Nucleic acid concatemers and methods for stabilizing and/or compacting the same

Patent metadata

Field	Value
Publication number	US-12553079-B2
Application number	US-202217816995-A
Country	US
Kind code	B2
Filing date	Aug 2, 2022
Priority date	Aug 3, 2021
Publication date	Feb 17, 2026
Grant date	Feb 17, 2026

Abstract

The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying multiple analytes present in a biological sample. In some aspects, the methods and compositions provided herein address one or more issues associated with the stability and/or size of nucleic acid structures, such as RCPs, in the biological sample without the use of exogenously added oligonucleotide compaction probes. In some embodiments, provided herein are methods involving the use of self-hybridizing hybridizing regions for compacting and/or stabilizing nucleic acid concatemers (e.g., RCPs). In some embodiments, dynamic inter-strand annealing between tandem units of an RCP is used for compaction and/or stabilization. In some embodiments, short palindromic regions in an RCP are used for compaction and/or stabilization.

First claim

The invention claimed is:

1 . A method comprising:
(a) contacting a biological sample with a circular probe or circularizable probe or probe set that hybridizes to a target nucleic acid in the biological sample, wherein the circular probe or circularizable probe or probe set comprises a first hybridizing region and a second hybridizing region capable of hybridizing to each other; (b) generating a rolling circle amplification product (RCP) at a location in the biological sample using the circular probe or a circularized probe generated from the circularizable probe or probe set as a template, wherein the RCP comprises multiple copies of complementary sequences of the first and second hybridizing regions, and wherein the complementary sequences of the first and second hybridizing regions from different copies hybridize to one another, thereby compacting and/or stabilizing the RCP at the location; and (c) detecting the RCP at the location in the biological sample.

2 . The method of claim 1 , wherein the first and second hybridizing regions are complementary to each other.

3 . The method of claim 1 , wherein the circular or circularizable probe or probe set comprises a palindromic sequence comprising the first and second hybridizing regions.

4 . The method of claim 1 , wherein the circular probe or circularizable probe or probe set further comprises a third hybridizing region and a fourth hybridizing region capable of hybridizing to each other.

5 . The method of claim 4 , wherein the first and second hybridizing regions are different in sequence from the third and fourth hybridizing regions, respectively.

6 . The method of claim 4 , wherein the first and second hybridizing regions do not hybridize to the third or fourth hybridizing region.

7 . The method of claim 4 , wherein the first and second hybridizing regions are the same in sequence as the third and fourth hybridizing regions, respectively.

8 . The method of claim 1 , wherein the circular probe or circularizable probe or probe set further comprises one or more barcode sequences.

9 . The method of claim 1 , wherein the first and second hybridizing regions are linked by a loop region.

10 . The method of claim 9 , wherein the loop region is no more than 5, no more than 10, no more than 15, no more than 20, no more than 25, or no more than 30 nucleotides in length.

11 . The method of claim 1 , wherein the melting temperature of a duplex of the first and second hybridizing regions is in thermodynamic equilibrium with the temperature at which the RCP is generated.

12 . The method of claim 11 , wherein the melting temperature is between about 15° C. and about 45° C. and/or wherein the temperature at which the RCP is generated is between about 15° C. and about 45° C.

13 . The method of claim 1 , wherein in the RCP, the complementary sequence of the first hybridizing region of a first copy hybridizes to the complementary sequence of the second hybridizing region of a second copy, and the complementary sequence of the second hybridizing region of the first copy hybridizes to the complementary sequence of the first hybridizing region of the second copy.

14 . The method of claim 13 , wherein the first and second copies are separated by 0, 1, 2, 5, 10, 20, 50, 100, or more copies in the RCP.

15 . The method of claim 1 , wherein: i) the compacted RCP has a diameter of between about 0.1 μm and about 3 μm, optionally between about 0.1 μm and about 0.5 μm, optionally between about 0.2 μm and about 0.3 μm, or between about 0.3 μm and about 0.4 μm, between about 0.5 μm and about 1 μm, between about 0.8 μm and about 1.3 μm, or between about 1 μm and about 1.5 μm; and/or ii) the compacted RCP is between about 1 and about 15 kilobases, between about 15 and about 25 kilobases, between about 25 and about 35 kilobases, between about 35 and about 45 kilobases, between about 45 and about 55 kilobases, between about 55 and about 65 kilobases, between about 65 and about 75 kilobases, or more than 75 kilobases in length, optionally between about 45 and about 70 kilobases.

16 . The method of claim 1 , wherein the target nucleic acid is an mRNA.

17 . The method of claim 1 , wherein the compacted RCP is immobilized in the biological sample.

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

19 . A method comprising:
(a) contacting the biological sample with a padlock probe comprising a sequence that hybridizes to a target nucleic acid in the biological sample, wherein the padlock probe comprises: (i) a first hybridizing region and a second hybridizing region capable of hybridizing to each other, wherein the first and second hybridizing regions are linked by a loop region and (ii) one or more barcode sequences; (b) generating a rolling circle amplification product (RCP) at a location in the biological sample using a circularized padlock probe as a template, wherein the padlock probe is circularized to generate the circularized padlock probe using the target nucleic acid and/or a splint as template, wherein the RCP comprises multiple copies of complementary sequences of the first and second hybridizing regions, and wherein the complementary sequences of the first and second hybridizing regions from different copies hybridize to one another, thereby compacting and/or stabilizing the RCP at the location in the biological sample; and (c) detecting the RCP at the location in the biological sample.

20 . A method comprising:
(a) contacting the biological sample with a padlock probe comprising a sequence that hybridizes to a target nucleic acid in the biological sample, wherein the padlock probe comprises: (i) a first hybridizing region and a second hybridizing region capable of hybridizing to each other, wherein the first and second hybridizing regions are linked by a first loop region; (ii) a third hybridizing region and a fourth hybridizing region capable of hybridizing to each other, wherein the third and fourth hybridizing regions are linked by a second loop region; and (iii) one or more barcode sequences in the first and/or second loop regions; (b) generating a rolling circle amplification product (RCP) at a location in the biological sample using a circularized padlock probe as a template, wherein the padlock probe is circularized to generate the circularized padlock probe using the target nucleic acid and/or a splint as template, wherein the RCP comprises multiple copies of complementary sequences of the first, second, third, and fourth hybridizing regions, and wherein the complementary sequences of the first and second hybridizing regions from two or more different copies hybridize to one another and the complementary sequences of the third and fourth hybridizing regions from two or more different copies hybridize to one another, thereby compacting and/or stabilizing the RCP at the location in the biological sample; and (c) detecting the RCP at the location in the biological sample.

Assignees

• 10X Genomics Inc

Inventors

• Costa Justin
• Hernández Neuta Jorge Iván
• Marks Patrick J

Classifications

• G01N21/6458

  Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16) · CPC title

• C12Q2563/107

  fluorescence · CPC title

• C12Q2543/101

  in situ amplification · CPC title

• C12Q2531/125

  Rolling circle · CPC title

• C12Q2527/156

  Permeability · CPC title