# US 12,110,515 B2 · Modified Thermococcus polymerases

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- Number: US 12,110,515 B2
- Document ID: US12110515B2
- Status: Granted
- Kind: B2
- Application number: 18/048,388
- Filed: 2022-10-20
- Published: 2023-07-20
- Granted: 2024-10-08
- Earliest claimed: 2019-09-12
- Assignees: Singular Genomics Systems, Inc.
- Applicants: Singular Genomics Systems, Inc.
- Inventors: Souad Naji; Carl W. Fuller; Eli N. Glezer; Andrew Spaventa
- Web page: https://patentgrep.com/patent/US12110515B2

## CPC

- C12N 9/1252: {DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12Q 1/6869: Methods for sequencing
- C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation
- 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)
- 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

Provided herein are modified Archaeal family B polymerases derived from the Archaeal microorganism Thermococcus sp. EP1 that exhibit improved incorporation of nucleotide analogues utilized in DNA sequences.

## Claims

### Claim 1

A method of extending a primer, said method comprising contacting a primer hybridized to a template polynucleotide with a nucleotide and extending said primer with a polymerase by incorporating the modified nucleotide to form an extended primer, wherein the polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1; and further comprises:
an alanine at amino acid position 411 or an amino acid functionally equivalent to amino acid position 411;
a glycine at amino acid position 412 or an amino acid functionally equivalent to amino acid position 412; and
a proline at amino acid position 413 or an amino acid functionally equivalent to amino acid position 413; and
an alanine or glycine at amino acid position 93 or an amino acid functionally equivalent to amino acid position 93.

### Claim 2 (depends on 1)

The method of claim 1, wherein the polymerase further comprises an alanine substitution mutation at position 141 and an alanine substitution mutation at position 143.

### Claim 3 (depends on 1)

The method of claim 1, wherein the polymerase further comprises an alanine substitution mutation at position 144.

### Claim 4 (depends on 1)

The method of claim 1, wherein the polymerase further comprises at least one of the following:
an alanine or histidine at amino acid position 36 or an amino acid position functionally equivalent to amino acid position 36;
an alanine at amino acid position 129 or an amino acid position functionally equivalent to amino acid position 129;
an alanine at amino acid position 141 or an amino acid position functionally equivalent to amino acid position 141;
an alanine at amino acid position 143 or an amino acid position functionally equivalent to amino acid position 143;
an alanine at amino acid position 144 or an amino acid position functionally equivalent to amino acid position 144;
a glutamic acid at amino acid position 152 or an amino acid position functionally equivalent to amino acid position 152;
a glutamic acid or threonine at amino acid position 153 or an amino acid position functionally equivalent to amino acid position 153;
a threonine at amino acid position 155 or an amino acid position functionally equivalent to amino acid position 155;
an aspartic acid at amino acid position 157 or an amino acid position functionally equivalent to amino acid position 157;
an alanine at amino acid position 215 or an amino acid position functionally equivalent to amino acid position 215;
an isoleucine at amino acid position 303 or an amino acid position functionally equivalent to amino acid position 303; or
a methionine at amino acid position 307 or an amino acid position functionally equivalent to amino acid position 307.

### Claim 5 (depends on 1)

The method of claim 1, wherein the polymerase further comprises at least one of the following:
an alanine at amino acid position 317 or an amino acid position functionally equivalent to amino acid position 317;
a phenylalanine at amino acid position 381 or an amino acid position functionally equivalent to amino acid position 381;
a tryptophan at amino acid position 479 or an amino acid position functionally equivalent to amino acid position 479;
an alanine at amino acid position 480 or an amino acid position functionally equivalent to amino acid position 480;
an alanine at amino acid position 481 or an amino acid position functionally equivalent to amino acid position 481;
a valine at amino acid position 488 or an amino acid position functionally equivalent to amino acid position 488;
a serine at amino acid position 517 or an amino acid position functionally equivalent to amino acid position 517;
an isoleucine at amino acid position 593 or an amino acid position functionally equivalent to amino acid position 593;
an alanine at amino acid position 605 or an amino acid position functionally equivalent to amino acid position 605;
or a leucine at amino acid position 642 or an amino acid position functionally equivalent to amino acid position 642.

### Claim 6 (depends on 1)

The method of claim 1, wherein template polynucleotide comprises DNA.

### Claim 7 (depends on 1)

The method of claim 1, wherein the nucleotide comprises a detectable label and a reversible terminator moiety.

### Claim 8 (depends on 1)

The method of claim 1, wherein nucleotide does not include a detectable label.

### Claim 9 (depends on 7)

The method of claim 7, wherein the reversible terminator moiety comprises an azido moiety or a disulfide moiety.

### Claim 10 (depends on 7)

The method of claim 7, wherein the nucleotide has the formula
wherein the Base is a nucleobase, R3 is a polyphosphate, and R′ is a reversible terminator having the formula:
wherein RA and RB are hydrogen or alkyl and RC is the remainder of the reversible terminator.

### Claim 11 (depends on 7)

The method of claim 7, wherein the reversible terminator moiety comprises

### Claim 12 (depends on 7)

The method of claim 7, wherein the reversible terminator moiety comprises

### Claim 13 (depends on 1)

The method of claim 1, wherein the nucleotide has the formula:
wherein
B is a nucleobase;
L2 and L3 are independently cleavable linker;
L4 is a bond, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;
R2 is hydrogen or —OH;
R3 is a polyphosphate;
R4 is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;
R5 is a detectable label, an anchor moiety, or affinity anchor moiety;
R12 is a complementary affinity anchor moiety binder;
R13 is a detectable label;
X1 and X2 are independently hydrogen, halogen, —N3, or —CN, wherein at least wherein at least one of X1 or X2 is halogen, —N3, or —CN; and
the symbol “----” is a non-covalent bond.

### Claim 14 (depends on 1)

The method of claim 1, wherein contacting the primer occurs at about 40° C. to about 80° C.

### Claim 15 (depends on 1)

The method of claim 1, wherein contacting the primer occurs at about 50° C. to about 70° C.

### Claim 16 (depends on 1)

The method of claim 1, wherein contacting the primer occurs at about 55° C. to about 65° C.

### Claim 17 (depends on 1)

The method of claim 1, wherein the polymerase further comprises an amino acid substitution mutation at position 36, 129, 141, 143, 144, 152, 153, 155, 157, 209, 210, 213, 214, 215, 276, 277, 303, 307, 313, 317, 479, 480, 481, 488, 517, 593, 605, or 642 of SEQ ID NO: 1.

### Claim 18 (depends on 1)

The method of claim 1, wherein the polymerase is capable of incorporating modified nucleotides at an increased rate of 1-fold to 10-fold relative to the rate of incorporation of its wild type polymerase.