# US 12,110,550 B2 · Methods of amplifying circular polynucleotides in situ

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- Number: US 12,110,550 B2
- Document ID: US12110550B2
- Status: Granted
- Kind: B2
- Application number: 18/588,765
- Filed: 2024-02-27
- Published: 2024-08-15
- Granted: 2024-10-08
- Earliest claimed: 2020-07-13
- Assignees: Singular Genomics Systems, Inc.
- Applicants: Singular Genomics Systems, Inc.
- Inventors: Daan Witters; Allen Lipson; Eli N. Glezer
- Web page: https://patentgrep.com/patent/US12110550B2

## CPC

- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by 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 complexes, kits, and efficient methods of sequencing two strands of a double stranded polynucleotide.

## Claims

### Claim 1

A method of incorporating nucleotides into a primer in situ, the method comprising:
(A) extending a first immobilized oligonucleotide hybridized to a circular polynucleotide with a strand-displacing polymerase to generate a first immobilized extension product comprising one or more complements of the circular polynucleotide, wherein said first immobilized oligonucleotide is attached to a protein in or on a cell or tissue;
(B) hybridizing a first primer to the first immobilized extension product, and extending the first primer with a polymerase to incorporate one or more modified nucleotides; and
(C) hybridizing the first immobilized extension product to a second immobilized oligonucleotide, wherein the second immobilized oligonucleotide is immobilized to a cellular component or a matrix in or on the cell or tissue.

### Claim 2 (depends on 1)

The method of claim 1, wherein the first immobilized extension product comprises three or more copies of the circular polynucleotide.

### Claim 3 (depends on 1)

The method of claim 1 prior to step (A), the method comprises forming the circular polynucleotide by ligating ends of a linear polynucleotide together.

### Claim 4 (depends on 1)

The method of claim 1, wherein the second immobilized oligonucleotide comprises one or more phosphorothioate nucleotides.

### Claim 5 (depends on 1)

The method of claim 1, wherein extending the first immobilized oligonucleotide comprises incubation with the strand-displacing polymerase (i) for about 10 seconds to about 30 minutes, and/or (ii) at a temperature of about 20° C. to about 50° C.

### Claim 6 (depends on 5)

The method of claim 5, wherein incubation with the strand-displacing polymerase is for about 0.5 minutes to about 10 minutes.

### Claim 7 (depends on 5)

The method of claim 5, wherein incubation with the strand-displacing polymerase is for about 1 minute to about 5 minutes.

### Claim 8 (depends on 5)

The method of claim 5, wherein incubation with the strand-displacing polymerase is at a temperature of about 35° C. to 42° C.

### Claim 9 (depends on 5)

The method of claim 5, wherein incubation with the strand-displacing polymerase is at a temperature of about 37° C. to 40° C.

### Claim 10 (depends on 5)

The method of claim 5, wherein the strand-displacing polymerase is phi29 polymerase, phi29 mutant polymerase or a thermostable phi29 mutant polymerase.

### Claim 11 (depends on 5)

The method of claim 5, wherein prior to step (A) the strand-displacing polymerase contacts the first immobilized oligonucleotide and circular polynucleotide in the absence of dNTPs; and optionally, excess strand-displacing polymerase is removed.

### Claim 12 (depends on 1)

The method of claim 1, further comprising contacting the cell or tissue with acetic acid, hydrochloric acid, nitric acid, formamide, guanidine, sodium salicylate, sodium hydroxide, dimethyl sulfoxide (DMSO), propylene glycol, urea, or a mixture thereof.

### Claim 13 (depends on 1)

The method of claim 1, wherein the circular polynucleotide is about 100 to about 1000 nucleotides, about 100 to about 300 nucleotides, about 300 to about 500 nucleotides, or about 500 to about 1000 nucleotides.

### Claim 14 (depends on 13)

The method of claim 13, wherein the circular polynucleotide is about 300 to about 600 nucleotides.

### Claim 15 (depends on 13)

The method of claim 13, wherein the circular polynucleotide includes at least one cleavable site.

### Claim 16 (depends on 2)

The method of claim 2, wherein the first extension product comprises a cleavable site.

### Claim 17 (depends on 1)

The method of claim 1, wherein the second immobilized oligonucleotide is covalently attached to a cellular component, wherein covalent attachment is formed via a reaction between an oligonucleotide comprising a first bioconjugate reactive moiety and a cellular component comprising a second bioconjugate reactive moiety.

### Claim 18 (depends on 1)

The method of claim 1, wherein the one or more modified nucleotides each comprise a reversible terminator moiety.

### Claim 19 (depends on 1)

The method of claim 1, wherein the one or more modified nucleotides each comprise a fluorophore.

### Claim 20 (depends on 1)

The method of claim 1, wherein the cell is a stem cell, an immune cell, or a cancer cell.

### Claim 21 (depends on 1)

The method of claim 1, wherein step (C) occurs before step (B).

### Claim 22 (depends on 1)

The method of claim 1, further comprising incorporating a dideoxynucleotide triphosphate (ddNTP) into the primer.

### Claim 23

A method of incorporating a modified nucleotide into a primer in situ, the method comprising:
extending a first immobilized oligonucleotide hybridized to a circular polynucleotide with a polymerase to generate an extension product comprising one or more complements of the circular polynucleotide, wherein said first immobilized oligonucleotide is attached to a cellular component of a cell;
hybridizing the extension product to a second oligonucleotide, wherein the second oligonucleotide tethers the extension product to a localized space; and
hybridizing a primer to the immobilized extension product, and extending the primer with a polymerase to incorporate the modified nucleotide.

### Claim 24 (depends on 23)

The method of claim 23, wherein the modified nucleotide comprises a 3′ blocking moiety preventing polymerase extension.

### Claim 25 (depends on 24)

The method of claim 24, wherein the modified nucleotide further comprises a fluorophore.

### Claim 26 (depends on 24)

The method of claim 24, wherein the 3′ blocking moiety comprises an azide moiety, a disulfide moiety, or an allyl moiety.

### Claim 27 (depends on 24)

The method of claim 24, further comprising removing the 3′ blocking moiety and incorporating a second modified nucleotide.

### Claim 28 (depends on 23)

The method of claim 23, wherein the second oligonucleotide comprises a 3′ blocking moiety preventing polymerase extension.

### Claim 29 (depends on 23)

The method of claim 23, wherein the cell is immobilized to a substrate.

### Claim 30 (depends on 29)

The method of claim 29, wherein the substrate comprises a polymer coating.

### Claim 31 (depends on 30)

The method of claim 30, wherein the substrate is a reaction chamber of a flow cell.

### Claim 32 (depends on 23)

The method of claim 23, wherein the localized space comprises a diameter of about 50 nm to about 350 nm.

### Claim 33 (depends on 23)

The method of claim 23, wherein the cell forms part of a tissue.

### Claim 34 (depends on 33)

The method of claim 33, wherein the tissue is kidney tissue, bladder tissue, colon tissue, brain tissue, bone tissue, or breast tissue.

### Claim 35 (depends on 23)

The method of claim 23, wherein the cellular component is a cell membrane.

### Claim 36 (depends on 23)

The method of claim 23, wherein the cellular component is a protein.

### Claim 37 (depends on 23)

The method of claim 23, wherein the cellular component is a nucleic acid molecule.

### Claim 38 (depends on 1)

The method of claim 1, wherein the first immobilized oligonucleotide is attached to a protein in a tissue and the second immobilized oligonucleotide is attached to a cellular component or a matrix in the tissue.