# US 11,299,739 B2 · Thermostable polymerase inhibitor compositions and methods

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- Number: US 11,299,739 B2
- Document ID: US11299739B2
- Status: Granted
- Kind: B2
- Application number: 16/305,858
- Filed: 2017-06-06
- Published: 2019-10-17
- Granted: 2022-04-12
- Earliest claimed: 2016-06-07
- Assignees: Cepheid
- Applicants: Cepheid
- Inventors: Sergey G. Lokhov; Alexander A. Gall; Vera Baraznenok; Ekaterina V. Viazovkina; Irina Shishkina; David H. Persing
- Web page: https://patentgrep.com/patent/US11299739B2

## CPC

- C12N 15/115: Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith {; Nucleic acids binding to non-nucleic acids, e.g. aptamers}
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/6853: using modified primers or templates
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12N 2310/16: Aptamers
- C12N 2310/531: Stem-loop; Hairpin
- C12N 2310/532: Closed or circular
- C12N 2320/52: modulating the physical stability, e.g. GC-content
- C12Q 2525/205: Aptamer
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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 disclosure relates to aptamers for temperature-dependent reversible inhibition of thermostable polymerase activity in order to improve sensitivity and specificity of various reactions and assays involving hot start polynucleotide synthesis. Methods for use of the aptamers and related compositions and kits are also provided.

## Claims

### Claim 1

An aptamer comprising:
a first sequence comprising SEQ ID NO:1 or a variant thereof or SEQ ID NO:2 or a variant thereof,
a second sequence comprising SEQ ID NO:3 or a variant thereof,
a third sequence comprising SEQ ID NO:1 or a variant thereof or SEQ ID NO:2 or a variant thereof,
and a fourth sequence comprising SEQ ID NO:3 or a variant thereof, wherein the second and fourth sequences are 12 nucleotides in length and are identical to each other;
wherein:
the variant of SEQ ID NO:1 comprises 1, 2, 3 or 4 nucleotide substitutions;
wherein each of the first and third sequences, comprising SEQ ID NOs:1 and 2, respectively, or variants thereof, are 16-18 nucleotides in length, are equal in length to each other, and are 100% complementary to each other along their entire length;
and
the variant of SEQ ID NO:3 contains 1 or 2 nucleotide substitutions; and
wherein:
there is no covalent bond between 2 of the nucleotides within the first sequence and wherein the aptamer comprises a 5′ terminus and a 3′ terminus; or
there is no covalent bond between 2 of the nucleotides within the third sequence and wherein the aptamer comprises a 5′ terminus and a 3′ terminus.

### Claim 2 (depends on 1)

The aptamer of claim 1, wherein there is no covalent bond between 2 of the nucleotides within the first sequence and wherein the aptamer comprises a 5′ terminus and a 3′ terminus.

### Claim 3 (depends on 1)

The aptamer of claim 1, wherein there is no covalent bond between 2 of the nucleotides within the third sequence and wherein the aptamer comprises a 5′ terminus and a 3′ terminus.

### Claim 4 (depends on 1)

The aptamer of claim 1, wherein the first sequence comprises SEQ ID NO:1, the third sequence comprises SEQ ID NO:2, and there is no covalent bond between nucleotides 8 and 9 of SEQ ID NO:1.

### Claim 5 (depends on 1)

The aptamer of claim 1, wherein the first sequence comprises SEQ ID NO:2, the third sequence comprises SEQ ID NO:1, and there is no covalent bond between nucleotides 8 and 9 of SEQ ID NO:2.

### Claim 6 (depends on 1)

The aptamer of claim 1, wherein the first sequence comprises SEQ ID NO:2 and the second and fourth sequences comprise SEQ ID NO:3.

### Claim 7 (depends on 1)

The aptamer of claim 1, wherein the first and third sequences are hybridized to each other along the entire length of each of the first and third sequences.

### Claim 8 (depends on 4)

The aptamer of claim 4, wherein the aptamer comprises SEQ ID NO:106.

### Claim 9 (depends on 5)

The aptamer of claim 5, wherein the aptamer comprises SEQ ID NO:110.

### Claim 10 (depends on 2)

The aptamer of claim 2, wherein:
the 5′ terminus is phosphorylated;
the 3′ terminus is linked to a 3′ capping moiety;
the 5′ terminus is linked to a 5′ capping moiety; or
the 3′ terminus is linked to a 3′ capping moiety and the 5′ terminus is linked to a 5′ capping moiety.

### Claim 11 (depends on 10)

The aptamer of claim 10, wherein the 3′ capping moiety is a propanediol spacer (C3) or a phosphate.

### Claim 12 (depends on 10)

The aptamer of claim 10, wherein the 5′ capping moiety is an amino group.

### Claim 13 (depends on 1)

A composition comprising an aptamer according to claim 1 and a thermostable polymerase.

### Claim 14 (depends on 13)

The composition of claim 13, wherein the molar ratio of the aptamer to the polymerase ranges from about 5:1 to 20:1.

### Claim 15 (depends on 1)

A method for extending a primer, comprising:
contacting a sample which might contain a target nucleic acid with a reagent composition to generate a reaction mixture, wherein the reagent composition comprises an aptamer according to claim 1, a thermostable polymerase, a forward primer specific for the target nucleic acid, deoxyribonucleotide triphosphates, and a divalent metal cation.

### Claim 16 (depends on 15)

The method of claim 15, wherein the ratio of the aptamer to the polymerase ranges from about 5:1 to 20:1.