# US 12,110,539 B2 · Metal ion-start DNA polymerase switch and isothermal polymerase amplification method using the same

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- Number: US 12,110,539 B2
- Document ID: US12110539B2
- Status: Granted
- Kind: B2
- Application number: 17/675,229
- Filed: 2022-02-18
- Published: 2023-03-09
- Granted: 2024-10-08
- Earliest claimed: 2021-09-09
- Assignees: Postech Research and Business Development Foundation
- Applicants: POSTECH Research and Business Development Foundation
- Inventors: Seung Soo Oh; Hye Bin Yoo
- Web page: https://patentgrep.com/patent/US12110539B2

## CPC

- C12Q 1/6823: Release of bound markers
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/6853: using modified primers or templates
- C12Q 1/686: Polymerase chain reaction [PCR]
- 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

The present invention relates to a metal ion-start DNA polymerase switch, a composition for isothermal polymerase amplification containing the same, and an isothermal amplification method using the metal ion-start DNA polymerase switch. The metal ion-start DNA polymerase switch according to the present invention may comprise: a binding module composed of TQ30 aptamer; a locking or unlocking module; and a catalytic module connecting between the binding module and the locking or unlocking module and composed of DNAzyme.

## Claims

### Claim 1

A metal ion-start DNA polymerase switch comprising:
a binding module composed of an aptamer for inhibiting DNA polymerase activity;
a locking or unlocking module; and
a catalytic module connecting between the binding module and the locking or unlocking module and composed of DNAzyme with a metal-ion recognition site,
wherein the aptamer comprises consensus sequence motif of TQ30, which is composed of a fixed loop that directly contacts a DNA polymerase, and a programmable stem that serves as a binding stabilizer,
wherein the metal ion-start DNA polymerase switch comprising the locking module comprises the nucleic acid sequence of SEQ ID NO: 1, and the metal ion-start DNA polymerase switch comprising the unlocking module comprises the nucleic acid sequence of SEQ ID NO: 4,
wherein the DNAzyme is Pb2+-responsive and RNA-cleaving GR5 DNAzyme, and
wherein the DNA polymerase is Taq polymerase.

### Claim 2 (depends on 1)

A composition for isothermal polymerase amplification containing: the metal ion-start DNA polymerase switch of claim 1; and a DNA polymerase,
wherein the DNA polymerase is Tag polymerase.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the locking module is cleaved by the DNAzyme to activate the DNA polymerase upon the addition of Pb2+ ions.

### Claim 4 (depends on 2)

The composition of claim 2, wherein the unlocking module is configured to deactivate the DNA polymerase in the presence of Pb2+ ions.

### Claim 5 (depends on 1)

A method for quantitative analysis of a target gene comprising steps of: preparing a mixture by mixing the metal ion-start DNA polymerase switch according to claim 1, Pb2+ ions, and a sample to be analyzed, and heating and incubating the mixture; and quantifying an amount of the target gene in the sample.