IP Library › Granted Patent US 12,735,738
Granted Patent B2
US 12,735,738 · App. 17/993,793 · Granted Sep 15, 2026

Circular probes and methods for sample analysis

Inventors: Zahra Kamila Belhocine (Pleasanton, CA); Justin Costa (Union City, CA); Eswar Prasad Ramachandran Iyer (Sunnyvale, CA)
Assignee: 10X GENOMICS, INC.
C12Q1/6841C12Q1/682C12Q1/6844
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Quick Facts
Patent No.
US 12,735,738
App. No.
17/993,793
Filed
Nov 23, 2022
Granted
Sep 15, 2026
Kind
B2
Art Unit
1681
USPC
435/6.12
Abstract

The present disclosure relates in some aspects to methods and compositions for analyzing a target nucleic acid, such as in situ detection of a region of interest in a polynucleotide in a cell or tissue sample. In some aspects, provided herein are circular probes (e.g., dumbbell probes) for analyzing a target nucleic acid, as well as methods comprising an enzymatic treatment to de-circularize unbound or non-specifically bound circular probes. Circular probes specifically bound to target nucleic acids remain intact during and after the enzymatic treatment and can be detected, e.g., via rolling circle amplification (RCA) of the circular probes and detection of the RCA products.

Claims (43)

1 . A method, comprising:

(a) contacting a biological sample comprising a plurality of ribonucleic acid (RNA) molecules with a plurality of circular probes, wherein a first circular probe of the plurality of circular probes comprises:

(i) a first loop region comprising a sequence complementary to a first sequence of a first RNA molecule of the plurality of RNA molecules,

(ii) a stem region comprising (1) a first strand complementary to the first RNA molecule, wherein the first strand comprises one or more ribonucleotides, and (2) a second strand comprising deoxyribonucleic acid (DNA) complementary to the first strand, and

(iii) a second loop region;

(b) hybridizing the first loop region to the first sequence of the first RNA molecule;

(c) displacing the second strand from the first strand and hybridizing the first strand to the first RNA molecule, thereby obtaining a hybridized first circular probe;

(d) after (c), contacting the biological sample with an RNAse, wherein the RNAse cleaves one or more ribonucleotides of a second circular probe of the plurality of circular probes, wherein the one or more ribonucleotides of the second circular probe are not hybridized to an RNA molecule of the plurality of RNA molecules;

(e) performing a rolling circle amplification reaction on the hybridized first circular probe to generate a rolling circle amplification (RCA) product; and

(f) detecting the RCA product, thereby detecting the first RNA molecule in the biological sample.

2 . The method of claim 1 , wherein the second circular probe comprises a stem region comprising (1) a first strand comprising one or more ribonucleotides, and (2) a second strand complementary to the first strand.

3 . The method of claim 2 , wherein the second circular probe comprises a loop region comprising a sequence complementary to the first sequence of the first RNA molecule.

4 . The method of claim 1 , wherein the first RNA molecule comprises a sequence of interest, wherein the sequence of interest or a portion thereof is complementary to one or more of the ribonucleotide(s) in the first strand of the first circular probe.

5 . The method of claim 3 , wherein the first strand of the stem region of the second circular probe does not hybridize to an RNA molecule of the plurality of RNA molecules.

6 . The method of claim 1 , wherein the first loop region or second loop region of the first circular probe comprises a first barcode sequence corresponding to the first RNA molecule or a sequence of interest therein.

7 . The method of claim 1 , wherein the RNAse is an RNAse H.

8 . The method of claim 7 , wherein the biological sample is treated with the RNAse H in the presence of one or more RNAse inhibitors that do not inhibit the RNAse H.

9 . The method of claim 1 , wherein the RNAse is an RNAse H1 or an RNAse H2.

10 . The method of claim 1 , wherein the RNAse does not cleave the first RNA molecule or a portion thereof hybridized to the first circular probe.

11 . The method of claim 1 , wherein the RNAse cleaves the first RNA molecule or a portion thereof hybridized to the first circular probe.

12 . The method of claim 1 , wherein performing the rolling circle amplification reaction on the hybridized first circular probe comprises contacting the biological sample with a primer that hybridizes to a sequence in the first loop region or second loop region.

13 . The method of claim 1 , wherein the detecting step comprises:

contacting the RCA product with one or more detectable probes that hybridize to the RCA product.

14 . The method of claim 1 , wherein the detecting step comprises:

contacting the RCA product with one or more intermediate probes that hybridize to the RCA product, wherein each intermediate probe comprises a sequence that hybridizes to the RCA product and a sequence that hybridizes to one or more detectable probes.

15 . The method of claim 14 , wherein the detecting step comprises contacting the RCA product with detectable probes and intermediate probes in sequential cycles.

16 . The method of claim 1 , wherein a circular probe of the plurality of circular probes is provided by ligating a first hairpin probe to a second hairpin probe, any one or both of which comprise one or more ribonucleotides at the 5′ and/or the 3′ end thereby circularizing the first and second hairpin probes to provide the circular probe.

17 . The method of claim 16 , wherein:

the first hairpin probe comprises a first loop region, a first stem region, and a first 3′ single-stranded region comprising one or more ribonucleotides;

the second hairpin probe comprises a second loop region, a second stem region, and a second 3′ single-stranded region that is DNA; and

the first 3′ single-stranded region or a portion thereof is complementary to the second 3′ single-stranded region or a portion thereof.

18 . The method of claim 1 , wherein the biological sample is a tissue sample.

19 . The method of claim 1 , wherein the biological sample is embedded in a matrix.

20 . A method, comprising:

(a) contacting a biological sample comprising a ribonucleic acid (RNA) molecule with a circular probe, wherein:

the circular probe comprises a stem region, a first loop region, and a second loop region,

the stem region comprises (i) a first strand comprising one or more ribonucleotides and (ii) a second strand complementary to the first strand, and

a sequence in the first loop region hybridizes to a first sequence in the RNA molecule;

(b) hybridizing a second sequence of the RNA molecule to the first strand of the stem region, wherein:

the second strand is displaced from the first strand, the circular probe hybridizes to at least the first and second sequences in the RNA molecule, and the one or more ribonucleotides in the circular probe hybridize to complementary ribonucleotide(s) in the RNA molecule;

(c) after (b), contacting the biological sample with an RNAse;

(d) performing a rolling circle amplification reaction on the circular probe to generate a rolling circle amplification (RCA) product; and

(e) detecting a signal or absence thereof, wherein the signal is associated with the RCA product in the biological sample.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 29, 2022
From: BELHOCINE, ZAHRA KAMILA; COSTA, JUSTIN; RAMACHANDRAN IYER, ESWAR PRASAD
To: 10X GENOMICS, INC.
Reel/Frame 062240/0171 →
Continuity (2)
Provisional Application 63283213 · Nov 25, 2021
Related Publication 20230159997A1 · May 25, 2023
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