# US 12,006,518 B2 · Engineered polymerases with reduced sequence-specific errors

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 12,006,518 B2
- Document ID: US12006518B2
- Status: Granted
- Kind: B2
- Application number: 18/160,951
- Filed: 2023-01-27
- Published: 2023-08-24
- Granted: 2024-06-11
- Earliest claimed: 2022-02-18
- Assignees: Element Biosciences, Inc.
- Applicants: Element Biosciences, Inc.
- Inventors: Jendrik Hentschel; Tyler Lopez; Michael Klein; Virginia Saade; Matthew Kellinger; Mark Ambroso
- Web page: https://patentgrep.com/patent/US12006518B2

## CPC

- C12N 9/1252: {DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase}
- C12N 9/1241: {Nucleotidyltransferases (2.7.7)}
- 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)
- 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 engineered variants of archaeal polymerases that exhibit exonuclease-minus activity, enhanced thermostability, enhanced incorporation of 3′ modified nucleotides, improved uracil-tolerance and/or reduce sequence-specific errors in polymerase-catalyzed nucleotide binding and extension reactions relative to wild type polymerase enzymes. Also provided are uses of the engineered polymerases for forming complexed polymerases and forming binding complexes, and uses for conducting nucleic acid sequencing reactions.

## Claims

### Claim 1

A composition comprising: a plurality of engineered polymerases comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 1316, and a plurality of modified nucleotides comprising a removable chain terminating moiety at the 3′ carbon position of the sugar group, wherein the plurality of engineered polymerases comprises amino acid substitutions Asp168Ala and Glu170Ala relative to SEQ ID NO:1316.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the removable chain terminating moiety comprises an alkyl group, alkenyl group, alkynyl group, allyl group, aryl group, benzyl group, azide group, azido group, O-azidomethyl group, amine group, amide group, keto group, isocyanate group, phosphate group, thio group, disulfide group, carbonate group, urea group, silyl or acetal group, and wherein the removable chain terminating moiety is cleavable with a chemical compound to generate an extendible 3′OH moiety on the sugar group.

### Claim 3 (depends on 1)

The composition of claim 1, wherein the plurality of modified nucleotides comprise one type of modified nucleotide selected from a group consisting of dATP, dGTP, dCTP, dTTP and dUTP.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the plurality of modified nucleotides comprise a mixture of any combination of two or more types of modified nucleotides selected from a group consisting of dATP, dGTP, dCTP, dTTP and/or dUTP.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the plurality of modified nucleotides are labeled with a fluorophore.

### Claim 6 (depends on 1)

The composition of claim 1, wherein at least one modified nucleotide in the plurality of modified nucleotides lacks a fluorophore label.

### Claim 7 (depends on 1)

The composition of claim 1, further comprising a plurality of nucleic acid template molecules, and a plurality of oligonucleotide primers.

### Claim 8 (depends on 7)

The composition of claim 7, wherein the plurality of nucleic acid template molecules comprise linear nucleic acid molecules, circular nucleic acid molecules, or a mixture of linear and circular nucleic acid molecules.

### Claim 9 (depends on 7)

The composition of claim 7, wherein the plurality of nucleic acid template molecules comprises clonally amplified template molecules.

### Claim 10 (depends on 7)

The composition of claim 7, wherein at least one of the nucleic acid template molecules in the plurality of nucleic acid template molecules comprise one copy of a target sequence of interest.

### Claim 11 (depends on 7)

The composition of claim 7, wherein at least one of the nucleic acid template molecules in the plurality of nucleic acid template molecules comprise a concatemer having two or more tandem copies of a target sequence of interest.

### Claim 12 (depends on 7)

The composition of claim 7, wherein at least one of the nucleic acid template molecules in the plurality of nucleic acid template molecules comprise at least one uracil.

### Claim 13 (depends on 1)

The composition of claim 1, further comprising a plurality of catalytic divalent cations that promote polymerase-catalyzed nucleotide incorporation, wherein the catalytic divalent cations comprise magnesium or manganese.

### Claim 14 (depends on 7)

The composition of claim 7, wherein the plurality of polymerases, the plurality of nucleic acid template molecules, and the plurality of oligonucleotide primers, form a plurality of complexed polymerases each comprising an engineered polymerase bound to a nucleic acid duplex where the duplex comprises a nucleic acid template molecule hybridized to an oligonucleotide primer.

### Claim 15 (depends on 14)

The composition of claim 14, wherein the plurality of nucleic acid template molecules comprise the same target of interest sequence or different target of interest sequences.

### Claim 16 (depends on 14)

The composition of claim 14, wherein the plurality of complexed polymerases are immobilized to a coating on a support.

### Claim 17 (depends on 16)

The composition of claim 16, wherein the density of the plurality of complexed polymerases immobilized to the coating on the support comprises 102-1012 per mm2.

### Claim 18 (depends on 16)

The composition of claim 16, wherein the plurality of immobilized complexed polymerases are immobilized to pre-determined sites on the coating.

### Claim 19 (depends on 16)

The composition of claim 16, wherein the plurality of immobilized complexed polymerases are immobilized to random sites on the coating.

### Claim 20 (depends on 16)

The composition of claim 16, wherein the coating comprises at least one hydrophilic polymer coating layer which comprises unbranched polyethylene glycol (PEG), or wherein the coating comprises at least one hydrophilic polymer coating layer which comprises branched polyethylene glycol (PEG) having at least 4 branches.

### Claim 21 (depends on 20)

The composition of claim 20, wherein the hydrophilic polymer coating layer has a water contact angle of no more than 45 degrees.