IP Library Granted Patent US 12,454,683
Granted Patent B2
US 12,454,683 · App. 18/500,785 · Granted Oct 28, 2025

Modified archaeal family B polymerases

Inventors: Souad Naji (San Diego, CA); Carl W. Fuller (New Jersey, NJ); Eli N. Glezer (Del Mar, CA); Andrew Spaventa (San Diego, CA)
Assignee: Singular Genomics Systems, Inc.
C12N9/1252C12P19/34C12Y207/07007
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,454,683
App. No.
18/500,785
Filed
Nov 2, 2023
Granted
Oct 28, 2025
Kind
B2
Art Unit
1656
USPC
435/91.5
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.

Claims (37)

1. A polymerase comprising an amino acid sequence that is at least 85% identical to a continuous 500 amino acid sequence within SEQ ID NO: 1; comprising the following amino acids:

an alanine at amino acid position 409 or at a position corresponding to amino acid position 409;

a glycine at amino acid position 410 or at a position corresponding to amino acid position 410;

an alanine at amino acid position 144 or at a position corresponding to amino acid position 144; and

a serine at amino acid position 515 or at a position corresponding to amino acid position 515.

2. The polymerase of claim 1 , wherein the polymerase comprises an amino acid sequence that is at least 95% identical to a continuous 500 amino acid sequence within SEQ ID NO: 1.

3. The polymerase of claim 1 , wherein the polymerase further comprises an amino acid substitution mutation between amino acid position 129 and 316 of SEQ ID NO: 1, comprising amino acid positions 129 and 316.

4. The polymerase of claim 1 , wherein the polymerase further comprises an alanine at amino acid positions 129, 141, and 143 or at positions corresponding to amino acid positions 129, 141, and 143.

5. The polymerase of claim 1 , wherein the polymerase further comprises an isoleucine at amino acid position 411 or at a position corresponding to amino acid position 411.

6. The polymerase of claim 1 , wherein the polymerase further comprises a valine at amino acid position 486 or at a position corresponding to amino acid position 486.

7. The polymerase of claim 1 , wherein the polymerase further comprises an isoleucine at amino acid position 590 or at a position corresponding to amino acid position 590.

8. The polymerase of claim 1 , wherein the polymerase further comprises a methionine at amino acid position 477 or at a position corresponding to amino acid position 477.

9. The polymerase of claim 1 , wherein the polymerase further comprises a tryptophan at amino acid position 477 or at a position corresponding to amino acid position 477.

10. The polymerase of claim 1 , wherein the polymerase further comprises a glutamic acid at amino acid position 712 or at a position corresponding to amino acid position 712.

11. The polymerase of claim 1 , wherein the polymerase further comprises an alanine at amino acid position 705 or at a position corresponding to amino acid position 705.

12. The polymerase of claim 1 , wherein the polymerase further comprises an alanine at amino acid position 713 or at a position corresponding to amino acid position 713.

13. The polymerase of claim 1 , wherein the polymerase further comprises an alanine at amino acid position 718 or at a position corresponding to amino acid position 718.

14. The polymerase of claim 1 , wherein the polymerase further comprises an asparagine at amino acid position 718 or at a position corresponding to amino acid position 718.

15. The polymerase of claim 1 , wherein the polymerase further comprises an alanine at amino acid position 215 or at a position corresponding to amino acid position 215.

16. The polymerase of claim 1 , wherein the polymerase further comprises an alanine at amino acid position 315 or at a position corresponding to amino acid position 315.

17. The polymerase of claim 1 , wherein the polymerase comprises the following mutations:

a) M129A, D141A, E143A, T144A, L409A, Y410G, P411I, A486V, and T515S;

b) M129A, D141A, T144A, E143A, L409A, Y410G, P411I, A486V, T515S, and T590I;

c) M129A, D141A, T144A, E143A, L409A, Y410G, P411I, A486V, T515S, and K477M;

d) M129A, D141A, T144A, E143A, L409A, Y410G, P411I, A486V, T515S, and K477W;

e) M129A, D141A, T144A, E143A, L409A, Y410G, P411I, A486V, T515S, and G153E;

f) M129A, D141A, T144A, E143A, L409A, Y410G, P411I, A486V, T515S, and K712E;

g) M129A, D141A, E143A, T144A, L409A, Y410G, P411I, A486V, T515S, T590I, and R705A;

h) M129A, D141A, E143A, T144A, L409A, Y410G, P411I, A486V, T515S, T590I, and R713A;

i) M129A, D141A, E143A, T144A, L409A, Y410G, P411I, A486V, T515S, T590I, and E718N; or

j) M129A, D141A, E143A, T144A, L409A, Y410G, P411I, A486V, T515S, T590I, and E718A.

18. The polymerase of claim 1 , wherein the polymerase comprises the following mutations:

a) M129A, D141A, T144A, E143A, L409A, Y410G, P411I, A486V, T515S, and G153E;

b) M129A, D141A, T144A, E143A, L409A, Y410G, P411I, A486V, T515S, and K712E;

c) M129A, D141A, T144A, E143A, L409A, Y410G, P411I, A486V, T515S, and T590I; or

d) M129A, D141A, T144A, E143A, L409A, Y410G, P411I, A486V, T515S, and K477W.

19. The polymerase of claim 1 , wherein the polymerase further comprises an alanine at amino acid positions 141 and 143 or at positions corresponding to amino acid positions 141 and 143.

Assignments (2)
SECURITY INTEREST Recorded Mar 7, 2025
From: SINGULAR GENOMICS SYSTEMS, INC.
To: FIRST-CITIZENS BANK & TRUST COMPANY
Reel/Frame 070440/0465 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 20, 2024
From: FULLER, CARL W.; GLEZER, ELI; SPAVENTA, ANDREW; NAJI, SOUAD
To: SINGULAR GENOMICS SYSTEMS, INC.
Reel/Frame 067468/0883 →
Continuity (4)
Continuation 17369593 · Jul 7, 2021
Continuation 16568089 · Sep 11, 2019
Provisional Application 62729875 · Sep 11, 2018
Related Publication 20240101979A1 · Mar 28, 2024
References Cited (115)
US 5034506A · Summerton et al. · 1991 [cited by applicant]
US 5235033A · Summerton et al. · 1993 [cited by applicant]
US 5756334A · Perler et al. · 1998 [cited by applicant]
US 5763594A · Hiatt et al. · 1998 [cited by applicant]
US 5808045A · Hiatt et al. · 1998 [cited by applicant]
US 5872244A · Hiatt et al. · 1999 [cited by applicant]
US 6232465B1 · Hiatt et al. · 2001 [cited by applicant]
US 6255475B1 · Kwiatkowski · 2001 [cited by applicant]
US 6664079B2 · Ju et al. · 2003 [cited by applicant]
US 7057026B2 · Barnes et al. · 2006 [cited by applicant]
US 7541170B2 · Wang et al. · 2009 [cited by applicant]
US 7541444B2 · Milton et al. · 2009 [cited by applicant]
US 8114973B2 · Siddiqi et al. · 2012 [cited by applicant]
US 8460910B2 · Smith et al. · 2013 [cited by applicant]
US 8852910B2 · Smith et al. · 2014 [cited by applicant]
US 9085762B2 · Hogrefe et al. · 2015 [cited by applicant]
US 9447389B2 · Smith et al. · 2016 [cited by applicant]
US 9765309B2 · Chen et al. · 2017 [cited by applicant]
US 10017750B2 · Smith et al. · 2018 [cited by applicant]
US 10041115B2 · Stupi et al. · 2018 [cited by applicant]
US 10421996B2 · Bomati et al. · 2019 [cited by applicant]
US 10738072B1 · Graham et al. · 2020 [cited by applicant]
US 11034942B1 · Naji et al. · 2021 [cited by applicant]
US 11136565B2 · Naji et al. · 2021 [cited by applicant]
US 11845923B2 · Naji · 2023 [cited by examiner]
US 11851687B2 · Naji et al. · 2023 [cited by applicant]
US 11884943B2 · Naji et al. · 2024 [cited by applicant]
US 11891633B2 · Naji et al. · 2024 [cited by applicant]
US 20030157483A1 · Sorge et al. · 2003 [cited by applicant]
US 20040191825A1 · Wang et al. · 2004 [cited by applicant]
US 20060199214A1 · Jack et al. · 2006 [cited by applicant]
US 20060240439A1 · Smith et al. · 2006 [cited by applicant]
US 20110045489A1 · Gardner et al. · 2011 [cited by applicant]
US 20150140561A1 · Bergmann et al. · 2015 [cited by applicant]
US 20160090579A1 · Bomati et al. · 2016 [cited by applicant]
US 20170130051A1 · Marma et al. · 2017 [cited by applicant]
US 20170298327A1 · Bomati et al. · 2017 [cited by applicant]
US 20180274024A1 · Ju et al. · 2018 [cited by applicant]
US 20190077726A1 · Graham et al. · 2019 [cited by applicant]
US 20200080065A1 · Fuller et al. · 2020 [cited by applicant]
US 20210079364A1 · Naji et al. · 2021 [cited by applicant]
US 20210284975A1 · Naji et al. · 2021 [cited by applicant]
US 20220119779A1 · Naji et al. · 2022 [cited by applicant]
US 20220119780A1 · Naji et al. · 2022 [cited by applicant]
US 20220282230A1 · Naji et al. · 2022 [cited by applicant]
US 20230011240A1 · Fuller et al. · 2023 [cited by applicant]
WO WO1996007669A1 · 1996 [cited by applicant]
WO WO2004018497A2 · 2004 [cited by applicant]
WO WO2005024010A1 · 2005 [cited by applicant]
WO WO2008083393A2 · 2008 [cited by applicant]
WO WO2014142921A1 · 2014 [cited by applicant]
WO WO2015200693A1 · 2015 [cited by applicant]
WO WO2017058953A1 · 2017 [cited by applicant]
WO WO2017205336A1 · 2017 [cited by applicant]
WO WO2018148723A1 · 2018 [cited by applicant]
WO WO2019164977A1 · 2019 [cited by applicant]
WO WO2020056044A1 · 2020 [cited by applicant]
WO WO2021173711A2 · 2021 [cited by applicant]
Yamaji et al. Mammalian adaptive mutations of the PA protein of highly pathogenic Avian H5N1 Influenza virus. Journal of Virology (2015), 89(8): 4117-4125. (Year: 2015). [cited by examiner]
Arezi, B. et al. (Sep. 27, 2002, e-published Sep. 17, 2002). “Efficient and high fidelity incorporation of dyeterminators by a novel archaeal DNA polymerase mutant,” [cited by applicant]
Bentley, D. R. et al. (Nov. 6, 2008). “Accurate whole human genome sequencing using reversible terminator chemistry,” [cited by applicant]
Bergen, K. et al. (Jun. 17, 2013, e-published Jun. 3, 2013). “Structures of KOO and 9°N DNA polymerases complexed with primer template duplex,” [cited by applicant]
Canard, B. et al. (Oct. 11, 1994, e-published Jan. 17, 2003). “DNA polymerase fluorescent substrates with reversible s-taqs,” [cited by applicant]
Canard, B. et al. (Nov. 21, 1995). “Catalytic editing properties of DNA polymerases,” [cited by applicant]
Chen, F. et al. (Feb. 2, 2010, e-published Jan. 11, 2010). “Reconstructed evolutionary adaptive paths give polymerases accepting reversible terminators for sequencing and SNP detection,” [cited by applicant]
Cohen, G. N. et al. (Mar. 5, 2003). “An integrated analysis of the genome of the hyperthermophilic archaeon Pyrococcus abyssi,” [cited by applicant]
Dietrich, J. et al. (Nov. 1, 2002). “PCR performance of the highly thermostable proof-reading B-type DNA polymerase from Pyrococcus abyssi,” [cited by applicant]
Dong, Q. et al. (Nov. 15, 1993). “Mutational studies of human DNA polymerase alpha. Identification of residues critical for deoxynucleotide binding and misinsertion fidelity of DNA synthesis,” [cited by applicant]
Evans, S. J. et al. (Mar. 1, 2000). “Improving dideoxynucleotide-triphosphate utilisation by the hyper-thermophilic DNA polymerase from the archaeon Pyrococcus furiosus,” [cited by applicant]
Extended European Search Report mailed on May 11, 2022, for EP Application No. 19859131.5, 7 pages. [cited by applicant]
Filee, J. et al. (Jun. 2002, e-published Feb. 25, 2014). “Evolution of DNA polymerase families: evidences for multiple qene exchange between cellular and viral proteins,” [cited by applicant]
Fogg, M. J. et al. (Dec. 1, 2002, e-published Nov. 4, 2022). “Structural basis for uracil recognition by archaeal family B DNA polymerases,” [cited by applicant]
Földesi, A. et al. (Apr. 4, 2007). “The fluoride cleavable 2-(cyanoethoxy)methyl (CEM) group as reversible 3′-0-terminator for DNA sequencing-by-synthesis—synthesis, incorporation, and cleavaqe,” [cited by applicant]
Fuller, C. W. et al. (Nov. 2009, e-published Nov. 6, 2009). “The challenges of sequencing by synthesis,” [cited by applicant]
Fuller, C. W. et al. (May 10, 2016, e-published Apr. 18, 2016). “Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array,” [cited by applicant]
Gardner, A. F. et al. (Jun. 1, 1999). “Determinants of nucleotide sugar recognition in an archaeon DNA polymerase,” [cited by applicant]
Gardner, A. F. et al. (Jan. 15, 2002). “Acyclic and dideoxy terminator preferences denote divergent sugar recognition by archaeon and Taq DNA polymerases,” [cited by applicant]
Gueguen, Y. et al. (Nov. 2001, e-published Dec. 20, 2001). “Characterization of two DNA polymerases from the hyperthermophilic euryarchaeon Pyrococcus abyssi,” [cited by applicant]
Guo, J. et al. (Jul. 8, 2008, e-published Jun. 30, 2008). “Four-color DNA sequencing with 3′-0-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides,” [cited by applicant]
Guo, J. et al. (Apr. 20, 2010, e-published Feb. 3, 2010). “An integrated system for DNA sequencing by synthesis using novel nucleotide analoques,” [cited by applicant]
Horhota, A. et al. (May 25, 2005, e-published Apr. 28, 2005). “Kinetic analysis of an efficient DNA-dependent TNA polymerase,” [cited by applicant]
Hutter, D. et al. (Dec. 1, 2010). “Labeled nucleoside triphosphates with reversibly terrninating aminoalkoxyl groups,” [cited by applicant]
International Search Report and Written Opinion mailed on Jan. 28, 2020 for PCT Application No. PCT/US2019/50678, filed Sep. 11, 2019, 16 pages. [cited by applicant]
Ishino, S. et al. (Aug. 29, 2014). “DNA polymerases as useful reagents for biotechnology—the history of developmental research in the field,” [cited by applicant]
Ju, J. et al. (Dec. 26, 2006, e-published Dec. 14, 2006). “Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators,” [cited by applicant]
Killelea, T. et al. (Jun. 14, 2011, e-published May 20, 2011). “Role of Disulfide Bridges in Archaeal Family-B DNA Polymerases,” [cited by applicant]
Kim, T. S. et al. (Jan. 4, 2010, e-published Dec. 23, 2009). “Novel 3′-0-fluorescently modified nucleotides for reversible termination of DNA synthesis,” [cited by applicant]
Kisselev, L. (Jan. 17, 2002). “Polypeptide release factors in prokaryotes and eukaryotes: same function, different structure,” [cited by applicant]
Knapp, D. C. et al. (Mar. 1, 2011, e-published Feb. 3, 2011). “Fluoride-Cleavable, Fluorescently Labelled Reversible Terminators: Synthesis and Use in Primer Extension,” [cited by applicant]
Kumar, S. et al. (2012, e-published Sep. 21, 2012). “PEG-labeled nucleotides and nanopore detection for single molecule DNA sequencing by synthesis,” [cited by applicant]
Lasken, R. S. et al. (Jul. 26, 1996). “Archaebacterial DNA polymerases tightly bind uracilcontaining DNA,” [cited by applicant]
Liu, H. et al. (Dec. 15, 2000). “Identification of conserved residues contributing to the activities of adenovirus DNA polymerase,” [cited by applicant]
Metzker, M. L. et al. (Oct. 11, 1994). “Termination of DNA synthesis by novel 3′modified-deoxyribonucleoside 5′-triphosphates,” [cited by applicant]
Needleman, S. B. et al. (Mar. 28, 1970, e-published Oct. 28, 2004). “A general method applicable to the search for similarities in the amino acid sequence of two proteins,” [cited by applicant]
Nikiforov, T. T. (May 15, 2011, e-published Feb. 26, 2011). “Fluorogenic polymerase, endonuclease, and ligase assays based on DNA substrates labeled with a single fluorophore,” [cited by applicant]
Nikiforov, T. T. (Sep. 15, 2014, e-published Jun. 5, 2014). “Generic assay format for endo- and exonucleases based on fluorogenic substrates labeled with single fluorophores,” [cited by applicant]
Nørholm, M. H. “A mutant Pfu DNA polymerase designed for advanced uracil-excision DNA engineering,” [cited by applicant]
Pavlov, Y. I. et al. (Sep. 1, 2001.) “In vivo consequences of putative active site mutations in yeast DNA polymerases α, ε, δ, and ζ,” [cited by applicant]
Pearson, W. R. et al. (Apr. 1, 1988). “Improved tools for biological sequence comparison,” [cited by applicant]
Rosenblum, B. B. et al. (Nov. 1, 1997). “New dye-labeled terminators for improved DNA sequencing patterns,” [cited by applicant]
Ruparel, H. et al. (Apr. 13, 2005). “Design and synthesis of a 3′-0-allyl photocleavable fluorescent nucleotide as a reversible terminator for DNA sequencing by synthesis,” [cited by applicant]
Seo, T. S. et al. (Jan. 1, 2003, e-published Dec. 21, 2002). “Click chemistry to construct fluorescent oligonucleotides for DNA sequencing,” [cited by applicant]
Smith T. F. et al. (Dec. 1981). “Comparison biosequences,” [cited by applicant]
Southworth, M. W. et al. (May 28, 1996). “Cloning of thermostable DNA polymerases from hyperthermophilic marine Archaea with emphasis on [cited by applicant]
Tabor, S. et al. (Apr. 15, 1989, e-published Sep. 3, 2004). “Selective inactivation of the exonuclease activity of bacteriophage T7 DNA polymerase by in vitro rnutaqenesis,” [cited by applicant]
Truniger, V. et al. (Jan. 1, 2004, e-published Jan. 16, 2004). “Function of the C-terminus of phi29 DNA polymerase in DNA and terminal protein bindlnq,” [cited by applicant]
Uniprot Accession No. E2D778 (Nov. 2010). Located at https://www.uniprot.org/uniprotkb/E2D778/entry. Last accessed Jan. 20, 2023. [cited by applicant]
Welch, M. B. et al. (Mar. 1, 1999, e-published Feb. 24, 1999). “Synthesis of Nucleosides Designed for Combinatorial DNA Sequencing,” [cited by applicant]
Welch, M. B. et al. (Feb. 1999, e-published Oct. 4, 2006). “Synthesis of fluorescent, photolabile 3′-0-protected nucleoside triphosphates for the base addition sequencing scheme,” [cited by applicant]
Whisstock, J. C. et al. (Aug. 2003, e-published Jan. 26, 2004). “Prediction of protein function from protein sequence and structure,” [cited by applicant]
Witkowski, A. et al. (Sep. 1, 1999, e-published Aug. 18, 1999). “Conversion of a beta-ketoacyl synthase to a malonyl decarboxylase by replacement of the active-site cysteine with glutamine,” [cited by applicant]
Wu, J. et al. (Oct. 16, 2007, e-published Oct. 8, 2007). “3′-0-modified nucleotides as reversible terminators for pvrosequencinq,” [cited by applicant]
Yang, G. et al. (Aug. 1, 2002, e-published Jul. 16, 2002). “A conserved Tyr residue is required for sugar selectivity in a Pol α DNA polymerase,” [cited by applicant]
Zhu, Z. et al. (Aug. 25, 1994). “Directly labeled DNA probes using fluorescent nucleotides with different length linkers,” [cited by applicant]
Guo, H. H. et al. (Jun. 14, 2004). “Protein tolerance to random amino acid change,” [cited by applicant]
Cited By (1)
US 12,668,834