Polymerases engineered to reduce nucleotide-independent DNA binding

• US 10,597,643 B2
• Filed: 01/09/2018
• Issued: 03/24/2020
• Est. Priority Date: 04/29/2016
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Omniome, Inc.
• Inventors: Iyidogan, Pinar, Wallen, Mark C., Liu, Ying L., Vijayan, Kandaswamy
• Primary Examiner(s): Ekstrom, Richard C
• Application Number:
  US15/866,353
• Publication Number:
  US 20180155698A1
• Time in Patent Office: 805 Days
• Field of Search: None
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase

Abstract

Provided are engineered DNA polymerases exhibiting modified functionality, and polynucleotides encoding same. Modified features include: (1) reduced catalytic activity in the presence of magnesium ions and/or (2) reduced affinity for primed template nucleic acid molecules in the absence of cognate nucleotide, and an ability to discriminate between cognate and non-cognate nucleotides under low salt conditions. Sequencing By Binding™ procedures employing the engineered polymerases have certain advantages. The engineered polymerases can have other uses as well.

Claims

1. An engineered DNA polymerase, comprising a variant of the sequence of SEQ ID NO:

3, said variant being at least 80% identical to SEQ ID NO;

3 and comprising an amino acid substitution mutation at one or more of positions K250, Q281, D355, Q425, and D532.

2. The engineered DNA polymerase of claim 1, wherein the variant is at least 90% identical to SEQ ID NO:

3.

3. The engineered DNA polymerase of claim 2, wherein the variant is at least 95% identical to SEQ ID NO:

3.

4. The engineered DNA polymerase of claim 3, wherein the variant is at least 98% identical to SEQ ID NO:

3.

5. The engineered DNA polymerase of claim 2, further comprising the sequence of SEQ ID NO:

5 joined to the amino terminus thereof.

6. The engineered DNA polymerase of claim 2, further comprising the sequence of SEQ ID NO:

6 joined to the amino terminus thereof.

7. The engineered DNA polymerase of claim 1,

wherein the substitution mutation at position K250 comprises a mutation to a polar amino acid,

wherein the substitution mutation at position Q281 comprises a mutation to an acidic amino acid,

wherein the substitution mutation at position D355 comprises a mutation to a different acidic amino acid,

wherein the substitution mutation at position Q425 comprises a mutation to a different polar amino acid, and

wherein the substitution mutation at position D532 comprises a mutation to a different acidic amino acid.

8. The engineered DNA polymerase of claim 7,

wherein the substitution mutation at position K250 comprises a mutation to Cys,

wherein the substitution mutation at position Q281 comprises a mutation to Glu,

wherein the substitution mutation at position D355 comprises a mutation to Glu,

wherein the substitution mutation at position Q425 comprises a mutation to Cys, and

wherein the substitution mutation at position D532 comprises a mutation to Glu.

9. The engineered DNA polymerase of claim 1, wherein said variant comprises replacement of up to 10 amino acids of SEQ ID NO:

3.

10. The engineered DNA polymerase of claim 9, wherein said variant comprises replacement of up to 5 amino acids of SEQ ID NO:

3.

11. The engineered DNA polymerase of claim 1, wherein said variant is present in a ternary complex that further includes a primed template nucleic acid and a cognate nucleotide or analog thereof.

12. The engineered DNA polymerase of claim 11, wherein the cognate nucleotide or analog thereof comprises an exogenous fluorescent label.

13. The engineered DNA polymerase of claim 1, wherein the at least one amino acid substitution mutation is a substitution mutation at position Q281 that replaces Gln (Q) with Glu (E).

14. The engineered DNA polymerase of claim 1, wherein the at least one amino acid substitution mutation is a substitution mutation at position K250 that replaces Lys (K) with Cys (C), and a substitution mutation at position Q425 that replaces Gln (Q) with Cys (C).

15. The engineered DNA polymerase of claim 1, wherein the at least one amino acid substitution mutation is a substitution mutation at position Q281 that replaces Gln (Q) with Glu (E), a substitution mutation at position K250 that replaces Lys (K) with Cys (C), and a substitution mutation at position Q425 that replaces Gln (Q) with Cys (C).

16. The engineered DNA polymerase of claim 1, wherein the at least one amino acid substitution mutation is a substitution mutation at position D355 that replaces Asp (D) with Glu (E), and a substitution mutation at position Q281 that replaces Gln (Q) with Glu (E).

17. The engineered DNA polymerase of claim 1, wherein the at least one amino acid substitution mutation is a substitution mutation at position D355 that replaces Asp (D) with Glu (E), a substitution mutation at position K250 that replaces Lys (K) with Cys (C), and a substitution mutation at position Q425 that replaces Gln (Q) with Cys (C).

18. The engineered DNA polymerase of claim 1, wherein the at least one amino acid substitution mutation is a substitution mutation at position D355 that replaces Asp (D) with Glu (E), a substitution mutation at position Q281 that replaces Gln (Q) with Glu (E), a substitution mutation at position K250 that replaces Lys (K) with Cys (C), and a substitution mutation at position Q425 that replaces Gln (Q) with Cys (C).

19. The engineered DNA polymerase of claim 1, further comprising an exogenous label covalently joined thereto.

20. The engineered DNA polymerase of claim 19, wherein the exogenous label comprises a fluorescent label.

21. The engineered DNA polymerase of claim 1, wherein the engineered DNA polymerase comprises Mg²⁺-dependent phosphodiester bond forming activity.

22. The engineered DNA polymerase of claim 1,

wherein the differential affinity of the engineered DNA polymerase for a primed template nucleic acid in the presence and absence of a cognate nucleotide is greater than the differential affinity of the DNA polymerase of SEQ ID NO:

4 for the primed template nucleic acid in the presence and absence of cognate nucleotide.

23. An isolated mutant DNA polymerase comprising a variant of the sequence of SEQ ID NO:

2, said variant being at least 80% identical to SEQ ID NO;

2 and wherein the variant comprises Glu (E) at position 290.

31. The isolated mutant DNA polymerase of claim 23, bound to a primed template nucleic acid molecule in combination with a nucleotide that is a next correct nucleotide for the primed template nucleic acid molecule.

32. The isolated mutant DNA polymerase of claim 23, which binds to a blocked primed template nucleic acid molecule in combination with a nucleotide that is a next correct nucleotide for the blocked primed template nucleic acid molecule.

24. An isolated mutant DNA polymerase comprising a variant of the sequence of SEQ ID NO:

2, said variant being at least 80% identical to SEQ ID NO;

2 and wherein the variant comprises Cys (C) at position 259, and Cys (C) at position 434.

33. The isolated mutant DNA polymerase of claim 24, bound to a primed template nucleic acid molecule in combination with a nucleotide that is a next correct nucleotide for the primed template nucleic acid molecule.

34. The isolated mutant DNA polymerase of claim 24, which binds to a blocked primed template nucleic acid molecule in combination with a nucleotide that is a next correct nucleotide for the blocked primed template nucleic acid molecule.

25. An isolated mutant DNA polymerase comprising a variant of the sequence of SEQ ID NO:

2, said variant being at least 80% identical to SEQ ID NO;

2 and wherein the variant comprises Glu (E) at position 290, Cys (C) at position 259, and Cys (C) at position 434.

35. The isolated mutant DNA polymerase of claim 25, bound to a primed template nucleic acid molecule in combination with a nucleotide that is a next correct nucleotide for the primed template nucleic acid molecule.

36. The isolated mutant DNA polymerase of claim 25, which binds to a blocked primed template nucleic acid molecule in combination with a nucleotide that is a next correct nucleotide for the blocked primed template nucleic acid molecule.

26. An isolated mutant DNA polymerase comprising a variant of the sequence of SEQ ID NO:

2, said variant being at least 80% identical to SEQ ID NO;

2 and wherein the variant comprises Glu (E) at position 364, and further comprises Glu (E) at position 290.

37. The isolated mutant DNA polymerase of claim 26, bound to a primed template nucleic acid molecule in combination with a nucleotide that is a next correct nucleotide for the primed template nucleic acid molecule.

38. The isolated mutant DNA polymerase of claim 26, which binds to a blocked primed template nucleic acid molecule in combination with a nucleotide that is a next correct nucleotide for the blocked primed template nucleic acid molecule.

27. An isolated mutant DNA polymerase comprising a variant of the sequence of SEQ ID NO:

2, said variant being at least 80% identical to SEQ ID NO;

2 and wherein the variant comprises Glu (E) at position 364, and further comprises Cys (C) at position 259 and Cys (C) at position 434.

39. The isolated mutant DNA polymerase of claim 27, bound to a primed template nucleic acid molecule in combination with a nucleotide that is a next correct nucleotide for the primed template nucleic acid molecule.

40. The isolated mutant DNA polymerase of claim 27, which binds to a blocked primed template nucleic acid molecule in combination with a nucleotide that is a next correct nucleotide for the blocked primed template nucleic acid molecule.

28. An isolated mutant DNA polymerase comprising a variant of the sequence of SEQ ID NO:

2, said variant being at least 80% identical to SEQ ID NO;

2 and wherein the variant comprises Glu (E) at position 364, and further comprises Glu (E) at position 290, Cys (C) at position 259, and Cys (C) at position 434.

41. The isolated mutant DNA polymerase of claim 28, bound to a primed template nucleic acid molecule in combination with a nucleotide that is a next correct nucleotide for the primed template nucleic acid molecule.

42. The isolated mutant DNA polymerase of claim 28, which binds to a blocked primed template nucleic acid molecule in combination with a nucleotide that is a next correct nucleotide for the blocked primed template nucleic acid molecule.

29. A reaction mixture, comprising:

a DNA polymerase selected from the group consisting of,

(i) an engineered DNA polymerase that comprises a variant of the sequence of SEQ ID NO;

3, said variant being at least 80% identical to SEQ ID NO;

3 and comprising an amino acid substitution mutation at one or more of positions K250, Q281, D355, Q425, and D532,

(ii) an engineered DNA polymerase that comprises a variant of the sequence of SEQ ID NO;

2, said variant being at least 80% identical to SEQ ID NO;

2 and wherein the variant comprises Glu (E) at position 290,

(iii) an engineered DNA polymerase that comprises a variant of the sequence of SEQ ID NO;

2, said variant being at least 80% identical to SEQ ID NO;

2 and wherein the variant comprises Cys (C) at position 259, and Cys (C) at position 434, and

(iv) an engineered DNA polymerase that comprises a variant of the sequence of SEQ ID NO;

2, said variant being at least 80% identical to SEQ ID NO;

2 and wherein the variant comprises Glu (E) at position 290, Cys (C) at position 259, and Cys (C) at position 434;

a primed template nucleic acid molecule, optionally comprising a reversible terminator nucleotide at a 3′

-end thereof; and

at least one nucleotide.

30. A kit for identifying the cognate nucleotide for a primed template nucleic acid molecule, comprising:

a DNA polymerase selected from the group consisting of,

(i) an engineered DNA polymerase that comprises a variant of the sequence of SEQ ID NO;

3, said variant being at least 80% identical to SEQ ID NO;

3 and comprising an amino acid substitution mutation at one or more of positions K250, Q281, D355, Q425, and D532,

(ii) an engineered DNA polymerase that comprises a variant of the sequence of SEQ ID NO;

2, said variant being at least 80% identical to SEQ ID NO;

2 and wherein the variant comprises Glu (E) at position 290,

(iii) an engineered DNA polymerase that comprises a variant of the sequence of SEQ ID NO;

2, said variant being at least 80% identical to SEQ ID NO;

2 and wherein the variant comprises Cys (C) at position 259, and Cys (C) at position 434, and

(iv) an engineered DNA polymerase that comprises a variant of the sequence of SEQ ID NO;

2, said variant being at least 80% identical to SEQ ID NO;

2 and wherein the variant comprises Glu (E) at position 290, Cys (C) at position 259, and Cys (C) at position 434;

a plurality of nucleotides or analogs thereof; and

a plurality of reversible terminator nucleotides.