-strand cDNA by extending the capturing oligonucleotide. The hybrid DNA molecule can be modified into a sequenceable library molecule suitable for downstream sequencing, such as next-generation sequencing (NGS). The efficiency of capturing cellular components, such as mRNA, can be improved by adding an excess of the capturing oligonucleotide. However, an excess of the capturing oligonucleotide contains the limiting DNA sequence used to amplify the captured mRNA / cDNA, which may cause excessive interference in downstream applications. Excessive bead-capture oligonucleotides can be removed through exonuclease treatment, SPRI bead purification, and other methods. See, for example... Figure 2 However, as described herein, a novel method can be used to selectively release cDNA-containing bead-target oligonucleotides while allowing excess bead-target oligonucleotides without cDNA to remain attached to a substrate. This substrate can then be used to remove or retain the excess bead-target oligonucleotides, while the cellular component of interest remains in solution. Additionally, partitions without a sample (e.g., without cells) typically release bead-target oligonucleotides even when no cells or target nucleic acids are available for their capture. This method will also remove such excess bead-target oligonucleotides resulting from this situation from downstream steps.

[0030] Figure 5 A table showing the results of Example 1 is provided. 4,000 beads containing ssDNA-capturing oligonucleotides with the restriction enzyme sites shown were incubated with an excess of complementary oligonucleotides to generate double-stranded DNA recognition sites for the restriction enzymes shown, or if the above procedure did not generate the double-stranded DNA recognition sites. The beads were then incubated in a buffer suitable for either the restriction enzyme or USER to digest the bead-capturing oligonucleotides. The reaction mixture was then centrifuged at 1000 × g for 5 min to precipitate the beads, and a volume of supernatant was removed from the sample. The collected supernatant was subjected to ddPCR to quantify the number of bead-capturing oligonucleotides present in the supernatant. The restriction enzyme releases the bead-capturing oligonucleotides only if the oligonucleotide contains a complementary sequence and is therefore double-stranded. In contrast, the USER enzyme cleaves uracil present in the bead-capturing oligonucleotides regardless of the complementary sequence, i.e., whether it is ssDNA or dsDNA.

[0031] Figure 6 A table of results for Example 2 is provided. 4,000 beads were incubated with total K562 RNA to simulate cell lysis and mRNA capture. After RNA capture, the beads were incubated with an excess of complementary oligonucleotides to generate the double-stranded DNA recognition site for the restriction enzymes shown, or if the above procedure did not generate the double-stranded DNA recognition site. The beads were then subjected to RT reaction at 50°C for 40 min, followed by restriction enzyme digestion at 37°C for 20 min. After RT and restriction enzyme reactions, the beads were centrifuged from the solution, and the supernatant was recovered. The supernatant was then analyzed by ddPCR targeting the bead-capturing oligonucleotides. Beads incubated with the complementary sequence of the bead-capturing oligonucleotides, or those digested with the USER enzyme, released the entire complementary sequence of the bead-capturing oligonucleotides. However, beads not incubated with the complementary sequence of the bead-capturing oligonucleotides released only a small amount of bead-capturing oligonucleotides, presumably only the bead-capturing oligonucleotides containing the captured cDNA and the bead-capturing complementary sequence.

[0032] Figure 7 A table showing the results of Example 3 is provided. 200,000 beads containing capture oligonucleotides with the indicated restriction sites were separated from cells in an aqueous droplet surrounded by oil. The aqueous solution of the droplet consisted of reverse transcription reactants and the indicated restriction enzyme. The droplet was incubated at 50°C for 40 min, then at 37°C for 20 min. After the RT reaction, the droplet was broken up, the beads were centrifuged from the solution, and the supernatant was recovered. The supernatant was then analyzed using ddPCR targeting GAPDH, with primers specific to GAPDH, or with one primer targeting the bead-capturing oligonucleotides and another oligonucleotide targeting the GAPDH gene, to produce chimeric amplicon containing both the bead and GAPDH sequences. The GAPDH cDNA levels produced by the restriction enzyme-released oligonucleotides were similar to, but slightly lower than, those from USER-digested beads.

[0033] Figure 8 Data generated as described in Example 4 is provided. Restriction enzymes recognize and cleave bead-capture oligonucleotides having dsDNA sites produced by RT performed with the cells in the compartment. The level of released cDNA is comparable to the level of bead-capture oligonucleotides released by USER digestion.

[0034] definition

[0035] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the laboratory procedures described below in cell culture, molecular genetics, organic chemistry, analytical chemistry, and nucleic acid chemistry and hybridization are those well-known and commonly used in the art. Standard techniques are used for nucleic acid and peptide synthesis. Techniques and procedures are generally performed according to conventional methods in the art and various general references (see often Sambrook et al.). Molecular Cloning: A Laboratory Manual （ MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (the references are incorporated herein by reference), and the conventional methods and various general references are provided throughout this document.

[0036] As used herein, the terms “a,” “an,” or “the” include not only aspects having one member but also aspects having more than one member. For example, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” include plural indicators. Thus, for example, a reference to “bead” includes a plurality of such beads, and a reference to “the sequence” includes a reference to one or more sequences known to those skilled in the art, etc.

[0037] The term "amplification reaction" refers to any in vitro method that amplifies a target sequence copy of nucleic acid in a linear or exponential manner. Such methods include, but are not limited to, polymerase chain reaction (PCR); DNA ligase chain reaction (LCR); Qβ RNA replicase; and RNA transcription-based amplification reactions. For example This includes amplification involving RNA polymerization induced by T7, T3, or SP6, such as transcription amplification systems (TAS), nucleic acid sequence-based amplification (NASBA), and self-sustaining sequence replication (3SR); single primer isothermal amplification (SPIA), loop-mediated isothermal amplification (LAMP), strand substitution amplification (SDA); multiple substitution amplification (MDA); rolling circle amplification (RCA); and other methods known to those skilled in the art. See, for example, Fakruddin et al. J. Pharm Bioallied Sci. 20135(4):245-252.

[0038] "Amplification" refers to the step of placing a solution under conditions sufficient for amplifying polynucleotides, provided all components of the reaction are intact. Components of an amplification reaction include, for example, primers, polynucleotide templates, polymerases, nucleotides, etc. The term "amplification" typically refers to an "exponential" increase in the number of target nucleic acids. However, as used herein, "amplification" can also refer to a linear increase in the number of selected target sequences of nucleic acids, as obtained through cyclic sequencing or linear amplification.

[0039] As used herein, a “barcode” is a short nucleotide sequence (e.g., at least about 4, 6, 8, 10, 12, 15, 20, 50, 75, or 100 or more nucleotides in length) that identifies a molecule conjugated to or derived from a solid carrier or partition. Barcodes can be used, for example, to identify molecules derived from partitions and / or beads, as sequenced later from a body reaction. This barcode may be unique to said partition or bead compared to barcodes present in other partitions or beads. For example, a partition containing target RNA from a single cell can be subjected to reverse transcription conditions using primers containing different partition-specific barcode sequences in each partition, thereby incorporating a copy of the unique “cell barcode” (because different cells are in different partitions, and each partition has a unique partition-specific barcode) into the reverse-transcribed target nucleic acid of each partition. Thus, nucleic acids from each cell can be distinguished from nucleic acids from other cells due to the unique “cell barcode.” In some embodiments, the oligonucleotide may also contain a barcode that uniquely identifies the molecule it is conjugated to; that is, the barcode acts as a unique molecular identifier (UMI). The length of the possible barcode sequence determines how many unique samples can be distinguished. For example, depending on degeneracy, a 1-nucleotide barcode can distinguish 4 or fewer distinct partitions; a 4-nucleotide barcode can distinguish 4... 4 There may be 256 or fewer partitions; a 6-nucleotide barcode can distinguish 4096 or fewer distinct partitions; and an 8-nucleotide barcode can index 65,536 or fewer distinct partitions. The barcode sequence may be continuous or may include discontinuous portions, which, if separated, will typically include constant regions that can be used to identify the location of the barcode sequence.

[0040] "Polymerase chain reaction" or "PCR" refers to a method for amplifying a specific segment or subsequence of a target double-stranded DNA in geometric progression. PCR is well known to those skilled in the art; see, for example, U.S. Patents 4,683,195 and 4,683,202; and PCR Protocols: A Guide to Methods and Applications, edited by Innis et al., 1990. Exemplary PCR reaction conditions typically involve two or three cycles. A two-step cycle includes a denaturation step followed by a hybridization / extension step. A three-step cycle includes a denaturation step followed by a hybridization step, followed by a separate extension step.

[0041] As used herein, “nucleic acid” refers to DNA, RNA, single-stranded, double-stranded, or more highly aggregated hybridization motifs, and any chemical modifications thereof. Modifications include, but are not limited to, those that provide chemical groups that incorporate additional charges, polarizabilities, hydrogen bonds, electrostatic interactions, linker sites, and functions on nucleic acid ligand bases or on the entire nucleic acid ligand. Such modifications include, but are not limited to, peptide nucleic acids (PNAs), phosphodiesterase group modifications (e.g., thiophosphates, methylphosphonates), 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at the exocyclic amine, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodouracil; backbone modifications, methylation, unusual base pairing combinations such as isobases, isocytosine, and isoguanidine. Nucleic acids may also include non-natural bases, such as nitroindole. Modifications may also include 3' and 5' modifications, including but not limited to end-capping with a fluorophore (e.g., quantum dots) or another component.

[0042] As used herein, the terms “partition” or “partitioned” refer to separating a sample into multiple parts or “partitions.” Partitions can be solid or fluid. In some embodiments, a partition is a solid partition, such as a microchannel. In some embodiments, a partition is a fluid partition, such as a droplet. In some embodiments, a fluid partition (e.g., a droplet) is a mixture of immiscible fluids (e.g., water and oil). In some embodiments, a fluid partition (e.g., a droplet) is an aqueous droplet surrounded by an immiscible carrier fluid (e.g., oil).

[0043] "Oligonucleotide" is a polynucleotide. Typically, oligonucleotides will have fewer than 250 nucleotides, and in some embodiments, 4-200, such as 10-150 nucleotides.

[0044] A “clonal” copy of a polynucleotide means a copy that is sequence-identical (or identical except for the unique molecular identifier (UMI) sequence). In some embodiments, at least 100, 1000, or 10 oligonucleotides are used. 4One or more clone copies are attached to the bead.

[0045] The “3’ capture sequence” on an oligonucleotide refers to the 3’ terminal portion of the oligonucleotide. The capture sequence can be as short as 1-2 nucleotides, but more typically 6-12 nucleotides, and in some embodiments 4-20 or more nucleotides. The capture sequence can be perfectly complementary to the target nucleic acid (e.g., the 3’ end of the target nucleic acid), but as is known in some embodiments and under certain conditions, 1, 2, 3, 4, or more nucleotides may mismatch, while still allowing the 3’ capture sequence of the oligonucleotide to adhere to the target nucleic acid. In other embodiments, conditions can be selected such that only perfectly complementary sequences will adhere. The 3’ capture sequence can be a random sequence, a poly-T or poly-A sequence, a target-specific sequence, or a universal sequence.

[0046] The term "beads" refers to any solid-phase carrier that can be located within a partition, such as small particles or other solid-phase carriers. Exemplary beads may include hydrogel beads. In some cases, the hydrogel is in the form of... sol Form. In some cases, the hydrogel is in the form of... gel Form. An exemplary hydrogel is an agarose hydrogel. Other hydrogels include, but are not limited to, those described in, for example, U.S. Patent Nos. 4,438,258; 6,534,083; 8,008,476; 8,329,763; U.S. Patent Application Nos. 2002 / 0,009,591; 2013 / 0,022,569; 2013 / 0,034,592; and International Patent Publication Nos. WO / 1997 / 030092 and WO / 2001 / 049240.

[0047] A "restriction enzyme recognition sequence" refers to a nucleotide sequence that a restriction enzyme binds to, the presence of which is essential to trigger cleavage of the nucleic acid containing that sequence. In some embodiments, the restriction enzyme also cleaves within the restriction enzyme recognition sequence. In other embodiments, the restriction enzyme cleaves outside the restriction enzyme recognition sequence. Detailed Implementation

[0048] Methods and compositions are provided for the selective cleavage of oligonucleotides from a solid vector (or, in other embodiments, from an affinity agent or link-blocking moiety) using a restriction enzyme that selectively recognizes a double-stranded DNA recognition sequence and substantially does not recognize the single-stranded form of said sequence. The oligonucleotide is annealed to a target nucleic acid, which is extended by a polymerase (which may be a reverse transcriptase) to form a double-stranded restriction enzyme recognition sequence that can be used to cleave the oligonucleotide and anneal the target nucleic acid, but without releasing oligonucleotides that do not include the double-stranded restriction enzyme recognition sequence, i.e., oligonucleotides that have not yet been annealed to the target nucleic acid extended by the polymerase. Thus, the methods and compositions allow the annealing of the target nucleic acid to an oligonucleotide linked to a solid vector (or, in other embodiments, an affinity agent or link-blocking moiety), and the use of the oligonucleotide as a template to extend the 3' end of the target nucleic acid to form a double-stranded recognition sequence, which is then selectively cleaved. This is not intended to limit the scope of application of the methods, which may be particularly useful in the context of single-cell sequencing, where numerous clonal oligonucleotides linked to beads are delivered to partitions. The challenge of the current workflow lies in the fact that the captured oligonucleotides on the beads generate non-productive byproducts downstream, which increase interference and reduce sensitivity. The restriction enzyme method described in this paper bypasses this problem by selecting the desired products (those oligonucleotides annealed to the target nucleic acid) and easily removing the byproducts without excessive cleanup steps.

[0049] Multiple clonal oligonucleotides can be ligated to a solid vector and then delivered to a partition that may or may not contain the target nucleic acid. In some embodiments, at least 100, 1000, or 10 oligonucleotides ligated to the beads are present. 4 10 5 10 6 10 7 10 8 Multiple or more cloned copies. In many embodiments, multiple solid vectors are provided, and the oligonucleotides contain solid vector-specific barcode sequences, such that the solid vector-specific barcode sequences can be used, upon ligation to the target nucleic acid, to determine the solid vector from which the solid vector-specific barcode sequences originate (and, in some embodiments, partitioning).

[0050] Oligonucleotides can be covalently or non-covalently linked to a solid carrier. If desired, oligonucleotides can be linked to beads. Methods for linking oligonucleotides to beads are described, for example, in WO 2015 / 200541. In some embodiments, oligonucleotides configured to link hydrogel beads to barcodes are covalently linked to the hydrogel. Many methods for covalently linking oligonucleotides to one or more hydrogel matrices are known in the art. As an example, aldehyde-derived agarose can be covalently linked to the 5'-amine group of a synthetic oligonucleotide.

[0051] Beads of any useful size and composition for delivery to the partition can be used. The particles or beads can be any particles or beads having a solid support surface. Suitable solid supports for the particles include controlled-porosity glass (CPG) (available from Glen Research, Sterling, Va.), oxalyl-based controlled-porosity glass (see, for example, Alul et al., Nucleic Acids Research 1991, 19, 1527), TentaGel support—an amino-polyethylene glycol-derived support (see, for example, Wright et al., Tetrahedron Letters 1993, 34, 3373), polystyrene, Poros (a copolymer of polystyrene / divinylbenzene), or reversibly crosslinked acrylamide. Many other solid supports are commercially available and suitable for the methods of this invention. In some embodiments, the bead material is polystyrene resin or poly(methyl methacrylate) (PMMA). The bead material can also be metal. In some embodiments, the particles or beads comprise a hydrogel or another similar composition. In some cases, the hydrogel is... Dissolve glue Form. In some cases, the hydrogel is in the form of... gel Form. An exemplary hydrogel is an agarose hydrogel. Other hydrogels include, but are not limited to, those described in, for example, U.S. Patent Nos. 4,438,258; 6,534,083; 8,008,476; 8,329,763; U.S. Patent Application Nos. 20020009591; 20130022569; 20130034592; and International Patent Publications Nos. WO1997030092 and WO2001049240. Additional compositions and methods for preparing and using hydrogels (such as barcode-encoded hydrogels) include, for example, Klein. et al. , Cell Those described in , May 21, 2015; 161(5):1187-201.

[0052] Although oligonucleotides can be ligated to solid vectors in various ways, the 3' end, and usually the 3' capture sequence, which is the sequence that will anneal to the target nucleic acid at the 3' end, will be the free end. This means that the 3' end and the 3' capture sequence are not directly ligated to the solid vector. Instead, different parts of the oligonucleotide carrying the 3' end and the 3' capture sequence are ligated to the solid vector, so that the 3' capture sequence can be used to anneal to the complementary target nucleic acid while being ligated to the solid vector.

[0053] In other embodiments, the oligonucleotide is not linked to a solid carrier, but rather to an affinity agent. Exemplary affinity agents may be, for example, biotin, avidin, streptavidin, or an antibody. As further explained below in these embodiments, once the double-stranded restriction sequence has been formed and cleaved, the affinity agent can be used to separate the cleaved ends and the uncleavage sequence from the target nucleic acid.

[0054] In other embodiments, the oligonucleotide is not ligated to a solid carrier, but rather to a link-blocking portion. Exemplary link-blocking portions may be, for example, 3'-dideoxycytidine (ddC), 3'-reverse dT, 3'-C3 spacer, 3'-amino, and 3'-phosphorylated. As further explained below in these embodiments, once the double-stranded restriction sequence has been formed and cleaved, the link-blocking portion will remain on the uncleaved oligonucleotide and the cleaved ends, but the cleaved portion containing the target nucleic acid will be available for the ligation reaction, which can then be used to subsequently separate the target nucleic acid from the remainder of the nucleic acid, for example, by affinity for the linker, or where the linker contains biotin or other affinity agents.

[0055] The length and sequence of the 3' capture sequence of the oligonucleotide can vary depending on the target nucleic acid. In some embodiments, the target nucleic acid is mRNA, and the 3' capture sequence is a polyT sequence, such as 4-30 or more consecutive thymine nucleotides. In some embodiments, the target nucleic acid is RNA or DNA, and the 3' capture sequence is a random sequence, a target-specific sequence, a universal sequence (e.g., a sequence annealed to the end of a fragment introduced by a cleavage spiking enzyme), or any other desired capture sequence. Other desired capture sequences may be, but are not limited to, short or long scattered retrotransposon elements (SINES or LINES, respectively).

[0056] In any of the embodiments described herein, the oligonucleotide linked to the bead may comprise one or more barcode nucleotide sequences. In some embodiments, the oligonucleotide includes a barcode sequence that is unique to the solid carrier to which it is attached (e.g., a bead) and can therefore be used to distinguish oligonucleotides from different beads, for example, after the oligonucleotide has been released and used to generate sequencing reads. Additional barcodes, such as, but not limited to, unique molecular identifiers (UMIs) or sample-specific barcodes, may also be included in the oligonucleotide sequence.

[0057] In some embodiments, the oligonucleotide further comprises one or more PCR adapter sequences or their reverse complementary sequences, such that the downstream nucleic acid product of the method can be amplified in an amplification reaction by common (“universal”) primers.

[0058] In addition to the 3' end and 3' capture sequences as discussed herein, and other optional sequences, the oligonucleotide will also include an optional PCR ligation and barcoding sequence located at the 5' of the 3' capture sequence, one strand of the double-stranded restriction enzyme recognition sequence, and (if the oligonucleotide is ligated to a solid vector) a solid vector-specific barcoding sequence (both located at the 5' of the 3' capture sequence). In some embodiments, the oligonucleotide comprises one strand of two, three, or more identical or different double-stranded restriction enzyme recognition sequences; for example, in some embodiments, it is permissible to use two or more different restriction enzymes to cleave the double-stranded recognition sequence.

[0059] The restriction enzyme and the corresponding double-stranded recognition sequence can be selected as needed, provided that the restriction enzyme selectively acts on the double-stranded recognition sequence compared to a single strand of the same sequence (e.g., the activity against the dsDNA sequence is 2, 5, 10, 50, 100, or higher than that against the ssDNA sequence or an RNA / DNA heteroduplex). In some embodiments, the recognition sequence is 4-10 nucleotides long, such as 6-8 nucleotides long. Exemplary restriction enzymes may include, but are not limited to, ApaLI, PmeI, AflII, AflIII, and PacI.

[0060] In some embodiments, the 5' end of the oligonucleotide includes a plurality of nucleotides separating the restriction site recognition sequence from the 5' end of the oligonucleotide, for example, but not limited to, embodiments in which the oligonucleotide is ligated to a solid carrier via its 5' end. The exact number and identity of the nucleotides can be determined by the user, and spacers will be provided so that the recognition sequence is not so close to the solid carrier that subsequent restriction enzyme cleavage is inhibited. In some embodiments, the spacer nucleotides comprise 2-10, for example, 4-6 nucleotides located at the 5' end of one strand of the double-stranded restriction enzyme recognition sequence. The nucleotides can be determined as needed. In some embodiments, the nucleotide is T, i.e., containing a thymine base.

[0061] While in some embodiments the methods and steps described herein can be performed in bulk solution without separating different cells or samples in partitions, in many embodiments some of all the steps described herein are performed in partitions, allowing different cells or samples or other materials to be isolated and reacted in parallel in separate partitions. Therefore, in some embodiments, multiple oligonucleotides linked to a solid carrier (e.g., beads) can be introduced into the partitions as needed. Exemplary partitions may include, but are not limited to, droplets (e.g., in emulsions) or micropores. The introduction of the solid carrier into the partition can occur during or after partition formation. The partitions can be pre-formed, optionally with other reagents and optionally with single cells or target nucleic acids from a biological sample, and the solid carrier and linked oligonucleotides can be injected or otherwise introduced into the partition. Methods and compositions for delivering reagents to one or more partitions include microfluidic methods known in the art; droplet or microcapsule coalescence, aggregation, fusion, rupture, or degradation (e.g., as described in US 2015 / 0027,892; US 2014 / 0227,684; WO 2012 / 149,042; and WO 2014 / 028,537); droplet injection methods (e.g., as described in WO 2010 / 151,776); and combinations thereof. In other embodiments, such as where the partitions are droplets, droplets in the form of an emulsion can be formed using an immiscible fluid such as oil, thereby forming a bulk solution containing droplets containing beads and linked oligonucleotides, optionally with other reagents and / or sample nucleic acids or cells. For example, methods of emulsion formation are described in published patent applications WO 2011 / 109546 and WO 2012 / 061444.

[0062] In some embodiments, the distribution of beads (and single cells) in partitions (e.g., droplets) can be determined by a Poisson distribution. Depending on the end use, the average number of beads per partition can be less than 1 (e.g., 0.2–0.9), 1, or greater than 1 (e.g., 1–3, 1–10, or greater). In some embodiments, it is desirable to avoid having multiple beads in a single partition, and in these cases, many partitions may be left empty, such that most partitions containing beads contain only one bead. In other embodiments, such as where deconvolution methods can be used to decrypt sequencing results with multiple beads in a single partition, each partition can be loaded with more beads on average, and then deconvolution is used to parse the sequencing results. See, for example, PCT / US2017 / 012618, PCT / US2019 / 015638, and PCT / US2020 / 36699.

[0063] In some embodiments, the sample (e.g., single cells and / or target nucleic acids and beads linked to oligonucleotides) is partitioned into at least 500 partitions, at least 1000 partitions, at least 2000 partitions, at least 3000 partitions, at least 4000 partitions, at least 5000 partitions, at least 6000 partitions, at least 7000 partitions, at least 8000 partitions, at least 10,000 partitions, at least 15,000 partitions, or at least 20,000 partitions. 00 partitions, at least 30,000 partitions, at least 40,000 partitions, at least 50,000 partitions, at least 60,000 partitions, at least 70,000 partitions, at least 80,000 partitions, at least 90,000 partitions, at least 100,000 partitions, at least 200,000 partitions, at least 300,000 partitions, at least 400,000 partitions, at least 500,000 partitions, at least 60 0,000 partitions, at least 700,000 partitions, at least 800,000 partitions, at least 900,000 partitions, at least 1,000,000 partitions, at least 2,000,000 partitions, at least 3,000,000 partitions, at least 4,000,000 partitions, at least 5,000,000 partitions, at least 10,000,000 partitions, at least 20,000,000 partitions, at least 30 ,000,000 partitions, at least 40,000,000 partitions, at least 50,000,000 partitions, at least 60,000,000 partitions, at least 70,000,000 partitions, at least 80,000,000 partitions, at least 90,000,000 partitions, at least 100,000,000 partitions, at least 150,000,000 partitions, or at least 200,000,000 partitions.

[0064] Any type of cell can be used according to the methods and compositions described herein. In some embodiments, the cell is a mammalian cell, such as a human cell. In some embodiments, the cell is derived from a biological sample. The biological sample can be obtained from any biological organism, such as an animal, plant, fungus, pathogen (e.g., bacteria or virus), or any other organism. In some embodiments, the biological sample is derived from an animal, such as a mammal (e.g., a human or non-human primate, cow, horse, pig, sheep, cat, dog, mouse, or rat), a bird (e.g., a chicken), or a fish. The biological sample can be any tissue or body fluid obtained from a biological organism, such as blood, blood fractions or blood products (e.g., serum, plasma, platelets, erythrocytes, etc.), sputum or saliva, tissues (e.g., kidney, lung, liver, heart, brain, nerve tissue, thyroid, eye, skeletal muscle, cartilage, or bone tissue); cultured cells, such as primary cultures, explants and transformed cells, stem cells, or cells found in feces, urine, etc.

[0065] In some embodiments, cells are fixed and permeabilized. In some embodiments, the cells are formalin-fixed cells. Exemplary detergents may include, for example, Triton X-100, Brij-35, and / or NP-40 for permeabilization (e.g., at 0.1-0.5% (v / v, in PBS)). In some embodiments, steroidal saponins (or saponins) are used to dissolve lipids, thereby causing permeabilization. An exemplary saponin is digitin.

[0066] The methods described herein are used to contact oligonucleotides linked to a solid carrier, an affinity agent, or a linker blocking region with a target nucleic acid. The target nucleic acid can be, for example, RNA (e.g., but not limited to mRNA) or DNA. In some embodiments, a single cell is provided in a partition, and the single cell in the partition is permeated or lysed to allow other agents in the partition, such as oligonucleotides and / or enzymes, to contact the target nucleic acid from the cell. In the embodiments described herein, the oligonucleotide remains linked to the solid carrier under conditions that allow the target nucleic acid to anneal to the 3' capture sequence on the oligonucleotide. Thus, the solid carrier will be linked to multiple oligonucleotides, at least some (but typically not all) of which are annealed to the target nucleic acid.

[0067] Once the target nucleic acid anneals to some oligonucleotides, the polymerase contacts the annealed nucleic acid. The polymerase uses the target nucleic acid as a template to extend the oligonucleotide, resulting in a first-strand copy of the target nucleic acid (reverse complementary sequence) with the oligonucleotide sequence as its 5' sequence. This product can then be used for various downstream manipulations and detections, such as for nucleotide sequencing. The target nucleic acid also uses the oligonucleotide as a template in a polymerase-dependent reaction to extend, causing the single-stranded portion of the oligonucleotide containing the restriction enzyme recognition sequence to be copied, thereby generating a double-stranded recognition sequence. See, for example, Figure 4 .

[0068] The exemplary polymerase used will depend on whether the target nucleic acid is DNA or RNA. In embodiments where the target nucleic acid is RNA, a reverse transcriptase may be used to (1) extend the 3' end of an oligonucleotide using the target RNA as a template, and (2) extend the 3' end of RNA using an oligonucleotide as a template. Although only activity (1) was initially expected, the latter activity has been observed. Regarding activity (2), without intending to limit the scope of the invention, it may be that the mRNA polyA tail is modified, removed, or looped out to allow the 3' end of the tail bases to pair with the bead-capturing oligonucleotide and to allow the formation of a double-stranded recognition sequence. Exemplary target RNAs may include, for example, mRNA, miRNA, scRNA, etc. Exemplary reverse transcriptases may include, but are not limited to, HIV reverse transcription polymerase, murine leukemia virus (MLV) reverse transcriptase, avian myeloma virus (AMV) reverse transcriptase, respiratory syncytial virus (RSV) reverse transcriptase, equine infectious anemia virus (EIAV) reverse transcriptase, or Rous-associated virus 2 (RAV2) reverse transcriptase or mutants thereof. For example, Super Script IV™ (ThermoFisher Scientific) or Maxima H-™ (ThermoFisher Scientific) or any reverse transcriptase described in U.S. Patent Application Publication No. 2011 / 0065606 can be used. In embodiments where the target nucleic acid is DNA, a DNA polymerase can be used to (1) extend the 3' end of an oligonucleotide using the target DNA as a template, and (2) extend the 3' end of a DNA using an oligonucleotide as a template.

[0069] In some embodiments, the use of a multi-omics approach means that two or more of RNA, DNA, and proteins are detected in the method, wherein a reagent representing each target is annealed and linked to an oligonucleotide, allowing for later determination of which sample molecules originate from the same partition. In some embodiments, proteins in or on cells are detected by contacting cells with one or more antibodies, wherein the antibodies are linked to oligonucleotides. This may be referred to as CITE-seq. See, for example, Stoeckius, M., Hafemeister, C., Stephenson, W. et al., Nat Methods 14, 865-868 (2017). Antibodies targeting different antigens are linked to oligonucleotides with different sequences, such that the sequence identity of the oligonucleotide indicates the affinity of the antibody to which it is linked. In some embodiments of the methods described herein, the sample nucleic acid annealed to bead oligonucleotides may be an oligonucleotide linked to or cleaved from such antibodies, allowing for purification and eventual detection of the properties of the oligonucleotides associated with antibodies binding to target antigens in a partition of the sample.

[0070] One or more restriction enzymes, either (i) present in the reaction mixture or partition containing the solid support, or (ii) introduced after polymerase extension and annealing of the nucleic acid, are contacted with an oligonucleotide having the annealed and extended target nucleic acid, thereby allowing the restriction enzyme to recognize the double-stranded recognition sequence at the restriction enzyme recognition site and then cleave the double-stranded nucleic acid. In some embodiments, more than one restriction enzyme recognition sequence may be formed, and in such embodiments, one or more different restriction enzymes may be contacted with the nucleic acid. In some embodiments, forming more than one restriction enzyme recognition sequence and using more than one restriction enzyme can increase the likelihood that the annealed nucleic acid is indeed cleaved from the solid support. The result of restriction enzyme action is the generation of cleaved oligonucleotides that have been extended to have the reverse complementary sequence of the target nucleic acid, while leaving excess oligonucleotides that have not been annealed to the target nucleic acid uncleaved (still ligated to the solid support).

[0071] Any restriction enzyme can be used, as long as it recognizes a double-stranded recognition sequence generated during target nucleic acid extension using an oligonucleotide as a template, and wherein the restriction enzyme is selective for double-stranded DNA compared to single-stranded DNA and / or DNA / RNA heteroduplexes. In some embodiments, the restriction enzyme cleaves within the double-stranded DNA recognition sequence, but this is not required; it is sufficient that the restriction enzyme cleaves the annealed oligonucleotide from the solid vector. The restriction enzyme can be selected, for example, from restriction enzymes that recognize 4, 5, 6, 7, 8, 9, or 10-base-pair double-stranded sequences. Exemplary restriction enzymes may include, but are not limited to, ApaLI, PmeI, AflII, AflIII, and PacI.

[0072] After selectively cleaving those nucleic acids with double-stranded recognition sequences, the extended oligonucleotides cleaved from the solid carrier can be separated from the solid carrier and any remaining oligonucleotides linked thereto. Any desired method can be used to separate these populations, including, for example, using gravity, centrifugation, filtration, or other methods to separate the solublely released oligonucleotides from the solid carrier. In embodiments where oligonucleotides are linked to an affinity agent, the affinity agent is cleaved from the target nucleic acid by a restriction enzyme, and the affinity agent can then be used to remove uncleaved oligonucleotides retaining the affinity agent and the cleaved ends of the oligonucleotides containing the affinity agent from the cleaved portion containing the target nucleic acid. In this case, for example, a reagent with affinity for the affinity agent can be used to bind to and then separate the nucleic acid containing the affinity agent. For example, a reagent with affinity for the affinity agent can be linked to the solid carrier and used as described above to separate populations of nucleic acids. In embodiments where oligonucleotides are linked to a link-blocking portion, the link-blocking portion is cleaved from the target nucleic acid by a restriction enzyme, and a ligation reaction can be performed to selectively link polynucleotides to the cleaved target nucleic acids, as they will lack the link-blocking portion. Those nucleic acids containing the linked polynucleotides can then be separated from the rest of the nucleic acids in the mixture, for example, by affinity (complementarity between the polynucleotide and the polynucleotide on the solid support) or other methods that selectively separate the polynucleotide-containing nucleic acids from the mixture. Depending on the restriction enzyme used, the ligation can be blunt-end or sticky-end ligation.

[0073] Before or after separation of the extended oligonucleotide from the solid carrier and any remaining oligonucleotides linked thereto, the extended oligonucleotide chain can be used as a first strand to form a second strand. For example, in embodiments where the target nucleic acid is RNA, the extended oligonucleotide will be a first-strand cDNA containing an oligonucleotide sequence at its 5' end, and the first-strand cDNA can be used as a template to generate a second-strand cDNA. The second strand can be generated as needed. In some embodiments, the second strand can be formed by extending from random primers of the first strand, or template switching can be used to generate the second strand. See, for example, Zhu YY, Machleder EM et al., (2001). Biotechniques , 30(4):892-897;Ramskold D, Luo S et al., (2012) Nat Biotechnol , 30(8):777-78. In some embodiments, the second strand synthesis introduces a universal sequence at the 5' end of the second strand, which, when amplified together with the first strand, generates an amplicon with the universal sequence at either end, thereby allowing selective amplification of the captured target nucleic acid using primers annealed to the universal sequence or its complementary sequence. See, for example Figure 4 The bottom.

[0074] In some embodiments of any of the methods described herein, exonucleases, or uracil DNA glycosylases and endonucleases VIII (the latter two sometimes referred to as "uracil-specific excision reagents"), or all of them, are not used in the methods. For example, in some embodiments, the oligonucleotides linked to the solid vector do not include uracil.

[0075] In some embodiments, transposases carrying oligonucleotides are used to introduce breaks in DNA (e.g., amplicons containing a first and a second strand as described above) and introduce the carried oligonucleotides into the break site. The function of some transposases is sometimes referred to as "spiking," and the enzyme may be called a "spiking enzyme," and may involve introducing different adaptor sequences on different sides of the DNA break site, or the added adaptor sequences may be the same. Homologous adaptor-loaded spiking enzymes are spiking enzymes containing only one adaptor sequence, which is added to both ends of a spiking enzyme-induced break site in genomic DNA. Heterologous adaptor-loaded spiking enzymes are spiking enzymes containing two different adaptors, such that different adaptor sequences are added to the two DNA ends created by a spiking enzyme-induced break site in the DNA. The enzymes that spiking the adapter-loaded enzymes are further described, for example, in U.S. Patent Publications: 2010 / 0120098; 2012 / 0301925; and 2015 / 0291942; and U.S. Patents: 5,965,443; 6,437,109; 7,083,980; 9,005,935; and 9,238,671, the contents of each of which are hereby incorporated in their entirety by reference for all purposes.

[0076] Once the amplicon is prepared, any nucleotide sequencing method can be used to generate sequencing reads from the extended oligonucleotides, and said reads contain a solid-vector specific barcode or other sequences from the original oligonucleotide linked to the first strand.

[0077] A sequencing platform can be selected as needed to generate sequencing reads. In some embodiments, an Illumina™-enabled sequencing method is employed. See, for example, U.S. Patent Nos. 11,029,513, 11,150,179, 11,308,640, and 11,473,067, and their citations. Exemplary DNA sequencing technologies include fluorescence-based sequencing methods (…). See For example, Birren et al.Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, NY; incorporated herein by reference in its entirety. In some embodiments, automated sequencing techniques understood in the art are utilized. In some embodiments, the technique provides parallel sequencing of partitioned amplicon (PCT Publication WO 2006 / 0841,32, incorporated herein by reference in its entirety). In some embodiments, DNA sequencing is achieved by parallel oligonucleotide extension (see, for example, U.S. Patent Nos. 5,750,341 and 6,306,597, both of which are incorporated herein by reference in their entirety). Additional examples of sequencing techniques include Church polymerase colony technology (Mitra... et al. , 2003, Analytical Biochemistry 320, 55-65; Shendure et al. , 2005 Science 309, 1728-1732; and U.S. Patents 6,432,360, 6,485,944, and 6,511,803; which are incorporated herein by reference in their entirety), 454 picoliter pyrosequencing technology (Margulies et al. , 2005 Nature 437, 376-380; US Publication No. 2005 / 0130173; incorporated herein by reference in its entirety), Solexa single-base addition technology (Bennett et al. , 2005, Pharmacogenomics, 6, 373-382; U.S. Patents 6,787,308 and 6,833,246; incorporated herein by reference in their entirety), Lynx massively parallel signature sequencing technology (Brenner). et al. (2000). Nat. Biotechnol. 18:630-634; U.S. Patent Nos. 5,695,934 and 5,714,330; which are incorporated herein by reference in their entirety) and Adessi PCR colony technology (Adessi et al. (2000). Nucleic Acid Res. 28, E87; WO2000 / 018957; incorporated herein by reference in its entirety.

[0078] The methods described above can also be implemented using double-stranded targeting enzymes other than restriction enzymes. For example, using oligonucleotides as templates, extending the 3' end of the target nucleic acid with a polymerase can form a double-stranded portion, regardless of the presence of a restriction enzyme recognition sequence. In these embodiments, instead of using restriction enzymes to cleave the double-stranded portion, enzymes can be used to selectively label or modify the double-stranded portion without similarly labeling or modifying the single-stranded portion. A non-restrictive list of such enzymes may include, for example, Argonaute proteins, formamidopyrimidine DNA glycosyltransferases, methyltransferases, or TelN prokaryotic telomerases, which selectively modify the double-stranded portion, which can then be used to separate the labeled or modified nucleic acid from the unlabeled or modified nucleic acid.

[0079] In some embodiments, the Argonaute protein and the guide nucleic acid are used to target the double-stranded portion. Examples of targeting the Argonaute protein having the sequence of the guide nucleic acid can be found, for example, in Tabatabaei SK et al. (April 2020). Nature Communications 11 (1): 1742. Therefore, in some embodiments, a method for generating a captured target nucleic acid population is provided, comprising providing a plurality of oligonucleotides individually ligated to a solid carrier, wherein said oligonucleotides have a free 3' end; contacting said plurality of oligonucleotides with a sample containing a target nucleic acid having a 3' end and a 5' end, wherein the target nucleic acid annealed to some of said free 3' ends of said oligonucleotides, and wherein an excess of oligonucleotides is present such that at least some oligonucleotides still have a free 3' end; and then using said oligonucleotides as a template, extending the 3' end of said target nucleic acid with a polymerase to form a double-stranded portion containing an Argonaute protein recognition sequence. The Argonaute protein recognition sequence is then cleaved using Argonaute protein (and optionally a guide nucleic acid to which the Argonaute protein is directed) to form (i) a released target nucleic acid having a single-stranded 5' end and a double-stranded 3' end and (ii) cleaved oligonucleotide ends linked to the solid carrier, affinity agent, or link-blocking moiety, leaving (iii) some uncleaved oligonucleotides that are not cleaved from the solid carrier, affinity agent, or link-blocking moiety and still have free 3' ends; the uncleaved oligonucleotides are then separated from the released target nucleic acid to form a target nucleic acid solution.

[0080] As an example, a methyltransferase that selectively methylates double-stranded DNA can be applied to methylate the double-stranded DNA, which can then be separated from the unmethylated nucleic acid. In some embodiments, the methyltransferase protein is used to specifically methylate single-stranded DNA, leaving the double-stranded portion unmethylated. Subsequently, bisulfite conversion can be used to selectively convert the unmethylated (i.e., double-stranded) cytosine to uracil, and then the uracil-containing nucleic acid is cleaved with the USER reagent to selectively cleave the double-stranded portion, leaving the single-stranded oligonucleotide linked to the bead. Thus, in some embodiments, a method for generating a captured target nucleic acid population is provided, comprising providing a plurality of oligonucleotides individually linked to a solid carrier, wherein the oligonucleotides have a free 3' end; contacting the plurality of oligonucleotides with a sample containing a target nucleic acid having a 3' end and a 5' end, wherein the target nucleic acid anneals to some of the free 3' ends of the oligonucleotides, and wherein an excess of oligonucleotides is present such that at least some oligonucleotides still have a free 3' end; then using the oligonucleotides as a template, extending the 3' end of the target nucleic acid with a polymerase to form a double-stranded portion; and selectively cleaving the double-stranded portion with a methyltransferase. Cytosine in the strand portion is converted to uracil; then uracil is cleaved with uracil DNA glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII to form (i) a released target nucleic acid having a single-stranded 5' end and a double-stranded 3' end and (ii) cleaved oligonucleotide ends linked to the solid carrier, affinity agent or link-blocking moiety, leaving (iii) some uncleaved oligonucleotides that are not cleaved from the solid carrier, affinity agent or link-blocking moiety and still have free 3' ends; then the uncleaved oligonucleotides are separated from the released target nucleic acid to form a target nucleic acid solution.

[0081] In some embodiments, a formamidopyrimidine DNA glycosylation enzyme is used to target double-stranded portions. The formamidopyrimidine DNA glycosylation enzyme selectively cleaves double-stranded DNA containing oxoguanine bases (e.g., present in bead-linked oligonucleotides). A formamidopyrimidine DNA glycosylation enzyme (FPG) cleaves oxoguanine bases from the double-stranded portion rather than the ssDNA sequence. Removing guanine by FPG leaves breaks in the DNA backbone, so that, if properly designed, the oligonucleotides will dissociate from each other due to heat. Therefore, in some embodiments, a method for generating a captured target nucleic acid population is provided, comprising providing a plurality of oligonucleotides individually linked to a solid carrier, wherein said oligonucleotides have a free 3' end and one or more oxoguanine bases; contacting said plurality of oligonucleotides with a sample containing a target nucleic acid having a 3' end and a 5' end, wherein the target nucleic acid anneals to some of said free 3' ends of said oligonucleotides, and wherein an excess of oligonucleotides is present such that at least some oligonucleotides still have a free 3' end; and then using said oligonucleotides as a template, extending the 3' ends of said target nucleic acid with a polymerase. The double-stranded portion is formed by cleaving the double-stranded portion with one or more oxoguanine bases; then the double-stranded portion is cleaved with a formamidopyrimidine DNA glycosylase to form (i) a released target nucleic acid having a single-stranded 5' end and a double-stranded 3' end and (ii) cleaved oligonucleotide ends attached to the solid carrier, affinity agent or link-blocking portion, and leaving (iii) some uncleaved oligonucleotides that are not cleaved from the solid carrier, affinity agent or link-blocking portion and still have free 3' ends; then the uncleaved oligonucleotides are separated from the released target nucleic acid to form a target nucleic acid solution.

[0082] In some embodiments, TelN prokaryotic telomerase is used to target the double-stranded portion. TelN prokaryotic telomerase selectively cleaves the double-stranded sequence and ligates the 5' end of one strand to the 3' end of the complementary end. The resulting sequence can be targeted for PCR amplification. Since the 5' end of the newly formed sequence is now accessible (and the bead is covalently linked to another bead molecule), the 5' end of the newly formed sequence can be targeted as needed for modification and enrichment. Therefore, in some embodiments, a method for generating a captured target nucleic acid population is provided, comprising providing a plurality of oligonucleotides individually ligated to a solid carrier, wherein the oligonucleotides have a free 3' end; contacting the plurality of oligonucleotides with a sample containing a target nucleic acid having both a 3' end and a 5' end, wherein the target nucleic acid anneals to some of the free 3' ends of the oligonucleotides, and wherein an excess of oligonucleotides is present such that at least some oligonucleotides still have a free 3' end; and then using the oligonucleotides as a template, extending the 3' end of the target nucleic acid with a polymerase to form a sequence containing a TelN prokaryotic telomerase recognition sequence. The double-stranded portion is then cleaved with TelN prokaryotic telomerase to form (i) a released target nucleic acid having a single-stranded 5' end and a 3' end formed by the connection of the 3' end to the 5' end due to double-strand cleavage, and (ii) cleaved oligonucleotide ends connected to the solid carrier, affinity agent, or link-blocking portion, leaving (iii) some uncleaved oligonucleotides that are not cleaved from the solid carrier, affinity agent, or link-blocking portion and still have free 3' ends; the uncleaved oligonucleotides are then separated from the released target nucleic acid to form a target nucleic acid solution.

[0083] Multiple solid vectors or multiple partitions containing said multiple solid vectors are also provided, said multiple solid vectors being linked to multiple oligonucleotides as described herein in the context of the methods described above. For example, the oligonucleotides from 5' to 3' may comprise: one strand of a double-stranded restriction enzyme recognition sequence, a bead-specific barcode sequence, and a 3' target-specific sequence. In some embodiments, the partitions may also contain a restriction enzyme that cleaves at the double-stranded restriction enzyme recognition sequence. In some embodiments, the partitions may also contain a polymerase for extending the target nucleic acid, which will anneal to the oligonucleotide as described herein. For example, in some embodiments, the polymerase is a reverse transcriptase.

[0084] Kits are also provided that provide one or more reagents for performing the described methods. For example, in some embodiments, the kit comprises (i) a plurality of solid carriers (e.g., beads) linked to a plurality of oligonucleotides, the plurality of oligonucleotides comprising, from 5' to 3': one strand of a double-stranded restriction enzyme recognition sequence, a bead-specific barcode sequence, and a 3' target-specific sequence; and (ii) a restriction enzyme, or Argonaute protein, formamidopyrimidine DNA glycosylase, methyltransferase, or TelN prokaryotic telomerase, that cleaves at the double-stranded restriction enzyme recognition sequence. In some embodiments, the kit may also comprise a polymerase for extending a target nucleic acid, which will be annealed to the oligonucleotide as described herein. For example, in some embodiments, the polymerase is a reverse transcriptase.

[0085] Example

[0086] Example 1

[0087] Figure 5 A table of results for Example 1 is provided. 4,000 beads containing ssDNA-capturing oligonucleotides with the restriction enzyme shown were incubated with an excess of complementary oligonucleotides to generate double-stranded DNA recognition sites for the restriction enzyme shown, or if the above procedure did not generate the double-stranded DNA recognition sites. The beads were then incubated in a buffer suitable for either the restriction enzyme or USER to digest the bead-capturing oligonucleotides. The reaction mixture was then centrifuged at 1000 × g for 5 min to precipitate the beads, and a volume of supernatant was removed from the sample. The collected supernatant was subjected to ddPCR to quantify the number of bead-capturing oligonucleotides present in the supernatant. The restriction enzyme releases the bead-capturing oligonucleotides only if the oligonucleotide contains a complementary sequence and is therefore double-stranded. In contrast, the USER enzyme cleaves uracil present in the bead-capturing oligonucleotides regardless of the complementary sequence, i.e., whether it is ssDNA or dsDNA.

[0088] Example 2

[0089] Figure 6A table of results from Example 2 is provided. 4,000 beads containing the capturing oligonucleotide with the restriction enzyme shown were incubated with an excess of complementary oligonucleotides to generate a double-stranded DNA recognition site for the restriction enzyme shown, or the above operation did not generate the double-stranded DNA recognition site. The beads were then separated from the cells in an aqueous droplet in oil. The droplet contained reverse transcription reactants and the restriction enzyme shown, and was incubated at 37°C for one hour. After the RT reaction, the droplets were broken, the beads were centrifuged from the solution, and the supernatant was recovered. The supernatant was then analyzed by ddPCR targeting the bead-capturing oligonucleotide. Only beads incubated with the complementary sequence of the bead-capturing oligonucleotide, or those digested with the USER enzyme, released the entire complementary sequence of the bead-capturing oligonucleotide. However, beads not incubated with the complementary sequence of the bead-capturing oligonucleotide released only a small amount of bead-capturing oligonucleotide, presumably only the bead-capturing oligonucleotide with the captured cDNA and the bead-complementary sequence.

[0090] Example 3

[0091] Figure 7 A table of results from Example 3 is provided. 200,000 beads containing capture oligonucleotides with the restriction enzyme shown were separated from cells in an aqueous droplet in oil. The aqueous solution of the droplet consisted of reverse transcription reactants and the restriction enzyme shown, and the droplet was incubated at 37°C for one hour. After the RT reaction, the droplet was broken up, the beads were centrifuged from the solution, and the supernatant was recovered. The supernatant was then analyzed using ddPCR targeting GAPDH, either with primers specific to GAPDH or with one primer targeting the bead-capturing oligonucleotide and another oligonucleotide targeting the GAPDH gene, to produce chimeric amplicon containing both the bead and GAPDH sequences. The GAPDH levels produced by the restriction enzyme-released oligonucleotides were similar to, but slightly lower than, those from USER-digested beads.

[0092] Example 4

[0093] 200,000 beads containing bis(acryloyl)cysteine ​​(a cross-linking chemical containing disulfide bonds reduced in the presence of DTT) and capturing oligonucleotides with the restriction enzyme (ApaLI) shown were separated from 10,000 cells in an oil-enclosed aqueous droplet. The aqueous droplet consisted of a reverse transcription reactant containing either the restriction enzyme or the USER enzyme shown. The sample was first incubated at 4°C for 25 min for cell lysis and mRNA capture, followed by incubation at 50°C for 45 min for oligonucleotide release and RT, then at 65°C for 30 min for second-strand synthesis, and finally at 80°C for 5 min for enzyme inactivation. After the RT reaction, the droplets were ruptured, and the sample was purified using SPRI reagent at a ratio of 1.8:1. cDNA was recovered and then analyzed by ddPCR targeting the GAPDH, HPRT1, or PTEN genes. See [link to PCR description]. Figure 8 .

[0094] Although the foregoing disclosure has been described in detail by way of illustration and example for the purpose of clarity, those skilled in the art will understand that certain changes and modifications may be made within the scope of the appended claims. Furthermore, each reference provided herein, including patents, patent applications, non-patent documents, and Genbank accession numbers, is incorporated herein by reference in its entirety to the same extent as if it were individually incorporated by reference. In the event of any conflict between this application and the references provided herein, this application shall prevail.

Claims

1. A method for generating a population of captured target nucleic acids, the method comprising: Provided multiple oligonucleotides individually linked to a solid carrier, wherein said oligonucleotides have a free 3' end; The plurality of oligonucleotides are contacted with a sample containing a target nucleic acid having 3' and 5' ends, wherein the target nucleic acid is annealed to some of the free 3' ends of the oligonucleotides, and wherein an excess of oligonucleotides is present such that at least some oligonucleotides still have free 3' ends; and then Using the oligonucleotide as a template, the 3' end of the target nucleic acid is extended with polymerase to form a double-stranded portion containing a restriction enzyme recognition sequence; And then The restriction enzyme recognition sequence is cleaved with a restriction enzyme to form (i) a released target nucleic acid having a single-stranded 5' end and a double-stranded 3' end and (ii) cleaved oligonucleotide ends linked to the solid carrier, affinity agent, or link-blocking moiety, leaving (iii) some uncleaved oligonucleotides that are not cleaved from the solid carrier, affinity agent, or link-blocking moiety and still have free 3' ends; and then The uncut oligonucleotides are separated from the released target nucleic acid to form a target nucleic acid solution.

2. The method according to claim 1, wherein the plurality of oligonucleotides are individually ligated to a solid carrier.

3. The method of claim 2, wherein the oligonucleotide comprises a 5' end, and the 5' end is attached to the solid carrier.

4. The method according to claim 1, wherein the oligonucleotide from 5' to 3' comprises: one strand of the double-stranded restriction enzyme recognition sequence, a solid-carrier-specific barcode sequence, and a 3' target-specific sequence.

5. The method according to claim 1, wherein the double-stranded restriction enzyme recognition sequence is selected from the group consisting of ApaLI, PmeI, AflII, AflIII and PacI.

6. The method of claim 1, wherein the oligonucleotide comprises a 5' end and the 5' end is linked to a bead, and the oligonucleotide further comprises 2-10 nucleotides at the 5' end of one strand of the double-stranded restriction enzyme recognition sequence.

7. The method according to any one of claims 1 to 6, wherein the double-stranded portion comprises two or more different restriction enzyme recognition sequences.

8. The method of claim 2, wherein the oligonucleotide is covalently linked to the solid support.

9. The method of claim 2, wherein the oligonucleotide is non-covalently linked to the solid support.

10. The method of claim 9, wherein the oligonucleotide is biotinylated and the solid carrier comprises streptavidin.

11. The method of claim 10, wherein, after cleavage, biotin-based streptavidin affinity separates the biotinylated nucleic acid comprising the cleaved oligonucleotide terminus from the released target nucleic acid.

12. The method according to any one of claims 1 to 9, wherein the target nucleic acid is an RNA molecule and the polymerase is a reverse transcription polymerase.

13. The method of claim 12, wherein the reverse transcription polymerase is HIV reverse transcription polymerase, M-MLV reverse transcription polymerase or AMV reverse transcription polymerase.

14. The method according to any one of claims 1 to 9, wherein the target nucleic acid is a DNA molecule and the polymerase is a DNA polymerase.

15. The method according to any one of claims 1 to 13, wherein the solid carrier is one or more beads, wherein copies of oligonucleotides attached to the beads contain bead-specific barcode sequences, and wherein oligonucleotides attached to different beads have different bead-specific barcode sequences.

16. The method according to any one of claims 1 to 15, wherein the provision includes providing the solid carrier in a partition.

17. The method of claim 16, wherein the partition is a droplet or micropore in an oil-based emulsion.

18. The method of claim 16, wherein at least some of the partitions further comprise single cells, and wherein the target nucleic acid is a nucleic acid derived from the cell.

19. The method of claim 18, further comprising lysing or permeabilizing the cells in the partition.

20. The method according to any one of claims 16 to 19, wherein the cutting occurs in the partition.

21. The method according to any one of claims 1 to 20, wherein the oligonucleotide comprises a solid vector-specific barcode sequence and an optional PCR handle sequence.

22. The method of claim 1, wherein the plurality of oligonucleotides are individually linked to the affinity agent, and The separation includes contacting the affinity agent on the cleaved oligonucleotide ends and the uncleaved oligonucleotides with a reagent that binds the affinity agent; and separating the reagent that binds the affinity agent from the released target nucleic acid.

23. The method of claim 22, wherein the affinity agent is biotin, and the reagent binding the affinity agent is avidin or streptavidin.

24. The method of claim 1, wherein the plurality of oligonucleotides are individually linked to the link-blocking portion.

25. The method of claim 24, wherein the connection blocking portion is selected from 3'-dideoxycytidine (ddC), 3'-reverse dT, 3'-C3 spacer, 3'-amino, and 3'-phosphorylation.

26. The method of any one of claims 24 or 25, wherein the separation comprises ligating the 3' end of the released nucleic acid to a polynucleotide, wherein the uncut oligonucleotide or the cleaved oligonucleotide end is not ligated to the polynucleotide; and The nucleic acid linked to the polynucleotide is separated from the oligonucleotide containing the link-blocking portion and the cleaved oligonucleotide ends.

27. The method of claim 26, wherein the polynucleotide is linked to a solid carrier or affinity agent, thereby allowing selective separation of the polynucleotide from other nucleic acids in the mixture.

28. The method according to any one of claims 1 to 21, wherein the oligonucleotide is a complete DNA oligonucleotide.

29. The method according to any one of claims 1 to 21, wherein the oligonucleotide comprises DNA and uracil bases.

30. The method of claim 18, wherein the single cell is a mammalian cell.

31. A plurality of beads, wherein each bead is linked to a plurality of oligonucleotides, the plurality of oligonucleotides comprising, from 5' to 3': one strand of a double-stranded restriction enzyme recognition sequence, a bead-specific barcode sequence, and a 3' target-specific sequence.

32. The plurality of beads according to claim 31, wherein the double-stranded restriction enzyme recognition sequence is selected from the group consisting of ApaLI, PmeI, AflII, AflIII and PacI.

33. The plurality of beads according to claim 31 or 32, wherein the oligonucleotide comprises one strand of two or more double-stranded restriction enzyme recognition sequences.

34. A plurality of beads according to any one of claims 31 to 32, wherein the oligonucleotide comprises a 5' end and the 5' end is attached to the bead, and the oligonucleotide further comprises 2-10 nucleotides at the 5' end of one strand of the double-stranded restriction enzyme recognition sequence.

35. A plurality of beads according to any one of claims 31 to 34, wherein the oligonucleotide is a complete DNA oligonucleotide.

36. A plurality of beads according to any one of claims 31 to 34, wherein the oligonucleotide comprises DNA and uracil bases.

37. A reagent kit comprising: A plurality of beads according to any one of claims 31 to 36; as well as The restriction enzyme that is cleaved at the double-stranded restriction enzyme recognition sequence.

38. The kit according to claim 34, further comprising reverse transcriptase.

39. Multiple partitions, wherein each partition comprises multiple oligonucleotides individually linked to a solid vector, wherein the oligonucleotides have a free 3' end and the oligonucleotides from 5' to 3' comprise: one strand of a double-stranded restriction enzyme recognition sequence, a solid vector-specific barcode sequence, and a 3' target-specific sequence.

40. The plurality of partitions according to claim 39, wherein the partitions are droplets or micropores in an oil-based emulsion.

41. The plurality of partitions according to claim 39 or 40, wherein the solid carrier is a bead.

42. The plurality of partitions according to any one of claims 39 to 41, wherein the double-stranded restriction enzyme recognition sequence is selected from the group consisting of ApaLI, PmeI, AflII, AflIII and PacI.

43. The plurality of partitions according to any one of claims 39 to 42, wherein the oligonucleotide comprises one strand of two or more double-stranded restriction enzyme recognition sequences.

44. The plurality of partitions according to any one of claims 39 to 43, wherein the oligonucleotide comprises a 5' end and the 5' end is attached to the bead, and the oligonucleotide further comprises 2-10 nucleotides at the 5' end of one strand of the double-stranded restriction enzyme recognition sequence.

45. The plurality of partitions according to any one of claims 39 to 44, wherein the oligonucleotide is a complete DNA oligonucleotide.

46. ​​The plurality of partitions according to any one of claims 39 to 44, wherein the oligonucleotide comprises DNA and uracil bases.

47. The plurality of partitions according to any one of claims 39 to 46, wherein some, but not all, of the partitions contain a target nucleic acid annealed to some of the free 3' ends of the oligonucleotide to form a double-stranded portion, and wherein there is an excess of oligonucleotide such that at least some of the oligonucleotide still have a free 3' end.

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