# US 12,006,519 B2 · Polymerase mutants and use with 3′-OH unblocked reversible terminators

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- Number: US 12,006,519 B2
- Document ID: US12006519B2
- Status: Granted
- Kind: B2
- Application number: 18/464,955
- Filed: 2023-09-11
- Published: 2024-01-18
- Granted: 2024-06-11
- Earliest claimed: 2021-06-29
- Assignees: Agilent Technologies, Inc.
- Applicants: Agilent Technologies, Inc.
- Inventors: Michelle Cayouette; Jeffrey Fox; Connie Hansen; Holly Hogrefe; Weidong Wu
- Web page: https://patentgrep.com/patent/US12006519B2

## CPC

- C12N 9/1252: {DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase}
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/6869: Methods for sequencing
- C12Y 207/07007: DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- 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
- C12Y: ENZYMES

## Abstract

Mutant polymerases are provided that have improved ability to incorporate modified nucleotides, including 3′-OH unblocked reversible terminators. The mutant polymerases may be used in a variety of applications, such as for polynucleotide sequencing, primer extension reactions, and template-independent enzymatic oligonucleotide synthesis.

## Claims

### Claim 1

A method for incorporating a single nucleotide into a priming strand in a template-independent reaction, the method comprising:
combining a priming strand with a 3′-OH-unmodified reversible terminator and a mutant polymerase, wherein the mutant polymerase is at least 96% identical to SEQ ID NO:2 and comprises:
a Y546H mutation at a position functionally equivalent to position 546 in Pfu polymerase;
a L409Y, L409H or L409F mutation at a position functionally equivalent to position 409 in Pfu polymerase; and
a A486X mutation at a position functionally equivalent to position 486 in Pfu polymerase, wherein X is any amino acid except alanine;
wherein incorporation of the terminator is at least 2-fold higher than for the mutant DNA polymerase of SEQ ID NO:11.

### Claim 2 (depends on 1)

The method of claim 1, wherein the polymerase further comprises one or more mutations at positions functionally equivalent to positions L270, E330, Q332, L333, P451, L453, L457, E476, L489, L490, N492, F494, Y497, and E581 in Pfu polymerase.

### Claim 3

The method of 1, wherein the 3′-OH-unmodified reversible terminator is a 2-nitrobenzyl-modified nucleotide.

### Claim 4 (depends on 1)

The method of claim 1, wherein the 3′-OH unmodified reversible terminator is a a C7- or C5-hydroxymethyl-α-tertbutyl-2-nitrobenzyl modified nucleotide and its α-thio derivative.

### Claim 5 (depends on 1)

The method of claim 1, wherein the mutant polymerase comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

### Claim 6 (depends on 1)

The method of claim 1, wherein the mutant polymerase is a derivative of Pyrococcus polymerase.

### Claim 7 (depends on 1)

The method of claim 1, wherein the mutant polymerase comprises the amino acid sequence of SEQ ID NO:2.

### Claim 8 (depends on 1)

The method of claim 1, wherein the mutant polymerase is a derivative of a Thermococcus polymerase.

### Claim 9 (depends on 1)

The method of claim 1, wherein the mutant polymerase further comprises a K477W mutation at a position functionally equivalent to position 477 in Pfu polymerase.

### Claim 10 (depends on 1)

The method of claim 1, wherein the mutant polymerase comprises a mutation at a position functionally equivalent to position F494 in Pfu polymerase, wherein the mutation is F494C, F494I, F494N, or F494T.

### Claim 11

A method of template-independent oligonucleotide synthesis comprising:
combining a priming strand, an 3′-OH-unmodified reversible terminator, and a mutant DNA polymerase, wherein mutant DNA polymerase comprises:
an amino acid sequences that is at least 96% identical to SEQ ID NO:2;
a Y546H mutation to histidine at a position functionally equivalent to position 546 in Pfu polymerase;
a L409Y, L409H, or L409F mutation at a position functionally equivalent to position 409 in Pfu polymerase; and
a A486X mutation at a position functionally equivalent to position 486 in Pfu polymerase, wherein X is any amino acid except alanine;
incorporating the 3′-OH-unmodified reversible terminator to the priming strand.

### Claim 12 (depends on 11)

The method of claim 11, wherein the polymerase further comprises one or more mutations at positions functionally equivalent to positions L270, E330, Q332, L333, P451, L453, L457, E476, L489, L490, N492, F494, Y497, and E581 in Pfu polymerase.

### Claim 13

The method of 11, wherein the 3′-OH-unmodified reversible terminator is a 2-nitrobenzyl-modified nucleotide.

### Claim 14 (depends on 11)

The method of claim 11, wherein the 3′-OH unmodified reversible terminator is a a C7- or C5-hydroxymethyl-a-tertbutyl-2-nitrobenzyl modified nucleotide and its α-thio derivative.

### Claim 15 (depends on 11)

The method of claim 11, wherein the mutant polymerase comprises the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5.

### Claim 16 (depends on 11)

The method of claim 11, wherein the mutant polymerase is a derivative of Pyrococcus polymerase.

### Claim 17 (depends on 11)

The method of claim 11, wherein the mutant polymerase comprises the amino acid sequence of SEQ ID NO:2.

### Claim 18 (depends on 11)

The method of claim 11, wherein the mutant polymerase is a derivative of a Thermococcus polymerase.

### Claim 19 (depends on 11)

The method of claim 11, wherein the mutant polymerase further comprises a K477W mutation at a position functionally equivalent to position 477 in Pfu polymerase.

### Claim 20 (depends on 11)

The method of claim 11, wherein the mutant polymerase comprises a mutation at a position functionally equivalent to position F494 in Pfu polymerase, wherein the mutation is F494C, F494I, F494N, or F494T.