# US 12,180,534 B2 · Conditional primer extension for single-molecule detection

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- Number: US 12,180,534 B2
- Document ID: US12180534B2
- Status: Granted
- Kind: B2
- Application number: 17/523,611
- Filed: 2021-11-10
- Published: 2022-03-03
- Granted: 2024-12-31
- Earliest claimed: 2016-05-27
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard College
- Inventors: Feng Xuan; Peng Yin; Mingjie Dai; Xi Chen
- Web page: https://patentgrep.com/patent/US12180534B2

## CPC

- C12Q 1/6804: Nucleic acid analysis using immunogens (immunoassay G01N33/53)
- C40B 70/00: Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
- 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
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## Abstract

The present disclosure provides, in some embodiments, methods and compositions for single-molecule detection.

## Claims

### Claim 1

A composition for producing a nucleic acid, the composition comprising:
(a) a strand displacing polymerase and a mixture of dNTPs;
(b) a nucleic acid probe linked to a first target-binding molecule, wherein the nucleic acid probe comprises, in the following order, a first toehold domain, a first double-stranded domain, a first stopper, a second double-stranded domain, a second stopper, a third double-stranded domain, and a second toehold domain, wherein the first stopper and the second stopper are molecules or modifications that terminate polymerization by the strand displacing polymerase; and
(c) a nucleic acid primer strand linked to a second target-binding molecule, wherein the nucleic acid primer strand comprises a first single-stranded primer domain that can bind to the first toehold domain of the nucleic acid probe.

### Claim 2 (depends on 1)

The composition of claim 1, further comprising:
(d) a target protein that binds to the first target-binding molecule and/or the second target-binding molecule.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the target protein binds to the first target binding molecule and the second target-binding molecule.

### Claim 4 (depends on 1)

The composition of claim 1, wherein:
(i) the mixture of dNTPs lacks one dNTP chosen from: dATP, dTTP, dCTP, and dGTP; and
(ii) the first stopper and/or second stopper are/is complements or a complement of the dNTP lacking from the mixture of dNTPs.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the first stopper and/or the second stopper are/is synthetic non-DNA linkers or modified bases or a synthetic non-DNA linker or a modified base.

### Claim 6 (depends on 1)

The composition of claim 1, wherein the strand displacing polymerase is phi29 DNA polymerase; Bst DNA polymerase; or Bsu DNA polymerase.

### Claim 7 (depends on 1)

The composition of claim 1, wherein (1) the mixture of dNTPs consists of dATP, dTTP, and dCTP, and the first stopper and/or second stopper are/is dC; (2) the mixture of dNTPs consists of dATP, dTTP, and dGTP, and the first stopper and/or second stopper are/is dG; (3) the mixture of dNTPs consists of dTTP, dCTP, and dGTP, and the first stopper and/or second stopper are/is dT; or (4) the mixture of dNTPs consists of dATP, dCTP, and dGTP, and the first stopper and/or second stopper are/is dA.

### Claim 8 (depends on 1)

The composition of claim 1, wherein the first target-binding molecule and/or the second target-binding molecule are/is antibodies or aptamers or an antibody or an aptamer.

### Claim 9 (depends on 1)

The composition of claim 1, wherein the reaction mixture further comprises a pair of primers that bind to the nucleic acid.