Multicomponent nucleic acid probes for sample analysis

Patent metadata

Field	Value
Publication number	US-12297499-B2
Application number	US-202117403169-A
Country	US
Kind code	B2
Filing date	Aug 16, 2021
Priority date	Aug 17, 2020
Publication date	May 13, 2025
Grant date	May 13, 2025

Abstract

The present disclosure provides methods, systems, compositions, and kits for analyzing target molecules, including using probes comprising a plurality of components for analyzing target molecules in situ in a sample.

First claim

The invention claimed is:

1. A method for analyzing a sample, comprising:
(a) forming a hybridization complex comprising a first probe, a second probe, a splint, and a target nucleic acid molecule in the sample, wherein:
the target nucleic acid molecule comprises a first target sequence and a second target sequence,
the splint comprises a first splint sequence and second splint sequence,
the first probe comprises, from one end to another: (i) a first splint-hybridizing region forming a first duplex with the first splint sequence and (ii) a first target-hybridizing region hybridized to the first target sequence,
the second probe comprises, from one end to another: (i) a second target-hybridizing region hybridized to the second target sequence, (ii) a barcode region corresponding to the target nucleic acid molecule or a sequence thereof, and (iii) a second splint-hybridizing region forming a second duplex with the second splint sequence, and
the first duplex and the second duplex differ in stability;
(b) generating a circular probe by (i) ligating the first target-hybridizing region and the second target-hybridizing region and (ii) ligating the first splint-hybridizing region and the second splint-hybridizing region; and
(c) determining a sequence of the barcode region or complement thereof in the circular probe or an amplification product thereof,
thereby detecting the target nucleic acid molecule in the sample.

2. The method of claim 1 , wherein the target nucleic acid molecule is a cDNA tethered to a three-dimensional (3D) matrix embedding the sample, and/or the first and second probes do not comprise ribonucleotides.

3. The method of claim 1 , wherein forming the hybridization complex comprises:
(i) contacting the sample with the first probe and the second probe to allow probe hybridization to the target nucleic acid molecule;
(ii) washing the sample; and
(iii) contacting the sample with the splint to allow splint hybridization to the first and second probes.

4. The method of claim 1 , wherein the first duplex is less stable than the second duplex.

5. The method of claim 1 , wherein the first duplex is more stable than the second duplex.

6. The method of claim 5 , wherein the melting temperature of the first duplex is at least 1° C., at least 2° C., at least 5° C., at least 10° C., or at least 15° C. higher than the melting temperature of the second duplex.

7. The method of claim 5 , wherein the first duplex is at least 1, at least 2, at least 5, at least 10, or at least 15 base pairs longer than the second duplex.

8. The method of claim 1 , wherein the first splint-hybridizing region is at least 10 nucleotides and the second splint-hybridizing region is shorter than 10 nucleotides in length.

9. The method of claim 1 , wherein the barcode region and the second splint-hybridizing region do not overlap.

10. The method of claim 9 , wherein the barcode region and the second splint-hybridizing region are connected by a phosphodiester bond.

11. The method of claim 1 , wherein the barcode region and the second splint-hybridizing region partially or wholly overlap with each other.

12. The method of claim 1 , wherein the first probe does not comprise a barcode region or a portion thereof.

13. The method of claim 1 , wherein the first splint-hybridizing region does not comprise a barcode region or a portion thereof.

14. The method of claim 1 , wherein the first splint-hybridizing region comprises a common primer binding site among a plurality of first probes each hybridizing to a different first target sequence.

15. The method of claim 1 , wherein determining the sequence of the barcode region comprises performing sequencing by ligation using the splint or a portion thereof as primer.

16. The method of claim 1 , further comprising performing rolling circle amplification (RCA) using the splint or a portion thereof as primer and the circular probe as template to generate an RCA product of the circular probe.

17. The method of claim 16 , wherein determining the sequence of a complement of the barcode region in the RCA product comprises performing sequencing by ligation, sequencing by hybridization, and/or sequencing by synthesis.

18. A method for analyzing a sample, comprising:
(a) reverse transcribing an RNA molecule in the sample to produce a cDNA molecule comprising a tethering moiety;
(b) embedding the sample containing the cDNA molecule in a three-dimensional (3D) matrix, wherein the tethering moiety is configured to tether the cDNA molecule to the 3D matrix;
(c) contacting the sample embedded in the 3D matrix with a first probe and a second probe, wherein:
the first probe comprises, in the 5′ to 3′ direction: (i) a first splint-hybridizing region, (ii) a linker region, and (iii) a first target-hybridizing region that hybridizes to a first target sequence in the cDNA molecule, and
the second probe comprises, in the 5′ to 3′ direction: (i) a second target-hybridizing region that hybridizes to a second target sequence in the cDNA molecule, (ii) a barcode region corresponding to the RNA molecule, and (iii) a second splint-hybridizing region;
(d) contacting the sample embedded in the 3D matrix with a splint comprising a first splint sequence and second splint sequence, wherein the first splint sequence and the first splint-hybridizing region form a first duplex and the second splint sequence and the second splint-hybridizing region form a second duplex, and wherein the first duplex is more stable than the second duplex;
(e) generating a circular probe hybridized to the cDNA molecule and the splint by (i) ligating the first target-hybridizing region and the second target-hybridizing region and (ii) ligating the first splint-hybridizing region and the second splint-hybridizing region;
(f) generating a rolling circle amplification (RCA) product of the circular probe in the sample, wherein the RCA product comprises one or more modified nucleotides configured to tether the RCA product to the 3D matrix; and
(g) sequencing a complement of the barcode region in the RCA product tethered to the 3D matrix,
thereby detecting the RNA molecule in the sample.

19. The method of claim 18 , wherein the sample is a tissue sample, and wherein the method further comprises, between (b) and (c):
i) clearing the sample embedded in the 3D matrix; and
ii) degrading the RNA molecule.

20. The method of claim 18 , wherein the RCA product comprises multiple copies of a unit sequence comprising a complement of the linker region, and the method further comprises contacting the RCA product with one or more oligonucleotide probes that hybridize to complements of the linker region in different copies of the unit sequence.

Assignees

• 10X Genomics Inc

Inventors

• Daugharthy Evan
• Terry Richard
• Lee Soo Hee
• Busby Michele A
• Droz Shoshoni
• Gilmore Ryan

Classifications

• C12Q1/6832

  Enhancement of hybridisation reaction · CPC title

• C12Q1/6876 Primary

  Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes · CPC title

• C12Q1/6816 Primary

  characterised by the detection means (C12Q1/6804 takes precedence) · CPC title