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US-20260125660-A1 - MODIFIED ARCHAEAL FAMILY B POLYMERASES

US20260125660A1US 20260125660 A1US20260125660 A1US 20260125660A1US-20260125660-A1

Abstract

Provided herein are modified Archaeal family B polymerases derived from the Archaeal microorganism Pyrococcus abyssi that exhibit improved incorporation of nucleotide analogues utilized in DNA sequencing.

Inventors

  • Souad NAJI
  • Carl W. Fuller
  • Eli N. Glezer
  • Andrew Spaventa

Assignees

  • Singular Genomics Systems, Inc.

Dates

Publication Date
20260507
Application Date
20251008

Claims (16)

  1. 1 . A polymerase-template complex, comprising: a polymerase bound to a primer, wherein said primer is bound to a template nucleic acid molecule, wherein said polymerase comprises an amino acid sequence that is at least 80% identical to a continuous 500 amino acid sequence within SEQ ID NO: 1; comprising: an alanine, serine, or histidine at amino acid position 409 or at a position corresponding to amino acid position 409; and a proline at amino acid position 411 or at a position corresponding to the amino acid position 411.
  2. 2 . The polymerase-template complex of claim 1 , wherein the polymerase comprises an alanine or glycine at amino acid position 410.
  3. 3 . The polymerase-template complex of claim 1 , wherein the polymerase comprises a glycine at amino acid position 410.
  4. 4 . The polymerase-template complex of claim 1 , wherein the polymerase comprises a serine at amino acid position 409 and an alanine at amino acid position 410.
  5. 5 . The polymerase-template complex of claim 1 , wherein the polymerase comprises an amino acid mutation at positions 141 and 143.
  6. 6 . The polymerase-template complex of claim 5 , wherein the amino acid mutation is an alanine.
  7. 7 . The polymerase-template complex of claim 1 , wherein the polymerase comprises a leucine or valine substitution mutation at position 486.
  8. 8 . The polymerase-template complex of claim 1 , wherein the polymerase comprises a serine at position 515.
  9. 9 . The polymerase-template complex of claim 1 , wherein the polymerase comprises an isoleucine at position 590.
  10. 10 . The polymerase-template complex of claim 1 , wherein the primer comprises a modified nucleotide.
  11. 11 . The polymerase-template complex of claim 10 , wherein the modified nucleotide comprises a fluorescent dye.
  12. 12 . The polymerase-template complex of claim 10 , wherein the modified nucleotide comprises a reversible terminator moiety.
  13. 13 . The polymerase-template complex of claim 10 , wherein the modified nucleotide comprises a biotin moiety.
  14. 14 . The polymerase-template complex of claim 10 , wherein the modified nucleotide comprises a reversible terminator moiety.
  15. 15 . The polymerase-template complex of claim 1 , wherein the primer comprises the sequence SEQ ID NO:208.
  16. 16 . A solid support comprising the polymerase-template complex of claim 1 , wherein said template nucleic acid molecule is attached to the solid support.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS This application is a continuation of U.S. Utility application Ser. No. 18/500,785, filed Nov. 2, 2023, which is a continuation of U.S. Utility application Ser. No. 17/369,593, filed Jul. 7, 2021, now issued as 11,845,923, which is a continuation of U.S. Utility application Ser. No. 16/568,089, filed Sep. 11, 2019, now issued as U.S. Pat. No. 11,136,565, which claims the benefit of U.S. Provisional Application No. 62/729,875, filed Sep. 11, 2018, each of which are incorporated herein by reference in their entirety and for all purposes. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said Sequence Listing, created on Jan. 15, 2025, is named 00511C03US.xml and is 518,575 bytes in size. BACKGROUND The present disclosure generally relates to modified polymerase enzymes that exhibit improved incorporation of nucleotide analogues utilized in DNA sequencing. DNA-polymerases add nucleotide triphosphate (dNTP) residues to the 3′-end of the growing DNA chain, using a complementary DNA as template. Three motifs, A, B and C, are seen to be conserved across all DNA-polymerases. Initial experiments with native DNA polymerases revealed difficulties incorporating modified nucleotides. There are several examples in which DNA polymerases have been modified to increase the rates of incorporation of nucleotide analogues. For example, in order to increase the efficiency of incorporation of modified nucleotides, DNA polymerases have been engineered such that they lack 3′-5′ exonuclease activity (designated exo-). The exo-variant of 9°N polymerase is described by Perler et al, 1998 U.S. Pat. No. 5,756,334 and by Southworth et al, 1996 Proc. Natl Acad. Sci USA 93:5281. As described in WO 2005/024010, modification to the motif A region of DNA polymerases effects on the ability of polymerases to incorporate nucleotide analogues having a substituent at the 3′ position which is larger than a hydroxyl group (i.e., a reversible terminator). In the context of this application, the term “motif A region” specifically refers to the three amino acids functionally equivalent or homologous to amino acids 409, 410, and 411 in wild type P. abyssi; these amino acids are functionally equivalent to 408, 409, 410 in 9°N polymerase. Despite ongoing research, current modifications to the DNA polymerase still do not show sufficiently high incorporation rates of modified nucleotides. In nucleic acid sequencing applications, the modified nucleotide typically has a reversible terminator at the 3′ position and a modified base (e.g., a base linked to a fluorophore via a cleavable linker). In case of cleavable linkers attached to the base, there is usually a residual spacer arm left after the cleavage. This residual modification may interfere with incorporation of subsequent nucleotides by polymerase. Therefore, it is highly desirable to have polymerases for carrying out sequencing by synthesis process (SBS) that are tolerable of these scars. In addition to rapid incorporation, the enzyme needs to be stable and have high incorporation fidelity. Discovering a polymerase that has suitable kinetics and low misincorporation error remains a challenge. Disclosed herein, inter alia, are solutions to these and other problems in the art. BRIEF SUMMARY Provided herein are modified Archaeal family B polymerases and methods of using the same. Provided herein are modified Archaeal family B polymerases derived from the Archaeal microorganism Pyrococcus abyssi. In an aspect, a modified Pyrococcus abyssi polymerase is provided. The polymerase includes an amino acid sequence that is at least 80% identical to a continuous 500 amino acid sequence within SEQ ID NO: 1. The polymerase has exonuclease activity that is reduced at least 80% relative to the exonuclease activity of a polymerase of SEQ ID NO: 1. Provided herein are methods of using modified Archaeal family B polymerases for improved incorporation of modified nucleotides into a nucleic acid sequence. In an aspect, a method of incorporating a modified nucleotide into a nucleic acid is provided. The method includes allowing the following components to interact: (i) a nucleic acid template, (ii) a nucleotide solution, and (iii) a polymerase. The polymerase used in the method includes an amino acid sequence that is at least 80% identical to a continuous 500 amino acid sequence within SEQ ID NO: 1. The polymerase used in the method has exonuclease activity that is reduced at least 80% relative to the exonuclease activity of a polymerase of SEQ ID NO: 1. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a chart showing the relative average halftime, Δt, for a series of mutants, A65, A61, A62, A49, A56, A59, SG1227, A69, SG1251, A70, SG1253, A39, SG1247, SG1210, SG1243, and SG1217 relative to the average half time of incorporation for SG1200 incorporating S-term