# US 2023/0057571 A1 · Nucleic acid concatemers and methods for stabilizing and/or compacting the same

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- Number: US 2023/0057571 A1
- Document ID: US20230057571A1
- Status: Published application
- Kind: A1
- Application number: 17/816,995
- Filed: 2022-08-02
- Published: 2023-02-23
- Earliest claimed: 2021-08-03
- Assignees: 10x Genomics, Inc.
- Applicants: 10x Genomics, Inc.
- Inventors: Justin COSTA; Jorge Iván HERNÁNDEZ NEUTA; Patrick J. MARKS
- Web page: https://patentgrep.com/patent/US20230057571A1

## CPC

- C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 takes precedence)
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/682: Signal amplification
- C12Q 1/6832: Enhancement of hybridisation reaction
- C12Q 1/6841: In situ hybridisation
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q 2525/161: incorporating target specific and non-target specific sites
- C12Q 2525/307: Circular oligonucleotides
- C12Q 2527/156: Permeability
- C12Q 2531/125: Rolling circle
- C12Q 2543/101: in situ amplification
- C12Q 2563/107: fluorescence
- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## 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.

## Claims

### Claim 1

A method for analyzing a biological sample, 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; and
(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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

The method of claim 1, wherein the first and/or second hybridizing regions each comprises a palindromic sequence.

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 4)

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

### Claim 6 (depends on 4)

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

### Claim 7 (depends on 4)

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.

### Claim 8 (depends on 1)

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

### Claim 9 (depends on 1)

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

### Claim 10 (depends on 9)

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.

### Claim at position 11

11-16. (canceled)

### Claim 17 (depends on 1)

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.

### Claim 18 (depends on 17)

The method of claim 17, 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.

### Claim at position 14

19-20. (canceled)

### Claim 21 (depends on 1)

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.

### Claim 22

(canceled)

### Claim 23 (depends on 21)

The method of claim 21, wherein the first and second copies are separated by 0, 1, 2, 5, 10, 20, 50, 100, or more copies in the RCP.

### Claim 24 (depends on 1)

The method of claim 1, wherein:
i) the compacted RCP has a diameter of between about 0.1 μm and about 3 μm, e.g., between about 0.1 μm and about 0.5 μm (e.g., 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, e.g., between about 45 and about 70 kilobases.

### Claim at position 19

25-33. (canceled)

### Claim 34 (depends on 1)

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

### Claim 35 (depends on 1)

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

### Claim 36

(canceled)

### Claim 37 (depends on 1)

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

### Claim at position 24

38-48. (canceled)

### Claim 49

A method for analyzing a biological sample, 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; and
(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.

### Claim 50

A method for analyzing a biological sample, 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; and
(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.

### Claim at position 27

51-65. (canceled)