CN-122029291-A - 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 separately attached to a solid support with a sample comprising a target nucleic acid having a3 'end and a 5' end, wherein the target nucleic acid anneals to some free 3 'end of the oligonucleotides, and wherein 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 the oligonucleotides as templates 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 the oligonucleotides uncleaved from the solid support and still having free 3' ends, and then optionally separating the solid support and uncleaved oligonucleotides from the released target nucleic acid to form a target nucleic acid solution.

Inventors

• J. PERRY
• T. Jodry

Assignees

• 生物辐射实验室股份有限公司

Dates

Publication Date

20260512

Application Date

20241015

Priority Date

20231016

Claims (20)

1. 1. A method of generating a population of captured target nucleic acids, the method comprising: Providing a plurality of oligonucleotides individually attached to a solid support, wherein the oligonucleotides have free 3' ends; Contacting the plurality of oligonucleotides with a sample comprising 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 of the oligonucleotides still have free 3' ends, and then Extending the 3' -end of the target nucleic acid with a polymerase using the oligonucleotide as a template to form a double-stranded portion comprising a restriction enzyme recognition sequence, and 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) a cleaved oligonucleotide end attached to the solid support, affinity agent, or ligation blocking moiety, and leaving (iii) some uncleaved oligonucleotide that is not cleaved from the solid support, affinity agent, or the ligation blocking moiety and still has a free 3' end, and then Separating the uncleaved oligonucleotides from the released target nucleic acid to form a target nucleic acid solution.
2. 2. The method of claim 1, wherein the plurality of oligonucleotides are individually attached to a solid support.
3. 3. The method of claim 2, wherein the oligonucleotide comprises a 5 'end, and the 5' end is attached to the solid support.
4. 4. The method of claim 1, wherein the oligonucleotide comprises, from 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.
5. 5. The method of claim 1, wherein the double stranded restriction enzyme recognition sequence is selected from the group consisting of ApaLI, pmeI, aflII, aflIII and PacI.
6. 6. The method of claim 1, wherein the oligonucleotide comprises a 5' end and the 5' end is attached to a bead, and the oligonucleotide further comprises 2-10 nucleotides 5' of one strand of the double-stranded restriction enzyme recognition sequence.
7. 7. The method of any one of claims 1 to 6, wherein the double stranded portion comprises two or more different restriction enzyme recognition sequences.
8. 8. The method of claim 2, wherein the oligonucleotide is covalently attached to the solid support.
9. 9. The method of claim 2, wherein the oligonucleotide is non-covalently attached to the solid support.
10. 10. The method of claim 9, wherein the oligonucleotide is biotinylated and the solid support comprises streptavidin.
11. 11. The method of claim 10, wherein after cleavage, the biotinylated nucleic acid comprising cleaved oligonucleotide ends is separated from the released target nucleic acid based on streptavidin affinity of the biotin.
12. 12. The method of 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. 13. The method of claim 12, wherein the reverse transcriptase polymerase is HIV reverse transcriptase polymerase, M-MLV reverse transcriptase polymerase, or AMV reverse transcriptase polymerase.
14. 14. The method of any one of claims 1 to 9, wherein the target nucleic acid is a DNA molecule and the polymerase is a DNA polymerase.
15. 15. The method of any one of claims 1 to 13, wherein the solid support is one or more beads, wherein the copies of the oligonucleotides attached to the beads comprise bead-specific barcode sequences, and wherein the oligonucleotides attached to different beads have different bead-specific barcode sequences.
16. 16. The method of any one of claims 1 to 15, wherein the providing comprises providing the solid support in a partition.
17. 17. The method of claim 16, wherein the partitions are droplets or micropores in an oil-based emulsion.
18. 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 from the cells.
19. 19. The method of claim 18, further comprising lysing or permeabilizing the cells in the partition.
20. 20. The method of any one of claims 16 to 19, wherein the cutting occurs in the partition.

Description

Enzymatic selection of nucleic acids Cross-reference to related patent applications The present application claims priority from U.S. provisional patent application No. 63/544,372, filed on 10/16 of 2023, which is incorporated by reference for all purposes. Background Single cell genomic analysis, including single cell sequencing, is performed in partitions (e.g., droplets). Sequencing is performed by attaching oligonucleotides comprising partition specific barcodes to target nucleic acids from cells, allowing differentiation of target nucleic acids from different cells based on the nature of the partition specific barcode sequences. In some methods, a solid support, such as a bead, is used to deliver a large number of copies of an oligonucleotide to a partition. The delivery of beads and single cells to the partition followed a poisson distribution (Poisson distribution). Because one goal is to avoid the presence of multiple cells in one partition, the user adjusts the number of partitions to be higher than the number of cells, resulting in many partitions that are devoid of cells and thus devoid of target nucleic acids, but typically contain beads and their oligonucleotides. Oligonucleotides can be released from the beads before or after annealing to the target nucleic acid, but in general, the release mechanism of the beads is independent of whether the partitions contain target nucleic acid. Examples of release mechanisms are e.g. uracil comprising one or more linked oligonucleotides to the bead and release of the oligonucleotide at uracil using Uracil DNA Glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII. In other cases, ultraviolet cleavable chemical moieties, disulfides, or other chemical components that can be released by the user are used. However, in general, these methods do not distinguish between oligonucleotides annealed to the target nucleic acid and non-annealed oligonucleotides, resulting in the presence of excess released oligonucleotides both in the region containing the sample and in the region lacking the sample, e.g., the region without single cells (and thus without target nucleic acid). The presence of released oligonucleotides that are not annealed to the target nucleic acid may cause background interference in subsequent sequencing reactions. Disclosure of Invention In some embodiments, a method of generating a population of captured target nucleic acids is provided. In some embodiments, the method includes providing a plurality of oligonucleotides individually attached to a solid support, wherein the oligonucleotides have free 3' ends, contacting the plurality of oligonucleotides with a sample comprising a target nucleic acid having 3' ends and 5' ends, wherein the target nucleic acid anneals to some of the free 3' ends of the oligonucleotides, and wherein there is an excess of oligonucleotides such that at least some of the oligonucleotides still have free 3' ends, then using the oligonucleotides as templates, extending the 3' ends of the target nucleic acid with a polymerase 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 (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 support, an affinity agent, or a ligation blocking moiety, and leaving (iii) some uncleaved oligonucleotides uncleaved from the solid support, affinity agent, or the ligation blocking moiety and still having free 3' ends, and then cleaving the target nucleic acid with a restriction enzyme to form the uncleaved target nucleic acid. In some embodiments, the double-stranded portion, but not the restriction enzyme recognition sequence, may be a sequence recognized by a different enzyme that selectively cleaves the double-stranded sequence, such as described elsewhere herein. In some embodiments, the plurality of oligonucleotides are individually attached to the solid support. In some embodiments, the oligonucleotide comprises a 5 'end, and the 5' end is attached to a solid support. In some embodiments, the oligonucleotide comprises, from 5' to 3', one strand of a double-stranded restriction enzyme recognition sequence, a solid support 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 a 5' end and the 5' end is attached to the bead, and the oligonucleotide further comprises 2-10 nucleotides 5' of one strand of the double-stranded restriction enzyme recognition sequence. In some embodiments, the double stranded portion comprises two or more different restriction enzyme recognition sequences. In some embodiments, the oligonucleotide is covalently linked to