Methods of depleting a target nucleic acid in a sample and kits for practicing the same
Provided are methods of depleting a target nucleic acid in a sample. The methods include contacting a target nucleic acid with two or more polymers that specifically hybridize to the target nucleic acid, and cleaving the hybridized regions of the target nucleic acid to deplete the target nucleic acid in the sample. Kits for practicing the subject methods are also provided.
1. A method of depleting an undesired ribosomal ribonucleic acid (rRNA) and enriching for a desired RNA in a sample, the method comprising:
contacting the sample with a polymer cocktail comprising polymers that collectively hybridize to less than 80% of the undesired rRNA, wherein each of the polymers specifically hybridizes to distinct regions of the undesired rRNA to generate hybridized regions of the rRNA that are separated by a gap;
cleaving the hybridized regions to deplete the undesired rRNA in the sample; and
collecting the desired RNA and/or removing the cleaved undesired target rRNA to enrich the for the desired RNA.
2. The method according to claim 1 , wherein the undesired rRNA and desired RNA are isolated from a biological sample.
3. The method according to claim 2 , wherein the biological sample is selected from the group consisting of: a tissue sample, a cell sample, and a bacterial sample.
4. The method according to claim 3 , wherein the biological sample is a fixed sample.
5. The method according to claim 1 , wherein the polymers are selected from the group consisting of: DNA oligonucleotides, RNA oligonucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA), and xeno nucleic acids (XNA).
6. The method according to claim 1 , wherein cleaving the hybridized regions comprises contacting the hybridized regions with an enzyme.
7. The method according to claim 6 , wherein cleaving the hybridized regions comprises contacting the hybridized regions with a ribonuclease.
8. The method according to claim 7 , wherein the ribonuclease specifically cleaves RNA present in an RNA/DNA duplex.
9. The method according to claim 8 , wherein the ribonuclease is RNase H.
10. The method according to claim 7 , wherein the ribonuclease specifically cleaves RNA present in an RNA/RNA duplex.
11. The method according to claim 10 , wherein the ribonuclease is RNase V.
12. The method according to claim 6 , wherein cleaving the hybridized regions comprises contacting the hybridized regions with a deoxyribonuclease.
13. The method according to claim 1 , wherein, after cleaving the hybridized regions, the method comprises removing the polymers.
14. The method according to claim 13 , wherein the polymers are removed by contacting the polymers with an enzyme.
15. The method according to claim 13 , wherein the polymers are removed by via a separation protocol.
16. The method according to claim 1 , wherein the rRNA is a mitochondrial rRNA.
17. The method according to claim 1 , wherein the polymers are 2 to 200 nucleotides in length.
18. The method according to claim 17 , wherein the polymers are 10 to 20, 11 to 30, 31 to 40, 41 to 50, 51-60 or 61 to 70 nucleotides in length.
19. The method according to claim 1 , wherein the polymers collectively hybridize to 75% or less of the undesired rRNA.
20. The method according to claim 19 , wherein the undesired rRNA is a 5S rRNA, a 18S rRNA, or a 28S rRNA.
21. The method according to claim 1 , wherein collecting the desired RNA and/or removing the cleaved undesired target rRNA comprises contacting the sample with a solid phase.
22. The method according to claim 21 , wherein the solid phase comprises Solid Phase Reversible Immobilization (SPRI) beads.