# US 12,065,645 B2 · Reverse transcriptases and uses thereof

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- Number: US 12,065,645 B2
- Document ID: US12065645B2
- Status: Granted
- Kind: B2
- Application number: 18/125,661
- Filed: 2023-03-23
- Published: 2023-10-19
- Granted: 2024-08-20
- Earliest claimed: 2017-02-16
- Assignees: Bio-Rad Laboratories, Inc.
- Applicants: Bio-Rad Laboratories, Inc.
- Inventors: Xiao-Song Gong; Yan Wang
- Web page: https://patentgrep.com/patent/US12065645B2

## CPC

- C12N 15/1096: {cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12Q 1/686: Polymerase chain reaction [PCR]
- C12N 2770/00022: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12Q 1/6844: Nucleic acid amplification reactions
- 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

Hybrid reverse transcriptases formed from portions of FLVRT and MLVRT are provided.

## Claims

### Claim 1

A nucleic acid comprising a polynucleotide encoding a hybrid reverse transcriptase, the hybrid reverse transcriptase comprising a finger domain, a palm domain, a thumb domain, a connection domain and an RNase H domain, wherein
(A) the hybrid reverse transcriptase comprises:
(i) a portion of feline leukemia virus reverse transcriptase (FLVRT) comprising the finger domain and the palm domain, wherein the portion of the FLVRT is at least 95% identical to SEQ ID NO:26, said portion of the FLVRT linked to a portion of mouse leukemia virus reverse transcriptase (MLVRT) comprising the thumb domain, the connection domain, and the RNase H domain, wherein the portion of the MLVRT is at least 95% identical to SEQ ID NO:28; or
(ii) a portion of feline leukemia virus reverse transcriptase (FLVRT) comprising the finger domain, the palm domain, the thumb domain, and the connection domain wherein the portion of the FLVRT is at least 95% identical to SEQ ID NO:27, said portion of the FLVRT linked to a portion of mouse leukemia virus reverse transcriptase (MLVRT) comprising the RNase H domain, wherein the portion of the MLVRT is at least 95% identical to SEQ ID NO:29; or
(B) the hybrid reverse transcriptase comprises:
a portion of mouse leukemia virus reverse transcriptase (MLVRT) comprising the finger domain and the palm domain, wherein the portion of the MLVRT is at least 95% identical to SEQ ID NO:1, said portion of the MLVRT linked to
a portion of feline leukemia virus reverse transcriptase (FLVRT) comprising the thumb domain, the connection domain, and the RNase H domain, wherein the portion of the FLVRT is at least 95% identical to SEQ ID NO:5.

### Claim 2 (depends on 1)

The nucleic acid of claim 1, comprising
a portion of feline leukemia virus reverse transcriptase (FLVRT) comprising the finger domain and the palm domain, wherein the portion of the FLVRT is at least 95% identical to SEQ ID NO:26, said portion of the FLVRT linked to
a portion of mouse leukemia virus reverse transcriptase (MLVRT) comprising the thumb domain, the connection domain, and the RNase H domain, wherein the portion of the MLVRT is at least 95% identical to SEQ ID NO:28.

### Claim 3 (depends on 2)

The nucleic acid of claim 2, wherein the hybrid reverse transcriptase comprises SEQ ID NO:30.

### Claim 4 (depends on 1)

The nucleic acid of claim 1,
a portion of feline leukemia virus reverse transcriptase (FLVRT) comprising the finger domain, the palm domain, the thumb domain, and the connection domain wherein the portion of the FLVRT is at least 95% identical to SEQ ID NO:27, said portion of the FLVRT linked to a portion of mouse leukemia virus reverse transcriptase (MLVRT) comprising the RNase H domain, wherein the portion of the MLVRT is at least 95% identical to SEQ ID NO:29.

### Claim 5 (depends on 4)

The nucleic acid of claim 4, wherein the hybrid reverse transcriptase comprises SEQ ID NO:31.

### Claim 6 (depends on 1)

The nucleic acid of claim 1, having at least one mutation that improves thermostability.

### Claim 7 (depends on 1)

The nucleic acid of claim 1, wherein the hybrid reverse transcriptase comprises:
a portion of mouse leukemia virus reverse transcriptase (MLVRT) comprising the finger domain and the palm domain, wherein the portion of the MLVRT is at least 95% identical to SEQ ID NO:1, said portion of the MLVRT linked to a portion of feline leukemia virus reverse transcriptase (FLVRT) comprising the thumb domain, the connection domain, and the RNase H domain, wherein the portion of the FLVRT is at least 95% identical to SEQ ID NO:5.

### Claim 8 (depends on 7)

The nucleic acid of claim 7, wherein the portion of the MLVRT comprises SEQ ID NO:1 and the portion of the FLVRT comprises SEQ ID NO:5.

### Claim 9 (depends on 8)

The nucleic acid of claim 8, wherein the portion of the FLVRT comprises SEQ ID NO:6 or SEQ ID NO:10.

### Claim 10 (depends on 9)

The nucleic acid of claim 9, wherein the portion of the FLVRT comprises SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:11.

### Claim 11 (depends on 8)

The nucleic acid of claim 8, wherein the portion of the MLVRT comprises SEQ ID NO:2.

### Claim 12 (depends on 8)

The nucleic acid of claim 8, wherein the portion of the MLVRT comprises SEQ ID NO:3 or SEQ ID NO:4.

### Claim 13 (depends on 8)

The nucleic acid of claim 8, wherein:
the portion of the MLVRT comprises SEQ ID NO:4 and the portion of the FLVRT comprises SEQ ID NO:7; or
the portion of the MLVRT comprises SEQ ID NO:4 and the portion of the FLVRT comprises SEQ ID NO:8; or
the portion of the MLVRT comprises SEQ ID NO:4 and the portion of the FLVRT comprises SEQ ID NO:9; or
the portion of the MLVRT comprises SEQ ID NO:3 and the portion of the FLVRT comprises SEQ ID NO:11; or
the portion of the MLVRT comprises SEQ ID NO:3 and the portion of the FLVRT comprises SEQ ID NO:10; or
the portion of the MLVRT comprises SEQ ID NO:4 and the portion of the FLVRT comprises SEQ ID NO:13.

### Claim 14 (depends on 8)

The nucleic acid of claim 8, wherein the hybrid reverse transcriptase comprises a sequence at least 95% identical to SEQ ID NO: 14, 15, 16, 17, 18, 19, 34, or 35.

### Claim 15 (depends on 1)

The nucleic acid of claim 1, comprising a promoter operably linked to the polynucleotide.

### Claim 16 (depends on 1)

An expression vector comprising the nucleic acid of claim 1.

### Claim 17 (depends on 1)

A cell comprising the nucleic acid of claim 1.