# US 12,006,534 B2 · Spatial sequencing

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- Number: US 12,006,534 B2
- Document ID: US12006534B2
- Status: Granted
- Kind: B2
- Application number: 18/356,724
- Filed: 2023-07-21
- Published: 2023-11-16
- Granted: 2024-06-11
- Earliest claimed: 2020-08-06
- Assignees: Singular Genomics Systems, Inc.
- Applicants: Singular Genomics Systems, Inc.
- Inventors: Eli N. Glezer
- Web page: https://patentgrep.com/patent/US12006534B2

## CPC

- C12Q 1/6841: In situ hybridisation
- 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

## Abstract

Disclosed herein, inter alia, are compositions and methods of use thereof for interrogating a sample comprising a cell.

## Claims

### Claim 1

A method of detecting an RNA molecule in a cell or tissue, said method comprising:
contacting a cell or tissue with a polynucleotide probe and hybridizing a first hybridization sequence of the polynucleotide probe to a first target sequence of the RNA molecule, and hybridizing a second hybridization sequence of the polynucleotide probe to a second target sequence of the RNA molecule, wherein said RNA molecule comprises a target sequence between the first target sequence and the second target sequence;
extending the polynucleotide probe along the target sequence to generate a complement of the target sequence, and ligating the complement of the target sequence to the polynucleotide probe thereby forming a circular oligonucleotide;
extending an amplification primer hybridized to the circular oligonucleotide with a polymerase to generate an extension product comprising the target sequence; and
hybridizing a sequencing primer to the extension product and sequencing the target sequence in the cell or tissue, thereby detecting the RNA molecule.

### Claim 2 (depends on 1)

The method of claim 1, wherein said polynucleotide probe comprises DNA, RNA, or a combination thereof.

### Claim 3 (depends on 1)

The method of claim 1, wherein said polynucleotide probe consists of DNA.

### Claim 4 (depends on 1)

The method of claim 1, wherein the cell is permeabilized and immobilized to a solid support.

### Claim 5 (depends on 1)

The method of claim 1, wherein the polynucleotide probe is about 50 to about 100 nucleotides in length.

### Claim 6 (depends on 1)

The method of claim 1, wherein the polynucleotide probe is a single-stranded polynucleotide comprising at least one amplification primer binding sequence, at least one sequencing primer binding sequence, or both one amplification primer binding sequence and one sequencing primer binding sequence.

### Claim 7 (depends on 1)

The method of claim 1, wherein said sequencing comprises sequencing-by-synthesis.

### Claim 8 (depends on 1)

The method of claim 1, wherein sequencing comprises extending the sequencing primer by incorporating a labeled nucleotide, or labeled nucleotide analogue, and detecting the label for each incorporated nucleotide or nucleotide analogue.

### Claim 9 (depends on 1)

The method of claim 1, wherein two or more polynucleotide probes hybridize to different complementary sequences of the same endogenous RNA molecule.

### Claim 10 (depends on 1)

The method of claim 1, wherein said target sequence is 1 to about 15 nucleotides.

### Claim 11 (depends on 1)

The method of claim 1, wherein the first hybridization sequence, the second hybridization sequence, or both the first and second hybridization sequences of the polynucleotide probe comprise one or more locked nucleic acid (LNA) nucleotides.

### Claim 12 (depends on 1)

The method of claim 1, wherein the first hybridization sequence is about 10 to 30 nucleotides and the second hybridization sequence is about 10 to 30 nucleotides.

### Claim 13 (depends on 1)

The method of claim 1, wherein the first hybridization sequence comprises more nucleotides than the nucleotides in the second hybridization sequence.

### Claim 14

A method of incorporating a modified nucleotide into a nucleic acid molecule in a cell or tissue, said method comprising:
contacting a cell or tissue with a polynucleotide probe and hybridizing a first hybridization sequence of the polynucleotide probe to a first target sequence of an RNA molecule, and hybridizing a second hybridization sequence of the polynucleotide probe to a second target sequence of the RNA molecule, wherein said RNA molecule comprises a target sequence between the first target sequence and the second target sequence;
extending the polynucleotide probe along the target sequence to generate a complement of the target sequence, and ligating the complement of the target sequence to the polynucleotide probe thereby forming a circular oligonucleotide;
amplifying the circular oligonucleotide by extending a first amplification primer hybridized to the circular oligonucleotide with a polymerase to generate an extension product comprising the target sequence; and
hybridizing a primer to the extension product and incorporating a modified nucleotide into the primer, wherein the modified nucleotide comprises a blocking group capable of preventing polymerase extension.

### Claim 15 (depends on 14)

The method of claim 14, wherein the blocking group is a reversible terminator moiety.

### Claim 16 (depends on 15)

The method of claim 15, further comprising removing the reversible terminator moiety and incorporating a second modified nucleotide into the primer.

### Claim 17 (depends on 14)

The method of claim 14, wherein the modified nucleotide comprises a detectable label.

### Claim 18 (depends on 14)

The method of claim 14, further comprising detecting the incorporated modified nucleotide.

### Claim 19 (depends on 1)

The method of claim 1, wherein extending the polynucleotide probe along the target sequence comprises extension with a reverse transcriptase.

### Claim 20 (depends on 17)

The method of claim 17, further comprising detecting the modified nucleotide, thereby identifying a complementary nucleotide of the extension product.