RNase H assisted in situ rolling circle amplification
The present disclosure relates in some aspects to methods for analyzing a target RNA in a biological sample, such as detection of a sequence of interest in an RNA. In some aspects, provided herein are methods of using an RNase such as RNase H to provide in situ RNA detection methods having high sensitivity, efficiency, and/or specificity.
1 . A method for analyzing a biological sample, comprising:
(a) contacting the biological sample with a circular or circularizable probe, wherein the circular or circularizable probe comprises a hybridization region that hybridizes to a target RNA sequence in a target RNA in the biological sample to form a duplex,
(b) contacting the biological sample comprising the target RNA hybridized to the circular or circularizable probe with a primer oligonucleotide, a ribonuclease H (RNase H), and a polymerase, wherein the primer oligonucleotide comprises a sequence complementary to a sequence in the circular or circularizable probe, and the RNase H cleaves the target RNA in the duplex at one or more sites in the target RNA sequence;
(c) performing rolling circle amplification (RCA) using the circular probe or a circularized probe formed by circularizing the circularizable probe as an RCA template; and
(d) detecting one or more products of the RCA, thereby detecting the target RNA or a sequence thereof in the biological sample.
2 . The method of claim 1 , wherein the method comprises ligating the circularizable probe to form the circularized probe and performing the RCA using the circularized probe as the RCA template.
3 . The method of claim 2 , wherein the ligating is performed using the target RNA sequence as a ligation template.
4 . The method of claim 2 , wherein the ligating is performed prior to contacting the biological sample with the RNase H.
5 . The method of claim 1 , wherein the biological sample is contacted with the primer oligonucleotide, the RNase H, and the polymerase simultaneously.
6 . The method of claim 1 , wherein the biological sample is contacted with the RNase H prior to contacting the biological sample with the primer oligonucleotide and the polymerase.
7 . The method of claim 1 , wherein the biological sample is contacted with the RNase H prior to contacting the biological sample with the polymerase.
8 . The method of claim 1 , wherein the polymerase is a Phi29 DNA polymerase or a Bst DNA polymerase.
9 . The method of claim 1 , wherein the RNase is an RNase H1 or an RNase H2.
10 . The method of claim 1 , wherein (b) comprises: contacting the biological sample with the RNase H
in a first buffer that is free or substantially free of a cofactor for the RNase H,
followed by incubating the biological sample with the RNase H and the cofactor.
11 . The method of claim 10 , further comprising:
incubating the biological sample with the RNase H in the first buffer, thereby allowing the RNase H to bind the target RNA, the circular or circularizable probe, or the duplex;
removing excess RNase H from the biological sample; and
incubating the biological sample in a second buffer which comprises the RNase H and the cofactor, thereby allowing the RNase H to cleave at the one or more sites in the target RNA sequence.
12 . The method of claim 1 , wherein the one or more products of the RCA comprise an RCA product generated by using the primer oligonucleotide as a primer.
13 . The method of claim 1 , wherein the biological sample is contacted with the RNase H at a concentration of between about 1×10 −3 U/μL and about 2×10 −2 U/μL.
14 . The method of claim 1 , wherein the detecting comprises contacting the one or more RCA products with a plurality of detectable probes in sequential cycles.
15 . A method for analyzing a biological sample, comprising:
(a) contacting the biological sample with a circularizable probe comprising a hybridization region that hybridizes to a target RNA sequence in a target RNA in the biological sample to form a duplex,
(b) ligating the circularizable probe to form a circularized probe;
(c) contacting the biological sample comprising the target RNA hybridized to the circularized probe with (i) a primer oligonucleotide that comprises a sequence complementary to a sequence in the circularized probe, (ii) a ribonuclease H (RNase H), and (iii) a Phi29 DNA polymerase,
wherein the contacting comprises contacting the biological sample with the RNase H in a first buffer that is free or substantially free of a cofactor for the RNase H, followed by incubating the biological sample in a second buffer comprising the RNase H and the cofactor;
(d) performing rolling circle amplification (RCA) using the circularized probe as an RCA template; and
(e) detecting one or more products of the RCA, thereby detecting the target RNA or a sequence thereof in the biological sample.
16 . The method of claim 10 , wherein the cofactor for the RNase H is Mg 2+ .
17 . The method of claim 1 , wherein the biological sample is a cell or tissue sample.
18 . The method of claim 17 , wherein the one or more products of the RCA are detected at location(s) in the biological sample immobilized on a solid support.
19 . The method of claim 1 , wherein the detecting comprises contacting the one or more RCA products with a plurality of intermediate probes each comprising: (i) a sequence that hybridizes to the one or more RCA products and (ii) a sequence that hybridizes to one or more detectable probes.