WO2025085431A1

WO2025085431A1 - Enzymatic selection of nucleic acid

Info

Publication number: WO2025085431A1
Application number: PCT/US2024/051392
Authority: WO
Inventors: Jeffrey Perry; Tasnim CHOUDHURY
Original assignee: Bio Rad Laboratories Inc
Current assignee: Bio Rad Laboratories Inc
Priority date: 2023-10-16
Filing date: 2024-10-15
Publication date: 2025-04-24
Anticipated expiration: 2026-04-16
Also published as: WO2025085431A9

Abstract

Methods and compositions for method of generating a population of captured target nucleic acids are provided. Exemplary methods can include contacting the plurality of oligonucleotides individually linked to a solid support with a sample comprising target nucleic acids having 3' ends and 5' ends, wherein target nucleic acids anneal to some of the free 3' ends of the oligonucleotides, and wherein there is an excess of oligonucleotides such that at least some oligonucleotides remain with free 3' ends; and then extending the target nucleic acid 3' end with a polymerase using the oligonucleotide as a template to form double-stranded portions that comprise a restriction enzyme recognition sequence; and then cleaving the restriction enzyme recognition sequence with a restriction enzyme to form released target nucleic acids having a single-stranded 5' end and a double-stranded 3' end and leaving oligonucleotides remain with free 3' ends uncleaved from the solid support; and optionally then separating the solid support and uncleaved oligonucleotides from the released target nucleic acids to form a solution of target nucleic acids.

Description

ENZYMATIC SELECTION OF NUCLEIC ACID

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS [0001] The present application claims benefit of priority to U.S. Provisional Patent Application No. 63/544,372, filed October 16. 2023, which is incorporated by reference for all purposes.

BACKGROUND OF THE INVENTION

[0002] Single cell genomic analyses, including single cell sequencing, occurs in partitions (e.g., droplets). Sequencing occurs by attaching oligonucleotides comprising partitionspecific barcodes to target nucleic acids from the cells, allowing target nucleic acids from different cells to be differentiated based on the identity of the partition-specific barcode sequence. In some approaches, a solid support, such as a bead, is used to deliver many copies of the oligonucleotides to the partition. Delivery' of the beads, as well as single cells, to the partitions is dictated by Poisson distributions. Because one goal is avoiding multiple cells in one partition, users adjust the number of partitions to be higher than the number of cells, thus resulting in many partitions lacking cells, and thus lacking target nucleic acids, but often containing beads and their oligonucleotides. Oligonucleotides can be released from the beads before or after being annealed to target nucleic acids, but in general, the mechanism of release of the beads is agnostic to whether the partitions contain a target nucleic acid. An example of a release mechanism is for example inclusion of one or more uracil linking the oligonucleotides to the beads and using uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII to release the oligonucleotides at the uracil. In other cases. UV cleavable chemical moieties, disulfides or other chemistry are used that can be released by the user. However, in general these methods do not distinguish between oligonucleotides annealed to target nucleic acids and those not annealed, resulting in excess released oligonucleotides in partitions containing sample and in partitions lacking sample, e.g., partitions without single cells (and thus without target nucleic acids). The presence of the released oligonucleotides not annealed to target nucleic acid can be a source of background noise in later sequencing reactions. BRIEF SUMMARY OF THE INVENTION

[0003] In some embodiments, a method of generating a population of captured target nucleic acids is provided. In some embodiments, the method comprises providing a plurality of oligonucleotides individually linked to a solid support, wherein the oligonucleotides have a free 3’ end; contacting the plurality' of oligonucleotides with a sample comprising target nucleic acids having 3‘ ends and 5’ ends, wherein target nucleic acids anneal to some of the free 3^? ends of the oligonucleotides, and wherein there is an excess of oligonucleotides such that at least some oligonucleotides remain with free 3’ ends; and then extending the target nucleic acid 3’ end with a polymerase using the oligonucleotide as a template to form doublestranded portions that comprise a restriction enzy me recognition sequence; and then cleaving the restriction enzyme recognition sequence with a restriction enzyme to form (i) released target nucleic acids having a single-stranded 5’ end and a double-stranded 3’ end and (ii) cleaved oligonucleotide ends linked to the solid support, affinity' agent, or ligation-blocking moiety' and leaving (iii) some uncleaved oligonucleotides remaining with free 3’ ends uncleaved from the solid support, affinity agent, or the ligation-blocking moiety’; and then separating the uncleaved oligonucleotides from the released target nucleic acids to form a solution of target nucleic acids. In some embodiments, the double stranded portion instead of being a restriction enzy me recognition sequence can be a sequence recognized by a different enzyme that selectively cuts the double-stranded sequence, for example as described elsewhere herein.

[0004] In some embodiments, the plurality of oligonucleotides are individually linked to a solid support. In some embodiments, the oligonucleotides comprise 5’ ends and the 5’ ends are linked to the solid support.

[0005] In some embodiments, the oligonucleotides comprise 5’ to 3’: one strand of the double-stranded restriction enzyme recognition sequence, a solid support-specific barcode sequence and a 3‘ target-specific sequence.

[0006] In some embodiments, the double-stranded restriction enzyme recognition sequence is selected from the group consisting of ApaLI, Pmel, Aflll, AfHII and Pad.

[0007] In some embodiments, the oligonucleotides comprise 5’ ends and the 5’ ends are linked to the beads and the oligonucleotides further comprise 2-10 nucleotides 5’ of the one strand of the double-stranded restriction enzyme recognition sequence. [0008] In some embodiments, the double-stranded portions comprise two or more different restriction enzyme recognition sequences.

[0009] In some embodiments, the oligonucleotides are covalently linked to the solid support. In some embodiments, the oligonucleotides are non-covalently linked to the solid support. In some embodiments, the oligonucleotides are biotinylated and solid support comprises streptavidin. In some embodiments, following the cleaving, separating biotinylated nucleic acids, including cleaved oligonucleotide ends, from released target nucleic acids based on streptavidin affinity of the biotin.

[0010] In some embodiments, the target nucleic acid is an RNA molecule and the polymerase is a reverse polymerase. In some embodiments, the reverse polymerase is a HIV reverse polymerase, M-MLV reverse polymerase, or an AMV reverse polymerase.

[0011] In some embodiments, the target nucleic acid is a DNA molecule and the polymerase is a DNA polymerase.

[0012] In some embodiments, the solid support is one or more bead, wherein copies of oligonucleotides linked to a bead comprise bead-specific barcode sequences and wherein oligonucleotides linked to different beads have different bead-specific barcode sequences.

[0013] In some embodiments, the providing comprises providing the solid supports in partitions. In some embodiments, the partitions are droplets in an oil-based emulsion or microwells. In some embodiments, at least some of the partitions further comprise single cells and wherein the target nucleic acids are nucleic acids from the cells. In some embodiments, the method further comprises lysing or permeabilizing the cells in the partitions.

[0014] In some embodiments, the cleaving occurs in the partitions.

[0015] In some embodiments, the oligonucleotides comprise a solid-support-specific barcode sequence and optionally a PCR handle sequence.

[0016] In some embodiments, the plurality of oligonucleotides individually are linked to an affinity agent and the separating comprises contacting the affinity agent on the cleaved oligonucleotide ends and uncleaved oligonucleotides with an agent that binds the affinity agent; and separating the agent bound to the affinity agent from the released target nucleic acids. In some embodiments, the affinity agent is biotin and the agent that binds the affinityagent is avidin or streptavidin.

[0017] In some embodiments, the plurality of oligonucleotides individually are linked to a ligation-blocking moiety. In some embodiments, the ligation-blocking moiety is selected from 3' dideoxy cytidine (ddC), 3' inverted dT, 3' C3 spacer, 3' amino, and 3' phosphory lation. In some embodiments, the separating comprises ligating released nucleic acid 3’ ends to a polynucleotide, wherein uncleaved oligonucleotides or cleaved oligonucleotide ends are not ligated to the polynucleotide; and separating nucleic acids ligated to the polynucleotide from oligonucleotides and cleaved oligonucleotide ends comprising the ligation-blocking moiety-. In some embodiments, the polynucleotide is linked to a solid support or an affinity- agent allowing for selective separation of the polynucleotides from other nucleic acids in a mixture.

[0018] In some embodiments, the oligonucleotides are fully DNA oligonucleotides. In some embodiments, the oligonucleotides comprise DNA and uracil bases.

[0019] In some embodiments, the single cells are mammalian cells.

[0020] In some embodiments, a plurality of beads are provided wherein individual beads are linked to a plurality- of oligonucleotides comprising 5’ to 3’: one strand of a doublestranded 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, Pmel, Aflll, AfUII and Pad. In some embodiments, the oligonucleotides comprise one strand of two or more double-stranded restriction enzyme recognition sequences.

[0021] In some embodiments, the oligonucleotides comprise 5’ ends and the 5‘ ends are linked to the beads and the oligonucleotides further comprise 2-10 nucleotides 5’ of the one strand of the double-stranded restriction enzyme recognition sequence.

[0022] In some embodiments, the oligonucleotides are fully DNA oligonucleotides. In some embodiments, the oligonucleotides comprise DNA and uracil bases.

[0023] In some embodiments, kits are provided. In some embodiments, the kits comprise the plurality- of beads as described above or elsewhere herein; and a restriction enzyme that cleaves at the double-stranded restriction enzyme recognition sequence. In some embodiments, the kit further comprises a reverse transcriptase. [0024] In some embodiments, a plurality of partitions is provided. In some embodiments, the partitions comprise plurality of oligonucleotides individually linked to a solid support, wherein the oligonucleotides have a free 3’ end and the oligonucleotides comprise 5’ to 3’: one strand of the double-stranded restriction enzyme recognition sequence, a solid supportspecific barcode sequence and a 3’ target-specific sequence. In some embodiments, the partitions are droplets in an oil-based emulsion or microwells. In some embodiments, the solid support is a bead. In some embodiments, the double-stranded restriction enzyme recognition sequence is selected from the group consisting of ApaLI, Pmel, Aflll, AfUII and Pad. In some embodiments, the oligonucleotides comprise one strand of two or more doublestranded restriction enzyme recognition sequences. In some embodiments, the oligonucleotides comprise 5’ ends and the 5' ends are linked to the beads and the oligonucleotides further comprise 2-10 nucleotides 5’ of the one strand of the doublestranded restriction enzyme recognition sequence. In some embodiments, the oligonucleotides are fully DNA oligonucleotides. In some embodiments, the oligonucleotides comprise DNA and uracil bases. In some embodiments, some but not all partitions comprise target nucleic acids that anneal to some of the free 3’ ends of the oligonucleotides to form double-stranded portions, and wherein there is an excess of oligonucleotides such that at least some oligonucleotides remain with free 3’ ends.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 depicts background options for oligonucleotide-based target nucleic acid capture. Hybridization-based capture of target nucleic acid requires an excess of capture molecule for every target molecule. The capture molecule can also include the tag or primer site necessary' for downstream application, for example, NGS library' creation, cloning constructs, etc.

[0026] FIG. 2 depicts a problem experienced in previous methods: removal of excess capture oligonucleotides from the reaction mixture. For example, in the past, methods for removal of excess capture oligonucleotides have included exonuclease digestion or biotinylation and streptavidin enrichment.

[0027] FIG. 3 depicts aspects of the methods described herein. Target nucleic acids are annealed to oligonucleotides comprising a single strand of a restriction enzyme recognition sequence and linked to the solid support. Following contact with a polymerase, and extension of the target nucleic acids using the oligonucleotides, including the single strand of a restriction enzyme recognition sequence, as a template (not depicted), a double-stranded restriction enzyme recognition sequence is generated. (Also not depicted: extension of the oligonucleotide 3’ end using the target nucleic acid as a template, thereby generating a sequence later to be sequenced.) A restriction enzyme cleaves in or near the double-stranded restriction enzyme recognition sequence, releasing those oligonucleotides, which have been extended by the polymerase to comprise a reverse complement of the target nucleic acid. The non-cleaved oligonucleotides remain linked to the solid support and can be separated from the released, extended, oligonucleotides, which can then be used for any desired downstream use, which can include but is not limited to nucleotide sequencing.

[0028] FIG. 4 depicts an exemplar}⁷ simplified workflow for the methods described herein. Droplet partitions are formed to contain (i) single cells and (ii) at least one bead linked to a plurality of copies of capture oligonucleotides (only one oligonucleotide is depicted for simplicity). Cells and beads can be compartmentalized in droplets surrounded by oil, microwells, or other partitions. Once compartmentalized, cells can be lysed to release cellular components of interest, for example mRNA. The released mRNA can be captured with 3’- end Poly T-containing oligonucleotides also having a diverse sequence used to barcode beads. Once the mRNA is captured, a reverse transcriptase (RT) reaction can create hybrid DNA molecules comprising a first strand cDNA by extending the capture oligonucleotide. The hybrid DNA molecules can be modified into sequenceable library molecules suitable for downstream sequencing such as next-generation sequencing (NGS). Efficiency of capturing the cellular components such as mRNA can be increased by adding excess capture oligos. However, excess capture oligos contain the defined DNA sequences used to amplify the captured mRNA/cDNA, which can cause excess noise in the downstream applications. Removing excess bead capture oligos can be accomplished by exonuclease treatments, SPRI bead cleanup, and other approaches. See, e.g., FIG. 2. However as described herein, one can use selective release of bead target oligonucleotides-containing cDNA by a novel approach that leaves excess bead oligonucleotide without cDNA attached to a substrate. That substrate can then be used to remove or retain excess bead oligonucleotides, while the cellular components of interest remain in solution. In addition, partitions without sample (e.g., without a cell) would typically release the bead oligonucleotides even with no cell or target nucleic acid to capture. This approach would also remove such excess bead oligonucleotides resulting from this situation from downstream steps. [0029] FIG. 5 provides a table of results from example 1. 4,000 beads containing ssDNA capture oligonucleotides with the restriction enzyme site indicated were incubated with an excess of complement oligonucleotide to create a double stranded DNA recognition site for restriction enzymes indicated, or not. The beads were then incubated in a buffer suitable for the restriction enzyme or USER to digest the bead capture oligonucleotide. The reactions were then centrifuged at 1000 x g for 5 minutes to pellet the beads, and a volume of supernatant was removed from the sample. A ddPCR assay was run on the collected supernatant to quantify the number of bead capture oligonucleotides present in the supernatant. The restriction enzy mes only released the bead capture oligonucleotide when the oligo contained complement and therefore was double stranded. In contrast the USER enzyme cleaved the uracil present in the bead capture oligonucleotide regardless of complement, so either ssDNA or dsDNA.

[0030] FIG. 6 provides a table of results from example 2. 4,000 beads were incubated with K562 total RNA to mimic cell lysis and mRNA capture. After RNA capture, beads were incubated with an excess of complement oligonucleotide to create a double strand DNA recognition site for restriction enzymes indicated, or not. The beads then underwent an RT reaction at 50°C for 40 minutes, followed by a restriction enzyme digestion reaction at 37°C for 20 minutes. After the RT and restriction enzyme reactions, beads were centrifuged out of solution, and the supernatant recovered. The supernatant was then assay ed with a ddPCR targeting the bead capture oligonucleotide. Beads incubated with bead capture oligonucleotide complement released the entire complement of bead capture oligonucleotides, or those beads digested with USER enzyme. However, the beads not incubated with bead capture oligonucleotide complement only released a small amount of bead capture oligo presumably only those bead capture oligonucleotides with captured cDNA and bead complement.

[0031] FIG. 7 provides a table of results from example 3. 200,000 beads containing capture oligonucleotides with the restriction site indicated were compartmentalized with cells in aqueous droplets surrounded by oil. The aqueous solution of the droplets consisted of a reverse transcription reaction and the restriction enzymes indicated. The droplets were incubated at 50°C for 40 minutes followed by 37°C for 20 minutes. After the RT reaction, droplets were broken, beads centrifuged out of solution, and the supernatant recovered. The supernatant was then assayed with a ddPCR targeting GAPDH either with primers specific to GAPDH or one primer targeting the bead capture oligonucleotide and the other oligo targeting the GAPDH gene creating a chimera amplicon with both bead and GAPDH sequence. Restriction enzyme released oligonucleotides yielded similar if slightly lower levels of GAPDH cDNA than the USER digested beads.

[0032] FIG. 8 provides data generated as described in Example 4. Restriction Enzymes recognize and cleave bead capture oligonucleotides with dsDNA sites created by RT with cells in compartments. Released cDNA is at comparable levels to USER digest releasing bead capture oligonucleotides.

DEFINITIONS

[0033] Unless defined otherwise, 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 belongs. Generally, the nomenclature used herein and the laboratoryprocedures in cell culture, molecular genetics, organic chemistry, analytical chemistry, and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art. Standard techniques are used for nucleic acid and peptide synthesis. The techniques and procedures are generally performed according to conventional methods in the art and various general references (see generally, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed. (1989) Cold Spnng Harbor Laboratory Press, Cold Spring Harbor, N.Y., which is incorporated herein by reference), which are provided throughout this document.

[0034] The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a.” “an.” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a bead” includes a plurality of such beads and reference to “the sequence” includes reference to one or more sequences know n to those skilled in the art, and so forth.

[0035] The term "amplification reaction" refers to any in vitro method for multiplying the copies of a target sequence 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); QBeta RNA replicase and RNA transcription-based amplification reactions (e.g, amplification that involves T7, T3, or SP6 primed RNA polymerization), such as the transcription amplification system (TAS), nucleic acid sequence based amplification (NASBA), and self-sustained sequence replication (3 SR); single-primer isothermal amplification (SPIA), loop mediated isothermal amplification (LAMP), strand displacement amplification (SDA); multiple displacement amplification (MDA); rolling circle amplification (RCA); as well as others known to those of skill in the art. See, e.g., Fakruddin et al., J. Pharm Bioallied Sci. 2013 5(4):245-252.

[0036] "Amplifying" refers to a step of submitting a solution to conditions sufficient to allow for amplification of a polynucleotide if all the components of the reaction are intact. Components of an amplification reaction include, e.g.. primers, a polynucleotide template, polymerase, nucleotides, and the like. The term "amplifying" typically refers to an "exponential" increase in target nucleic acid. However, "amplifying" as used herein can also refer to linear increases in the numbers of a select target sequence of nucleic acid, such as obtained with cycle sequencing or linear amplification.

[0037] As used herein a "‘barcode” is a short nucleotide sequence (e.g. , at least about 4. 6, 8, 10, 12, 15, 20, 50 or 75 or 100 nucleotides long or more) that identifies a molecule to which it is conjugated or from the solid support or partition in which it originated. Barcodes can be used, e.g., to identify⁷ molecules originating in a partition and/or bead as later sequenced from a bulk reaction. Such a barcode can be unique for that partition or bead as compared to barcodes present in other partitions or bead. For example, partitions containing target RNA from single-cells can be subject to reverse transcription conditions using primers that contain different partition-specific barcode sequence in each partition, thus incorporating a copy of a unique “cellular barcode” (because different cells are in different partitions and each partition has unique partition-specific barcodes) into the reverse transcribed target nucleic acids of each partition. Thus, nucleic acid from each cell can be distinguished from nucleic acid of other cells due to the unique “cellular barcode.” In some embodiments, oligonucleotides can further comprise barcodes that uniquely identify the molecule to which it is conjugated, i.e., the barcode acts as a unique molecular identifier (UMI). The length of the underlying barcode sequence determines how many unique samples can be differentiated. For example, a 1 nucleotide barcode can differentiate 4, or fewer depending on degeneracy, different partitions; a 4 nucleotide barcode can differentiate 4⁴ or 256 partitions or less; a 6 nucleotide barcode can differentiate 4096 different partitions or less; and an 8 nucleotide barcode can index 65,536 different partitions or less. Barcode sequences can be contiguous or can include non-contiguous portions, which if separated will often include a constant region that can be used to identify the position of the barcode sequences. [0038] "Polymerase chain reaction" or "PCR" refers to a method whereby a specific segment or subsequence of a target double-stranded DNA, is amplified in a geometric progression. PCR is well known to those of skill in the art; see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds, 1990. Exemplary PCR reaction conditions typically comprise either two or three step cycles. Two step cycles have a denaturation step followed by a hybridization/elongation step. Three step cycles comprise a denaturation step followed by a hybridization step followed by a separate elongation step.

[0039] As used herein, "nucleic acid" refers to DNA, RNA, single-stranded, doublestranded, or more highly aggregated hybridization motifs, and any chemical modifications thereof. Modifications include, but are not limited to, those providing chemical groups that incorporate additional charge, polarizability, hydrogen bonding, electrostatic interaction, points of attachment and functionality to the nucleic acid ligand bases or to the nucleic acid ligand as a whole. Such modifications include, but are not limited to, peptide nucleic acids (PNAs), phosphodiester group modifications (e.g.. phosphorothioates, methylphosphonates), 2'-position sugar modifications, 5-position pyrimidine modifications, 8-position purine modifications, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5 -iodo-uracil; backbone modifications, methylations, unusual base-pairing combinations such as the isobases, isocytidine and isoguanidine and the like. Nucleic acids can also include non-natural bases, such as, for example, nitroindole. Modifications can also include 3' and 5' modifications including but not limited to capping with a fluoroph ore (e.g., quantum dot) or another moiety.

[0040] As used herein, the term "partitioning" or "partitioned" refers to separating a sample into a plurality⁷ of portions, or "partitions." Partitions can be solid or fluid. In some embodiments, a partition is a solid partition, e.g., a microchannel. In some embodiments, a partition is a fluid partition, e.g., 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 that is surrounded by an immiscible carrier fluid (e.g., oil).

[0041] An "‘oligonucleotide” is a polynucleotide. Generally oligonucleotides will have fewer than 250 nucleotides, in some embodiments, between 4-200, e.g., 10-150 nucleotides. [0042] “Clonal” copies of a polynucleotide means the copies are identical in sequence (or identical aside from a unique molecular identifier (UMI) sequence). In some embodiments, there are at least 100, 1000, 10⁴ or more clonal copies of oligonucleotides in linked to a bead.

[0043] A “3’ capture sequence” on an oligonucleotide refers to the 3’ most portion of an oligonucleotide. The capture sequence can be as few as 1-2 nucleotides in length but is more commonly 6-12 nucleotides in length and in some embodiments is 4-20 or more nucleotides in length. The capture sequence can be completely complementary to a target nucleic acid (e.g., the 3’ end of the target nucleic acid), though as will be appreciated in some embodiments and certain conditions, 1, 2, 3, 4, or more nucleotides may be mismatched while still allowing the 3’ capture sequence of an oligonucleotide anneal to the target nucleic acid. In other embodiments, conditions can be selected such that only completely complementary sequences will anneal. The 3’ capture sequence can be a random sequence, a poly T or poly A sequence, a target-specific sequence, or a universal sequence.

[0044] The term “bead” refers to any solid support that can be in a partition, e.g., a small particle or other solid support. Exemplary⁷ beads can include hydrogel beads. In some cases, the hydrogel is in sol form. In some cases, the hydrogel is in gel form. An exemplary hydrogel is an agarose hydrogel. Other hydrogels include, but are not limited to, those described in, e.g, U.S. Patent Nos. 4,438,258; 6,534,083; 8,008,476; 8,329,763; U.S. Patent Appl. 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.

[0045] A “restriction enzyme recognition sequence” refers to a nucleotide sequence to which a restriction enzyme binds and whose presence is required to trigger cleavage of the nucleic acid in which the sequence resides. 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 DESCRIPTION OF THE INVENTION

[0046] Methods and compositions are provided that selectively cleave oligonucleotides from solid supports (or in other embodiments from an affinity agent or a ligation-blocking moiety) with a restriction enzy me selectively recognizes a double-stranded DNA recognition sequence and does not substantially recognize a single-stranded version of the sequence. The oligonucleotides are annealed to target nucleic acids that are extended by a polymerase (which can be a reverse transcriptase) to form a double-stranded restriction enzyme recognition sequence that can be used to cleave the oligonucleotides and annealed target nucleic acids, but does not release oligonucleotides that do not include the double-stranded restriction enzyme recognition sequence, i.e., oligonucleotides that have not been annealed to target nucleic acids that are extended by a polymerase. Accordingly, the methods and compositions allow one to anneal target nucleic acids to oligonucleotides linked to the solid support (or in other embodiments an affinity agent or a ligation-blocking moiety) and extend the target nucleic acid 3’ ends using the oligonucleotides as a template to form the doublestranded recognition sequence, which in turn are selectively cleaved. Without intending to limit the scope of applications of the methods, the methods can be particularly useful in the context of single-cell sequences in which many clonal oligonucleotides linked to a bead are delivered to a partition. A difficulty in current workflows is that capture oligonucleotides on the bead will create unproductive byproducts downstream that can increase noise and decrease sensitivity. The restriction enzy me approach described here bypasses this issue by selecting for the desired products (those oligonucleotides annealed to target nucleic acids) and allowing for the easy removal of byproducts without excessive cleanup steps.

[0047] A plurality of clonal oligonucleotides can be linked to a solid support and then delivered to a partition that may or may not contain a target nucleic acid. In some embodiments, there are at least 100, 1000, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸ or more clonal copies of oligonucleotides in linked to a bead. In many embodiments, a plurality of solid supports are provided and the oligonucleotides comprise a solid-support-specific barcode sequence such that the solid-support-specific barcode sequence, when linked to target nucleic acids, can be used to determine the solid support (and in some embodiments, the partition) from which the solid-support-specific barcode sequence originated.

[0048] The oligonucleotides can be covalently or non-covalently linked to the solid support(s). Oligonucleotides can be linked to beads as desired. Methods of linking oligonucleotides to beads are described in, e.g., WO 2015/200541. In some embodiments, the oligonucleotide configured to link a hydrogel bead to the barcode is covalently linked to the hydrogel. Numerous methods for covalently linking an oligonucleotide to one or more hydrogel matrices are known in the art. As but one example, aldehyde derivatized agarose can be covalently linked to a 5 '-amine group of a synthetic oligonucleotide.

[0049] Any bead of useful size and composition for delivery to partitions can be used. The particle or bead can be any particle or bead having a solid support surface. Solid supports suitable for particles include controlled pore glass (CPG)(available from Glen Research, Sterling, Va.), oxalyl-controlled pore glass (See. e.g., Alul, et al., Nucleic Acids Research 1991, 19, 1527), TentaGel Support— an aminopolyethyleneglycol derivatized support (See, e.g., Wright, et al., Tetrahedron Letters 1993, 34, 3373), polystyrene, Poros (a copolymer of polystyrene/divinylbenzene), or reversibly cross-linked acrylamide. Many other solid supports are commercially available and amenable to the present methods. In some embodiments, the bead material is a polystyrene resin or poly(methyl methacrylate) (PMMA). The bead material can be metal. In some embodiments, the particle or bead comprises hydrogel or another similar composition. In some cases, the hydrogel is in sol form. In some cases, the hydrogel is in gel form. An exemplary hydrogel is an agarose hydrogel. Other hydrogels include, but are not limited to, those described in, e.g.. U.S. Patent Nos. 4,438,258; 6,534,083; 8,008,476; 8,329,763; U.S. Patent Appl. Nos. 20020009591;

20130022569; 20130034592; and International Patent Publication Nos. W01997030092; and WO2001049240. Additional compositions and methods for making and using hydrogels, such as barcoded hydrogels, include those described in, e.g, Klein et al., Cell, 2015 May 21;161(5):1187-201.

[0050] While the oligonucleotides can be linked to the solid support(s) in a number of ways, the 3’ end, and in general the 3’ capture sequence, i.e., the sequence at the 3’ end that will anneal to the target nucleic acid, will be a free end, meaning the 3’ end and the 3’ capture sequence are not directly linked to the solid support and instead a different portion of the oligonucleotide carrying the 3’ end and 3’ capture sequence is linked to the solid support, leaving the 3’ capture sequence to be available to anneal to a complementary target nucleic acid while being linked to the solid support.

[0051] In other embodiments, the oligonucleotides are not linked to a solid support and instead are linked to an affinity agent. Exemplary affinity agents can be for example, biotin, avidin, streptavidin or an antibody. As explained further below in these embodiments, once a double-stranded restriction sequence has been formed and cleaved, the affinity agent can be used to separate cleaved ends and uncleaved sequences from the target nucleic acids.

[0052] In other embodiments, the oligonucleotides are not linked to a solid support and instead are linked to a ligation-blocking moiety. Exemplary ligation-blocking moi eties can be for example, a 3' dideoxycytidine (ddC), 3' inverted dT, 3' C3 spacer, 3' amino, and 3' phosphorylation. As explained further below in these embodiments, once a double-stranded restriction sequence has been formed and cleaved, the ligation-blocking moiety will remain on uncleaved oligonucleotides as well as the cleaved ends, but the cleaved portion comprising the target nucleic acids will be available for a ligation reaction, which can be used to subsequently separate the target nucleic acids from the remainder of the nucleic acids, for example by affinity to the ligation partner or wherein the ligation partner comprises a biotin or other affinity agent,

[0053] The 3’ capture sequences of the oligonucleotide can vary in length and sequence depending on the target nucleic acid. In some embodiments, the target nucleic acids are mRNA and the 3’ capture sequence is a polyT sequence, for example 4-30 or more contiguous thymine nucleotides. In some embodiments, the target nucleic acids are RNA or DNA and the 3’ capture sequence is a random sequence, a target-specific sequence, a universal sequence (e.g., that anneals to sequences at the end of fragments introduced by a tagmentase) or any other desired capture sequence. Other desired captures sequences can be, but are not limited to, short or long interspersed retrotransposable elements (SINES or LINES, respectively).

[0054] In any of the embodiments described herein, the oligonucleotides linked to the beads can comprise one or more barcode nucleotide sequences. In some embodiments, the oligonucleotides include a barcode sequence that is unique to the solid support (e g., bead) to which it is attached and thus can be used to distinguish oligonucleotides from different beads, e.g., after the oligonucleotides are released and used to generate sequencing reads. Additional barcodes, such as but not limited to, unique molecule identifiers (UMIs) or sample-specific barcodes can also be included in the oligonucleotide sequence.

[0055] In some embodiments, the oligonucleotides further comprise one or more PCR handle sequence, or a reverse complement thereof, such that downstream nucleic products of the method can be amplified by a common (“universal”) primer in an amplification reaction.

[0056] In addition to the 3‘ end and the 3’ capture sequence and other optional sequences as discussed herein , the oligonucleotides will also include 5’ of the 3 ’ capture sequence and optional PCR handle and barcode sequences, , one strand of a double-stranded restriction enzyme recognition sequence and, if the oligonucleotides are linked to a solid support, a solid-support specific barcode sequence (both being 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 allowing cleavage of the double-stranded recognition sequences using two or more different restriction enzymes.

[0057] The restriction enzyme and corresponding double-stranded recognition sequence can be selected as desired so long as the restriction enzyme selectively acts on the doublestranded recognition sequence compared to a single-strand of the same sequence (e.g., 2X, 5X, 10X, 5 OX, 100X or more active on the dsDNA sequence than ssDNA sequence or RNA/DNA heteroduplexes, or both). In some embodiments, the recognition sequence is 4-10 nucleotides long, e.g., 6-8 nucleotides long. Exemplary restriction enzymes can include but are not limited to ApaLI, Pmel, AHU, AfUII, and Pacl.

[0058] In some embodiments, the 5’ end of the oligonucleotides comprise a number of nucleotides that separate the restriction site recognition sequence from the very 5 ’ end of the oligonucleotides, for example but not limited to in embodiments in which the oligonucleotides are linked to the solid support by their 5’ ends. The precise number and identity of nucleotides can be determined by the user and will provide a spacer such that the recognition sequence is not so close to the solid support that the later restriction enzyme cleavage step is inhibited. In some embodiments, the spacer nucleotides comprise 2-10, e.g., 4-6 nucleotides 5‘ of the one strand of the double-stranded restriction enzyme recognition sequence. The nucleotides can be determined as desired. In some embodiments, the nucleotides are Ts, i.e., comprising thymine bases.

[0059] While in some embodiments the methods and steps thereof described herein can be performed in a bulk solution, without different cells or samples compartmentalized in partitions, in many embodiments, some of all of the steps described herein are performed in partitions such that different cells or samples or other materials can be separated and reacted in separate partitions in parallel. Thus, in some embodiments, the plurality of oligonucleotides linked to a solid support (for example beads) can be introduced into partitions as desired. Exemplary partitions can include but are not limited to droplets (e.g., in an emulsion) or microwells. Introduction of the solid supports into the partitions can occur during or after formation of the partitions. Partitions can be pre-formed, optionally w ith other agents and optionally single cells or target nucleic acids from a biological sample and the solid supports and linked oligonucleotides can be injected or otherwise introduced into the partitions. Methods and compositions for delivering reagents to one or more partitions include microfluidic methods as known in the art; droplet or microcapsule merging, coalescing, fusing, bursting, or degrading (e.g., as described in U.S. 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, for example in which the partitions are droplets, one can form droplets as an emulsion with an immiscible fluid such as oil such that the bulk solution forms droplets that contain the beads and linked oligonucleotides, optionally with other reagents and/or a sample nucleic acid or cells. Methods of emulsion formation are described, for example, in published patent applications WO 2011/109546 and WO 2012/061444.

[0060] Distribution of beads (as well as single cells) into partitions (e.g., such as droplets) can be dictated by a Poisson distribution, in some embodiments. 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 more than 1 (e.g., 1-3. 1-10 or more). In some embodiments, it is desirable to avoid multiple beads in a partition and in these cases many partitions may be left empty such that a majority of partitions that contain a bead only contain one bead. In other embodiments, e.g., in which deconvolution methods can be used to decipher sequencing results where multiple beads occur in a single partition, more beads can be loaded on average per partition, with deconvolution being used after to resolve sequencing results. See, e.g., PCT/US2017/012618; PCT/US2019/015638; PCT/US2020/36699.

[0061] In some embodiments, the sample (e.g., single cells and/or target nucleic acids and beads linked to oligonucleotides) are 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, at least 20,000 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 600,000 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.

[0062] Any type of cells can be used according to the methods and compositions described herein. In some embodiments, the cells are mammalian, for example human cells. In some embodiments, the cells are from a biological sample. Biological samples can be obtained from any biological organism, e.g.. an animal, plant, fungus, pathogen (e.g., bacteria or virus), or any other organism. In some embodiments, the biological sample is from an animal, e.g., a mammal (e.g., a human or anon-human primate, a cow, horse, pig, sheep, cat, dog, mouse, or rat), a bird (e.g., chicken), or a fish. A biological sample can be any tissue or bodily fluid obtained from the biological organism, e.g., blood, a blood fraction, or a blood product (e.g., serum, plasma, platelets, red blood cells, and the like), sputum or saliva, tissue (e.g., kidney, lung, liver, heart, brain, nervous tissue, thyroid, eye. skeletal muscle, cartilage, or bone tissue); cultured cells, e g., primary cultures, explants, and transformed cells, stem cells, or cells found in stool, urine, etc.

[0063] In some embodiments the cells are fixed and permeabilized. In some embodiments, the cells are formalin-fixed cells. Exemplary detergents can include, for example, Triton X-100, Brij-35and/or NP-40 are used for permeabilization (for example, at 0. 1-0.5% (v/v, in PBS). In some embodiments, a steroidal saponin (or saraponin) is used to solubilize lipid, resulting in permeabilization. An exemplary saraponin is Digitonin.

[0064] The methods described herein provide for contacting the oligonucleotides linked to the solid support, affinity agent or ligation-blocking moiety, to target nucleic acids. Target nucleic acids can be for example, RNA (for example but not limited to mRNA) or DNA. In some embodiments, single cells are provided in partitions and the single cells in the partitions are permeabilized or lysed to allow for other reagents such as the oligonucleotides and/or enzy mes in the partition to contact the target nucleic acids from the cells. In the embodiments described herein the oligonucleotides remain linked to the solid support under conditions to allow for the target nucleic acids to anneal to the 3’ capture sequences on the oligonucleotides. Thus, the solid support at this point will be linked to a plurality⁷ of the oligonucleotides, at least some of which (but not generally all) are annealed to a target nucleic acid.

[0065] Once the target nucleic acids are annealed to some of the oligonucleotides, a polymerase is contacted to the annealed nucleic acids. The polymerase will extend the oligonucleotide using the target nucleic as a template, thereby generating a first strand copy (reverse complement) of the target nucleic acid having as a 5’ sequence the oligonucleotide sequence. This product can later be used for various downstream manipulation and detection, for example for nucleotide sequencing. The target nucleic acid is also extended in a polymerase-dependent reaction using the oligonucleotide as a template such that the portion of the oligonucleotide comprising the single strand of the restriction enzyme recognition sequence (s) is copied, generating double-stranded recognition sequence(s). See. e.g., FIG. 4.

[0066] Exemplary polymerases used will depend on whether the target nucleic acid is DNA or RNA. In embodiments in which the target nucleic acids are RNA, a reverse transcriptase can be used to (1) extend the oligonucleotide 3’ end using the target RNA as a template and (2) extending the RNA 3’ end using the oligonucleotide as a template. The latter activity was observed in spite of only activity (1) being initially expected. With regard to activity (2), without intending to the 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 base to be paired to the bead capture oligonucleotide, and allowing for formation of the double-stranded recognition sequence. Exemplary target RNAs can include for example mRNA. miRNA, scRNA, etc. Exemplary reverse transcriptase can include, but are not limited to an HIV reverse polymerase, murine leukemia virus (MLV) reverse transcriptase, Avian Myeloblastosis Virus (AMV) reverse transcriptase, Respirator}' Syncytial Virus (RSV) reverse transcriptase, Equine Infectious Anemia Virus (EIAV) reverse transcriptase, or Rous-associated Virus-2 (RAV2) reverse transcriptase or a mutant thereof. For example. Super Script IV™ (ThermoFisher Scientific) or Maxima H-™ (ThermoFisher Scientific) or any reverse transcriptase described in U.S. Published Paten Application No. 2011/0065606 can be used. In embodiments in which the target nucleic acids are DNA, a DNA polymerase can be used to (1) extend the oligonucleotide 3‘ end using the target DNA as a template and (2) extending the DNA 3’ end using the oligonucleotide as a template.

[0067] In some embodiments, a multi-omics approach is used, meaning two or more of RNA, DNA and protein is detected in the method, wherein agents representative of each sort of target are annealed and linked to based oligonucleotides, allowing for later determination which sample molecules originated in the same partition. In some embodiments, protein in or on a cell is detected by contacting the cell with one or more antibodies, wherein the antibodies are linked to an oligonucleotide. This can be referred to as CITE-seq. See, e.g., Stoeckius, M., Hafemeister, C., Stephenson, W. et al. Nat Methods 14, 865-868 (2017). Antibodies that target different antigens are linked to oligonucleotides of 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 acids annealed to the bead oligonucleotides can be oligonucleotides linked to, or cleaved from, such antibodies, allowing for purification and ultimate detection of the identity of oligonucleotides associated with antibodies that bind target antigens in a sample in a partition.

[0068] One or more restriction enzyme(s), which are either (i) present in the reaction mixture or partition where the solid support resides, or (ii) introduced after the polymerase has extended annealed nucleic acids, is contacted to the oligonucleotides having annealed and extended target nuclease acids, allowing the restriction enzyme to recognize the doublestranded recognition sequence of the restriction enzyme recognition site and then cleave the double-stranded nucleic acid. In some embodiments, more than one restriction enzy me recognition sequence can be formed and in such embodiments, one or more different restriction enzymes can be contacted to the nucleic acids. In some embodiments, more than one restriction enzyme recognition sequence is formed and the use of more than one restriction enzyme can improve the chances that annealed nucleic acids are indeed cleaved from the solid support. The result of the restriction enzyme(s) action is to generate cleaved oligonucleotides that have been extended to have a reverse complement of the target nucleic acid, while leaving excess oligonucleotides not annealed to target nucleic acids uncleaved (still linked to the solid support).

[0069] Any restriction enzyme can be used so long as the restriction enzyme recognizes the double-stranded recognition sequence generated upon extension of the target nucleic acids using the oligonucleotide as a template and where in the restriction enzy me is selective for double-stranded DNA compared to single-stranded DNA and/or DNA/RNA heterodupexes. In some embodiments, the restriction enzyme cleaves within the double stranded DNA recognition sequence, though this is not necessary so long as the restriction enzyme cleaves the annealed oligonucleotide from the solid support. The restriction enzyme can be selected from, for example, restriction enzymes that recognized 4, 5, 6, 7, 8, 9. or 10 base-pair doublestranded sequences. Exemplary restriction enzymes can include but are not limited to, ApaLI, Pmel, AfHI, AfUII and Pad. [0070] Following selective cleavage of those nucleic acids having the double-stranded recognition sequence, the extended oligonucleotides cleaved from the solid support can be separated from the solid support and any remaining oligonucleotides linked thereto. Any method desired can separate these populations, including for example using gravity, centrifugation, filtration or other methods to separate soluble released oligonucleotides from a solid support. In embodiments in which the oligonucleotides are linked to an affinity agent, the affinity agent will be cleaved from target nucleic acids by the restriction enzyme(s), and the affinity agent can then be used to remove uncleaved oligonucleotides that retain the affinity⁷ agents as well as the cleaved end portion of the oligonucleotides having the affinity⁷ agent from the cleaved portion comprising the target nucleic acids. In this case, for example, an agent having affinity⁷ for the affinity agent can be used to bind and then separate the affinity-agent comprising nucleic acids. For example, the agent having affinity for the affinity agent can be linked to a solid support and be used as described above to separate the populations of nucleic acids. In embodiments in which the oligonucleotides are linked to a ligation-blocking moiety, the ligation-blocking moiety will be cleaved from target nucleic acids by the restriction enzyme(s), and a ligation reaction can be performed to ligate a polynucleotide selectively to the cleaved target nucleic acids as they will lack the ligationblocking moiety⁷. Those nucleic acids comprising the ligated polynucleotide can then be separated from the remainder of the nucleic acids in the mixture, for example based on affinity (complementarity of the polynucleotide to a polynucleotide on a solid support or other methods of selective separation of the polynucleotide-containing nucleic acids from the mixture. The ligation can be a blunt end or sticky-end ligation, depending on the restriction enzymes used.

[0071] Before or after the extended oligonucleotides are separated from the solid support and any remaining oligonucleotides linked thereto, a second strand can be formed, using the extended oligonucleotide strand as a first strand. For example, in embodiments in which the target nucleic acid RNA, the extended oligonucleotide will be a first strand cDNA comprising the oligonucleotide sequence at its 5’ end, and a second strand cDNA can be generated using the first strand cDNA as a template. Second strands can be generated as desired. In some embodiments, the second strand can be formed via random priming from the first strand or template-switching can be used to generate the second strand. See, e.g., 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 on the 5’ end of the second strand, which when amplified with the first strand generates an amplicon having universal sequences on either end, allowing for selective amplification of the captured target nucleic acids using primers that anneal to the universal sequences or a complement thereof. See e.g., bottom of FIG. 4.

[0072] In some embodiments of any of the methods described herein, exonuclease, or Uracil DNA glycosylase and endonuclease VIII (the latter two are sometimes referred to as “Uracil-Specific Excision Reagent⁷'), or all are not used in the methods. For example, in some embodiments, the oligonucleotides linked to the solid supports do not include a uracil.

[0073] In some embodiments, a transposase carrying oligonucleotides is used to introduce breaks in DNA (e.g., the amplicon comprising the first and second strands as described above) and introduce the carried oligonucleotides into the break sites. The action of some transposases is sometimes referred to as "tagmentation" and the enzyme can be referred to as a “tagmentase” and can involve introduction of different adaptor sequences on different sides of a DNA breakage point or the adaptor sequences added can be identical. Homoadaptor- loaded tagmentases are tagmentases that contain adaptors of only one sequence, which adaptor is added to both ends of a tagmentase-induced breakpoint in the genomic DNA. Heteroadaptor-loaded tagmentases are tagmentases that contain two different adaptors, such that a different adaptor sequence is added to the two DNA ends created by a tagmentase- induced breakpoint in the DNA. Adaptor loaded tagmentases are further described, e.g., in U.S. Patent Publication Nos: 2010/0120098; 2012/0301925: and 2015/0291942 and U.S.

Patent Nos: 5,965,443; U.S. 6,437.109; 7,083,980; 9,005,935; and 9,238,671. the contents of each of which are hereby incorporated by reference in the entirety for all purposes.

[0074] Once the amplicons are prepared any nucleotide sequencing methods can be used to generate sequencing reads from the extended oligonucleotide, and containing a solid-support- specific barcode or other sequences from the original oligonucleotide linked to the first strands.

[0075] Sequencing platforms can be selected as desired to generate sequencing reads. In some embodiments, Illumina™-supported sequencing methods are employed. See, e.g., U.S. Patent Nos 11,029,513; US 11,150,179; 11,308,640; and 11,473,067 and citations therein. Exemplary DNA sequencing techniques include fluorescence-based sequencing methodologies See, e.g.. Birren et al., Genome Analysis: Analyzing DNA, 1, Cold Spring Harbor, N.Y.; herein incorporated by reference in its entirety). In some embodiments. automated sequencing techniques understood in that art are utilized. In some embodiments, the present technology provides parallel sequencing of partitioned amplicons (PCT Publication No. WO 2006/0841,32, herein incorporated by reference in its entirety). In some embodiments, DNA sequencing is achieved by parallel oligonucleotide extension (See, e.g., U.S. Pat. Nos. 5,750,341; and 6,306,597, both of which are herein incorporated by reference in their entireties). Additional examples of sequencing techniques include the Church polony technology (Mitra et al., 2003. Analytical Biochemistry 320, 55-65; Shendure et al., 2005 Science 309, 1728-1732; and U.S. Pat. Nos. 6,432,360; 6,485,944; 6,511,803; herein incorporated by reference in their entireties), the 454 picotiter pyrosequencing technology⁷ (Margulies et al., 2005 Nature 437, 376-380; U.S. Publication No. 2005/0130173; herein incorporated by reference in their entireties), the Solexa single base addition technology (Bennett et al., 2005, Pharmacogenomics, 6, 373-382; U.S. Pat. Nos. 6,787,308; and 6,833,246; herein incorporated by reference in their entireties), the Lynx massively parallel signature sequencing technology (Brenner et al. (2000). Nat. Biotechnol. 18:630-634; U.S. Pat. Nos. 5.695,934; 5,714.330; herein incorporated by reference in their entireties), and the Adessi PCR colony technology' (Adessi et al. (2000). Nucleic Acid Res. 28, E87; WO 2000/018957; herein incorporated by reference in its entirety).

[0076] The above method can also be implemented using a double-stranded targeting enzyme other than a restriction enzyme. For example, the extending of the target nucleic acid 3' end yvith a polymerase using the oligonucleotide as a template can form a double-stranded portions regardless of the presence of a restriction enzyme recognition sequence. In these embodiments, instead of using a restriction enzyme to cleave the double-stranded portion, an enzyme can be used to selectively tag or modify the double-stranded portion without similarly tagging or modifying single-stranded portions. A non-limiting list of such enzymes can include, for example. Argonaute proteins, Formamidopyrimidine DNA Glycosylase, Methyltransferases, or TelN Protelomerase, which selectively modify the double-stranded portion that can subsequently be used to separate tagged or modified nucleic acids from nontagged or modified nucleic acids.

[0077] In some embodiments, an Argonuate protein and a guide nucleic acid is used to target the double-stranded portion. Examples of Argonaute proteins that target sequences with guide nucleic acids can be found in, e.g., Tabatabaei SK, et al. (April 2020). Nature Communications 11 (1): 1742. Thus in some embodiments, a method of generating a population of captured target nucleic acids is provided comprising providing a plurality of oligonucleotides individually linked to a solid support, wherein the oligonucleotides have a free 3^? end; contacting the plurality’ of oligonucleotides with a sample comprising target nucleic acids having 3’ ends and 5' ends, wherein target nucleic acids anneal to some of the free 3’ ends of the oligonucleotides, and wherein there is an excess of oligonucleotides such that at least some oligonucleotides remain with free 3' ends; and then extending the target nucleic acid 3’ end with a polymerase using the oligonucleotide as a template to form doublestranded portions that comprise an Argonaute protein recognition sequence; and then cleaving the Argonaute protein recognition sequence with an Argonaute protein (and optionally a guide nucleic acid that guides the Argonaute protein to the sequence) to form (i) released target nucleic acids having a single-stranded 5’ end and a double-stranded 3‘ end and (ii) cleaved oligonucleotide ends linked to the solid support, affinity agent, or ligationblocking moiety and leaving (iii) some uncleaved oligonucleotides remaining with free 3’ ends uncleaved from the solid support, affinity⁷ agent, or the ligation-blocking moiety⁷; and then separating the uncleaved oligonucleotides from the released target nucleic acids to form a solution of target nucleic acids.

[0078] As one example, a methyltransferase that selectively methylates double-stranded DNA can be applied to methylate the double-stranded DNA and then methylated DNA can be separated from unmethylated nucleic acids. In some embodiments, a methyltransferases protein is used to specifically methylate single-stranded DNA, leaving the double-stranded portion unmethylated. Subsequently, bisulfite conversion can be used to selectively convert unmethylated (i. e. , double-stranded) cytosines to uracil and then cleaving the uracil- containing nucleic acid with USER reagents to selectively⁷ cleave the double-stranded portions, leaving single-stranded oligonucleotides linked to the beads. Thus in some embodiments, a method of generating a population of captured target nucleic acids is provided comprising providing a plurality of oligonucleotides individually linked to a solid support, wherein the oligonucleotides have a free 3’ end; contacting the plurality of oligonucleotides with a sample comprising target nucleic acids having 3’ ends and 5’ ends, wherein target nucleic acids anneal to some of the free 3’ ends of the oligonucleotides, and wherein there is an excess of oligonucleotides such that at least some oligonucleotides remain with free 3’ ends; and then extending the target nucleic acid 3’ end with a polymerase using the oligonucleotide as a template to form double-stranded portions; selectively converting cytosines in the double-stranded portion to uracils with a methyltransferase; and then cleaving the uracils with uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII to form (i) released target nucleic acids having a single-stranded 5’ end and a double-stranded 3’ end and (ii) cleaved oligonucleotide ends linked to the solid support, affinity agent, or ligation-blocking moiety and leaving (iii) some uncleaved oligonucleotides remaining with free 3’ ends uncleaved from the solid support, affinity agent, or the ligationblocking moiety'; and then separating the uncleaved oligonucleotides from the released target nucleic acids to form a solution of target nucleic acids.

[0079] In some embodiments, a formamidopyrimidine DNA Glycosylase is used to target the double-stranded portion. Formamidopyrimidine DNA Glycosylase would selectively cleave the double stranded DNA that contained an oxo-guanine base (present for example in the bead-linked oligonucleotide). Formamidopyrimidine DNA Glycosylase (FPG) will cleave the oxo-guanine base out of the double-stranded portion and not a ssDNA sequence. Removal of the guanine by FPG leaves a break in the DNA backbone, so the oligonucleotides will disassociate with each other due to heat if designed properly. Thus in some embodiments, a method of generating a population of captured target nucleic acids is provided comprising providing a plurality of oligonucleotides individually linked to a solid support, wherein the oligonucleotides have a free 3’ end and one or more oxo-guanine base; contacting the plurality of oligonucleotides with a sample comprising target nucleic acids having 3’ ends and 5’ ends, wherein target nucleic acids anneal to some of the free 3’ ends of the oligonucleotides, and wherein there is an excess of oligonucleotides such that at least some oligonucleotides remain with free 3' ends; and then extending the target nucleic acid 3' end with a polymerase using the oligonucleotide as a template to form double-stranded portions that comprise the one or more oxo-guanine base; and then cleaving the doublestranded portion yvith a formamidopyrimidine DNA Glycosylase to form (i) released target nucleic acids having a single-stranded 5‘ end and a double-stranded 3’ end and (ii) cleaved oligonucleotide ends linked to the solid support, affinity agent, or ligation-blocking moiety and leaving (iii) some uncleaved oligonucleotides remaining with free 3’ ends uncleaved from the solid support, affinity agent, or the ligation-blocking moiety; and then separating the uncleaved oligonucleotides from the released target nucleic acids to form a solution of target nucleic acids.

[0080] In some embodiments, a TelN Protelomerase is used to target the double-stranded portion. TelN Protelomerase selectively cleaves double-stranded sequences and connects the 5’ end of one strand to the 3’ end of the complementary end. The resulting sequence can be targeted for PCR amplification. As the 5’ end of the newly -formed sequence is now accessible (and the other bead molecule is covalently linked to the bead), one can target the 5’ end of the newly-formed sequence for modification and enrichment as desired. Thus in some embodiments, a method of generating a population of captured target nucleic acids is provided comprising providing a plurality of oligonucleotides individually linked to a solid support, wherein the oligonucleotides have a free 3’ end; contacting the plurality⁷ of oligonucleotides with a sample comprising target nucleic acids having 3’ ends and 5‘ ends, wherein target nucleic acids anneal to some of the free 3’ ends of the oligonucleotides, and wherein there is an excess of oligonucleotides such that at least some oligonucleotides remain with free 3’ ends; and then extending the target nucleic acid 3’ end with a polymerase using the oligonucleotide as a template to form double-stranded portions that comprise a TelN Protelomerase recognition sequence; and then cleaving the TelN Protelomerase recognition sequence with a TelN Protelomerase to form (i) released target nucleic acids having a singlestranded 5’ end and a 3’ end formed by linkage of a 3’ and 5’ end caused by the doublestranded cleavage and (ii) cleaved oligonucleotide ends linked to the solid support, affinityagent, or ligation-blocking moiety and leaving (iii) some uncleaved oligonucleotides remaining with free 3’ ends uncleaved from the solid support, affinity agent, or the ligationblocking moiety; and then separating the uncleaved oligonucleotides from the released target nucleic acids to form a solution of target nucleic acids.

[0081] Also provided is a plurality of solid supports or a plurality of partitions containing the plurality⁷ of solid supports linked to a plurality of oligonucleotides as described herein in the context of the above-described methods. For example, the oligonucleotides can comprise 5’ to 3’: one strand of a double-stranded restriction enzy me recognition sequence, a beadspecific barcode sequence and a 3’ target-specific sequence. In some embodiments, the partitions can further comprise a restriction enzyme that cleaves at the double-stranded restriction enzyme recognition sequence. In some embodiments, the partition can further comprise a polymerase for extending target nucleic acids that will be annealed to the oligonucleotides as described herein. For example, in some embodiments, the polymerase is a reverse transcriptase.

[0082] Kits providing one or more reagent for performing the described methods are also provided. For example, in some embodiments, the kit comprises (i) a plurality of solid supports (e.g., beads) linked to a plurality of oligonucleotides comprising 5’ to 3’: one strand of a double-stranded restriction enzy me recognition sequence, a bead-specific barcode sequence and a 3’ target-specific sequence; and (ii) a restriction enzyme that cleaves at the double-stranded restriction enzyme recognition sequence or an Argonaute protein, Formamidopyrimidine DNA Glycosylase, Methyltransferase, or TelN Protelomerase. In some embodiments, the kit can further comprise a polymerase for extending target nucleic acids that Mil be annealed to the oligonucleotides as described herein. For example, in some embodiments, the polymerase is a reverse transcriptase.

EXAMPLES

Example 1

[0083] FIG. 5 provides a table of results from example 1. 4,000 beads containing ssDNA capture oligonucleotides with the restriction enzyme indicated were incubated with an excess of complement oligonucleotide to create a double stranded DNA recognition site for restriction enzymes indicated, or not. The beads were then incubated in a buffer suitable for the restriction enzyme or USER to digest the bead capture oligonucleotide. The reactions were then centrifuged at 1000 x g for 5 minutes to pellet the beads, and a volume of supernatant was removed from the sample. A ddPCR assay was run on the collected supernatant to quantify the number of bead capture oligonucleotides present in the supernatant. The restriction enzymes only released the bead capture oligonucleotide when the oligonucleotide contained complement and therefore double stranded. In contrast the USER enzy me cleaved the uracil present in the bead capture oligonucleotide regardless of complement, so either ssDNA or dsDNA.

Example 2

[0084] FIG. 6 provides a table of results from example 2. 4,000 beads containing capture oligonucleotides with the restriction enzyme indicated were incubated with an excess of complement oligonucleotide to create a double stranded DNA recognition site for restriction enzymes indicated, or not. The beads were then compartmentalized with cells in aqueous droplets in oil. The droplets contained reverse transcription reactions and restriction enzymes indicated and were incubated at 37 C for an hour. After the RT reaction, droplets were broken, beads were centrifuged out of solution, and the supernatant recovered. The supernatant was then assayed with a ddPCR targeting the bead capture oligonucleotide. Only beads incubated with bead capture oligonucleotide complement released the entire complement of bead capture oligonucleotides, or those beads digested with USER enzyme. However, the beads not incubated with bead capture oligo complement only released a small amount of bead capture oligonucleotide presumably only those bead capture oligonucleotides with captured cDNA and bead complement.

Example 3

[0085] FIG. 7 provides a table of results from example 3. 200,000 beads containing capture oligonucleotides with the restriction enzyme indicated were compartmentalized with cells in aqueous droplets in oil. The aqueous solution of the droplets consisted of a reverse transcription reaction and restriction enzymes indicated and the droplet were incubated at 37 C for an hour. After the RT reaction, droplets were broken, beads were centrifuged out of solution, and the supernatant recovered. The supernatant was then assayed with a ddPCR targeting GAPDH either with primers specific to GAPDH or one primer targeting the bead capture oligonucleotide and the other oligonucleotide targeting the GAPDH gene creating a chimera amplicon with both bead and GAPDH sequence. Restriction enzyme-released oligos yielded similar if slightly lower levels of GAPDH than the USER digested beads.

Example 4

[0086] 200,000 beads containing bis-acrylylcystamine (a crosslinking chemical containing a disulfide bond which is reduced in the presence of DTT) containing capture oligos with the restriction enzyme indicated (ApaLI) were compartmentalized with 10,000 cells in aqueous droplets surrounded by oil. The aqueous droplets consisted of reverse transcription reactions containing either restriction enzymes or USER enzymes as indicated. Samples were first incubated at 4C for 25 min for cell lysis and mRNA capture, followed by a 50C incubation for 45 minutes for bead oligo release and RT, followed by a 65C incubation for 30 min for second strand synthesis, and lastly a 5-minute enzy me inactivation at 80C. After the RT reaction, droplets were broken and a SPRI cleanup with a ratio of 1.8 SPRI reagent to 1 sample was performed on the samples. The cDNA was recovered and was then assayed with ddPCR targeting GAPDH, HPRT1, or PTEN genes. See, FIG. 8.

[0087] Although the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity' of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein, including patents, patent applications, non-patent literature, and Genbank accession numbers, is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference. Where a conflict exists between the instant application and a reference provided herein, the instant application shall dominate.

Claims

WHAT IS CLAIMED IS:

1. A method of generating a population of captured target nucleic acids, the method comprising, providing a plurality of oligonucleotides individually linked to a solid support, wherein the oligonucleotides have a free 3’ end; contacting the plurality of oligonucleotides with a sample comprising target nucleic acids having 3’ ends and 5' ends, wherein target nucleic acids anneal to some of the free 3’ ends of the oligonucleotides, and wherein there is an excess of oligonucleotides such that at least some oligonucleotides remain with free 3‘ ends; and then extending the target nucleic acid 3' end with a polymerase using the oligonucleotide as a template to form double-stranded portions that comprise a restriction enzyme recognition sequence; and then cleaving the restriction enzyme recognition sequence with a restriction enzyme to form (i) released target nucleic acids having a single-stranded 5' end and a doublestranded 3’ end and (ii) cleaved oligonucleotide ends linked to the solid support, affinity agent, or ligation-blocking moiety and leaving (iii) some uncleaved oligonucleotides remaining w ith free 3’ ends uncleaved from the solid support, affinity agent, or the ligationblocking moiety; and then separating the uncleaved oligonucleotides from the released target nucleic acids to form a solution of target nucleic acids.

2. The method of claim 1, wherein the plurality of oligonucleotides are individually linked to a solid support.

3. The method of claim 2, wherein the oligonucleotides comprise 5’ ends and the 5‘ ends are linked to the solid support.

4. The method of claim 1, wherein the oligonucleotides comprise 5’ to 3’: one strand of the double-stranded restriction enzyme recognition sequence, a solid supportspecific barcode sequence and a 3’ target-specific sequence.

5 . The method of claim 1, wherein the double-stranded restriction enzyme recognition sequence is selected from the group consisting of ApaLI, Pmel, Aflll, AfUII and Pacl.

6. The method of claim 1, wherein the oligonucleotides comprise 5^? ends and the 5’ ends are linked to the beads and the oligonucleotides further comprise 2-10 nucleotides 5’ of the one strand of the double-stranded restriction enzyme recognition sequence.

7. The method of any one of claims 1-6, wherein the double-stranded portions comprise two or more different restriction enzy me recognition sequences.

8. The method of claim 2, wherein the oligonucleotides are covalently linked to the solid support.

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

10. The method of claim 9, wherein the oligonucleotides are biotinylated and solid support comprises streptavidin.

11. The method of claim 10, wherein following the cleaving, separating biotinylated nucleic acids, including cleaved oligonucleotide ends, from released target nucleic acids based on streptavidin affinity of the biotin.

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

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

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

15. The method of any one of claims 1-13, wherein the solid support is one or more bead, wherein copies of oligonucleotides linked to a bead comprise bead-specific barcode sequences and wherein oligonucleotides linked to different beads have different bead-specific barcode sequences.

16. The method of any one of claims 1-15, wherein the providing comprises providing the solid supports in partitions.

17. The method of claim 16, wherein the partitions are droplets in an oilbased emulsion or microwells.

18. The method of claim 16, wherein at least some of the partitions further comprise single cells and wherein the target nucleic acids are nucleic acids from the cells.

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

20. The method of any one of claims 16-19, wherein the cleaving occurs in the partitions.

21. The method of any of claims 1-20, wherein the oligonucleotides comprise a solid-support-specific barcode sequence and optionally a PCR handle sequence.

22. The method of claim 1 , wherein the plurality of oligonucleotides individually are linked to an affinity agent and the separating comprises contacting the affinity agent on the cleaved oligonucleotide ends and uncleaved oligonucleotides with an agent that binds the affinity agent; and separating the agent bound to the affinity agent from the released target nucleic acids.

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

24. The method of claim 1, wherein the plurality of oligonucleotides individually are linked to a ligation-blocking moiety.

25. The method of claim 24, wherein the ligation-blocking moiety is selected from 3' dideoxy cytidine (ddC), 3' inverted dT, 3' C3 spacer, 3' ammo, and 3' phosphorylation.

26. The method of any one of claims 24 or 25, wherein the separating comprises ligating released nucleic acid 3’ ends to a polynucleotide, wherein uncleaved oligonucleotides or cleaved oligonucleotide ends are not ligated to the polynucleotide; and separating nucleic acids ligated to the polynucleotide from oligonucleotides and cleaved oligonucleotide ends comprising the ligation-blocking moiety.

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

28. The method of any of claims 1-21, wherein the oligonucleotides are fully DNA oligonucleotides.

29. The method of any of claims 1-21, wherein the oligonucleotides comprise DNA and uracil bases.

30. The method of claim 18, wherein the single cells are mammalian cells.

31. A plurality of beads wherein individual beads are linked to a plurality of oligonucleotides comprising 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 of claim 31 , wherein the double-stranded restriction enzyme recognition sequence is selected from the group consisting of ApaLI, Pmel, Aflll, AfUII and Pad.

33. The plurality’ of beads of claim 31 or 32, wherein the oligonucleotides comprise one strand of t vo or more double-stranded restriction enzyme recognition sequences.

34. The plurality’ of beads of any one of claims 31-32, wherein the oligonucleotides comprise 5’ ends and the 5’ ends are linked to the beads and the oligonucleotides further comprise 2-10 nucleotides 5’ of the one strand of the doublestranded restriction enzyme recognition sequence.

35. The plurality of beads of any one of claims 31-34, wherein the oligonucleotides are fully DNA oligonucleotides.

36. The plurality’ of beads of any one of claims 31-34, wherein the oligonucleotides comprise DNA and uracil bases.

37. A kit comprising, the plurality of beads of any one of claims 31-36; and a restriction enzyme that cleaves at the double-stranded restriction enzyme recognition sequence.

38. The kit of claim 34, further comprising a reverse transcriptase.

39. A plurality of partitions, wherein the partitions comprise plurality' of oligonucleotides individually linked to a solid support, wherein the oligonucleotides have a free 3’ end and the oligonucleotides comprise 5’ to 3’: one strand of the double-stranded restriction enzy me recognition sequence, a solid support-specific barcode sequence and a 3’ target-specific sequence.

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

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

42. The plurality' of partitions of any one of claims 39-41, wherein the double-stranded restriction enzyme recognition sequence is selected from the group consisting of ApaLI, Pmel. Aflll, Afllll and Pad.

43. The plurality' of partitions of any one of claims 39-42, wherein the oligonucleotides comprise one strand of two or more double-stranded restriction enzyme recognition sequences.

44. The plurality' of partitions of any one of claims 39-43, wherein the oligonucleotides comprise 5‘ ends and the 5‘ ends are linked to the beads and the oligonucleotides further comprise 2-10 nucleotides 5’ of the one strand of the doublestranded restriction enzyme recognition sequence.

45. The plurality of partitions of any one of claims 39-44, wherein the oligonucleotides are fully DNA oligonucleotides.

46. The plurality of partitions of any one of claims 39-44, wherein the oligonucleotides comprise DNA and uracil bases.

47. The plurality’ of partitions of any one of claims 39-46, wherein some but not all partitions comprise target nucleic acids that anneal to some of the free 3’ ends of the oligonucleotides to form double-stranded portions, and wherein there is an excess of oligonucleotides such that at least some oligonucleotides remain with free 3’ ends.