Methods and systems for targeted RNA cleavage and target RNA-primed rolling circle amplification

Patent metadata

Field	Value
Publication number	US-12319956-B2
Application number	US-202418789541-A
Country	US
Kind code	B2
Filing date	Jul 30, 2024
Priority date	Jul 31, 2023
Publication date	Jun 3, 2025
Grant date	Jun 3, 2025

Abstract

The present disclosure relates in some aspects to methods, systems, and kits for analyzing a biological sample comprising generating a rolling circle amplification product (RCP) using a target ribonucleic acid (RNA) as a primer. In some aspects, RNase H and a nucleic acid oligonucleotide are used to generate a free 3′ end of the target RNA to prime RCA.

First claim

The invention claimed is:

1. A method of analyzing a biological sample, comprising:
a) hybridizing a nucleic acid oligonucleotide to an oligonucleotide hybridization region in a target ribonucleic acid (RNA) in the biological sample, wherein the oligonucleotide hybridization region is adjacent to the 3′ end of a target sequence in the target RNA or is overlapping with the 3′ end of a target sequence in the target RNA;
b) cleaving the target RNA with an RNase H in the oligonucleotide hybridization region to generate a cleaved target RNA, wherein the cleaved target RNA comprises at least a portion of the target sequence;
c) subsequent to b), hybridizing a circular probe or a circularizable probe or probe set to the cleaved target RNA, wherein the circular probe or circularizable probe or probe set comprises a target recognition sequence complementary to the target sequence;
d) performing rolling circle amplification of the circular probe or of a circularized probe generated from the circularizable probe or probe set to generate a rolling circle amplification product (RCP) using the cleaved target RNA as a primer; and
e) detecting the RCP in the biological sample.

2. The method of claim 1 , wherein the biological sample is contacted with the nucleic acid oligonucleotide and with the RNase H simultaneously before contacting the biological sample with the circular probe or the circularizable probe or probe set.

3. The method of claim 1 , wherein the method comprises washing the biological sample after cleaving the target RNA with the RNase H and before contacting the biological sample with the circular probe or the circularizable probe or probe set.

4. The method of claim 1 , wherein the oligonucleotide hybridization region and the target sequence overlap by between 1 and 20 nucleotides.

5. The method of claim 1 , wherein the method does not comprise contacting the biological sample with a DNA primer that hybridizes to the circular probe or the circularized probe.

6. The method of claim 1 , wherein the target recognition sequence of the circularizable probe or probe set is a split recognition sequence comprising a first hybridization region having a first ligatable end and a second hybridization region having a second ligatable end,
wherein the first hybridization region hybridizes to a 5′ portion of the target sequence, and the second hybridization region hybridizes to a 3′ portion of the target sequence, and the method comprises ligating the first ligatable end to the second ligatable end to generate the circularized probe.

7. The method of claim 1 , wherein the RNase H comprises an RNase H1 and/or an RNase H2.

8. The method of claim 1 , wherein contacting the biological sample with the RNase H comprises contacting the biological sample with between 0.5 enzyme units (U) and 50 U of the RNase H.

9. The method of claim 1 , wherein the method comprises imaging the biological sample to detect the RCP.

10. The method of claim 9 , wherein the imaging comprises detecting a signal associated with a fluorescently labeled probe that directly or indirectly binds to the RCP.

11. The method of claim 10 , wherein a sequence of the RCP is analyzed by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof.

12. The method of claim 1 , wherein the oligonucleotide hybridization region and the 3′ portion of the target sequence overlap by about 6 to about 10 nucleotides.

13. The method of claim 1 , wherein the method comprises contacting the biological sample with a polymerase in a first reaction mixture comprising a non-catalytic metal of the polymerase, and then contacting the biological sample with a second reaction mixture comprising a catalytic cofactor of the polymerase to perform the rolling circle amplification.

14. The method of claim 13 , wherein the non-catalytic metal of the polymerase is barium, strontium, iron, cobalt, nickel, tin, zinc, or europium.

15. The method of claim 13 , wherein the non-catalytic cofactor of the polymerase is calcium or strontium.

16. The method of claim 13 , wherein the non-catalytic cofactor of the polymerase is strontium.

17. The method of claim 1 , wherein a sequence of the RCP is analyzed at a location in the biological sample or a matrix embedding the biological sample.

18. The method of claim 1 , comprising reacting at least one RNA in the biological sample with a polynucleotide kinase (PNK).

19. The method of claim 18 , wherein the method comprises reacting at least one RNA in the biological sample with the PNK after contacting the biological sample with the nucleic acid oligonucleotide and the RNase H.

20. The method of claim 19 , wherein the PNK is a T4 Polynucleotide Kinase (T4 PNK) or a T7 Polynucleotide Kinase (T7-PNK).

21. The method of claim 1 , wherein the nucleic acid oligonucleotide is 20 to 35 nucleotides in length.

22. A method of analyzing a biological sample, comprising:
a) contacting the biological sample with a plurality of nucleic acid oligonucleotides, wherein a first oligonucleotide of the plurality hybridizes to a first oligonucleotide hybridization region in a first target ribonucleic acid (RNA) in the biological sample, and a second oligonucleotide of the plurality hybridizes to a second oligonucleotide hybridization region in a second target RNA in the biological sample;
b) cleaving the first and second target RNAs with an RNase H in their respective oligonucleotide hybridization regions to generate a first cleaved target RNA and a second cleaved target RNA;
c) contacting the biological sample with a plurality of circular probes or circularizable probes or probe sets,
wherein a first circular probe or first circularizable probe or probe set of the plurality comprises a first target recognition sequence complementary to a first target sequence in the first target RNA,
wherein a second circular probe or second circularizable probe or probe set of the plurality comprises a second target recognition sequence complementary to a second target sequence in the second target RNA,
wherein the first and second circular probe or the first and second circularizable probe or probe set hybridize to their respective target RNAs,
wherein the first oligonucleotide hybridization region is overlapping with the 3′ end of the first target sequence, and
wherein the second oligonucleotide hybridization region is overlapping with the 3′ end of the second target sequence;
d) performing rolling circle amplification of the first and second circular probe or of a first and second circularized probe generated from the first and second circularizable probes or probe sets to generate a first and second rolling circle amplification product (RCP) using the first cleaved target RNA and the second cleaved target RNA as primers; and
e) detecting the first and second RCPs in the biological sample.

23. The method of claim 22 , wherein the method comprises imaging the biological sample to detect the first and second RCPs.

24. The method of claim 22 , wherein a sequence of the first and second RCPs is analyzed by sequential hybridization, sequencing by hybridization, sequencing by ligation, sequencing by synthesis, sequencing by binding, or a combination thereof.

25. The method of claim 22 , wherein the first oligonucleotide hybridization region and the 3′ portion of the first target RNA overlap by about 6

Assignees

• 10X Genomics Inc

Inventors

• Wang Su
• Henley Robert

Classifications

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q1/6841 Primary

  In situ hybridisation · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title