ENGINEERED POLYMERASES FOR IMPROVED SEQUENCING

• US 20200087638A1
• Filed: 09/11/2019
• Published: 03/19/2020
• Est. Priority Date: 09/17/2018
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Omniome, Inc.
• Inventors: IYIDOGAN, Pinar
• Granted Patent: US 10,731,141 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Y 207/07049 RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase

Abstract

Provided are nucleic acids encoding engineered polymerases comprising at least one modification in a motif A and/or at least one modification in a motif B of the polymerase and engineered polymerases encoded by the nucleic acids. Also provided are engineered DNA polymerases comprising a variant of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3, the variant being at least 80% identical to SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 and comprising an amino acid substitution at one or more positions selected from the group consisting of L408, Y409, P410, R484, A/L485, and I486. Methods, vectors, kits, and compositions comprising the nucleic acids and compositions, methods and kits comprising the engineered polymerases are also provided.

Claims

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

1, the variant being at least 95% identical to SEQ ID NO;

1 and comprising an amino acid substitution at L408, Y409 and P410.

4. The engineered polymerase of claim 1, wherein the modifications are selected from the group consisting of L408F, Y409F, Y409H, P410T, P410V.

5. The engineered polymerase of claim 1, wherein the polymerase comprises modifications L408F/Y409F/P410T or L408F/Y409H/P410V.

6. The engineered polymerase of claim 1, wherein the polymerase further comprises modifications at amino acid positions R484 and A/L485, R484 and I486, A/L485 and I486, or R484, A/L485 and I486.

7. The engineered polymerase of claim 1, wherein the polymerase further comprises a modification at amino acid position D141 and/or E143.

8. The engineered polymerase of claim 7, wherein the modifications are D141A and E143A.

9. The engineered polymerase of claim 1, wherein the polymerase is a B-type family polymerase.

10. The engineered polymerase of claim 1, wherein the polymerase has increased accuracy in pairing nucleotides to template bases as compared to a control polymerase.

11. The engineered polymerase of claim 1, wherein the polymerase has increased stability as compared to a control polymerase.

12. The engineered polymerase of claim 1, wherein the polymerase has improved polymerization kinetic rates as compared to a control polymerase.

13. The engineered polymerase of claim 1, wherein the polymerase has decreased polymerization error rates as compared to a control polymerase.

14. The engineered polymerase of claim 13, wherein the control polymerase has the amino acid sequence of SEQ ID NO:

1.

15. The engineered polymerase of claim 13, wherein the control polymerase has the amino acid sequence of SEQ ID NO:

2 or 3.

16. The engineered polymerase of claim 1, wherein the polymerase has an average error rate of less than 0.75 at 100 cycles of a sequencing by binding process.

17. The engineered polymerase of claim 1, wherein the polymerase has an average error rate of less than 8 at 150 cycles of a sequencing by binding process.

18. The engineered polymerase of claim 1, wherein the polymerase has an average error rate of between 1 and 5 at 150 cycles of a sequencing by binding process.

19. A method of identifying a base in a primed template nucleic acid, comprising

(a) contacting a primer-template nucleic acid hybrid with the engineered polymerase of claim 1 and a test nucleotide, thereby forming a ternary complex;

(b) detecting the complex; and

(c) identifying the next base of the primed template nucleic acid from the detected complex.

20. The method of claim 19, wherein detection occurs while precluding incorporation of the test nucleotide into the primer.

21. The method of claim 19, wherein the contacting occurs under conditions that stabilize the ternary complex formed between the polymerase, primer-template nucleic acid hybrid and test nucleotide when the test nucleotide is complementary to the next base of the primed template nucleic acid.

22. The method of claim 19, wherein the detecting comprises detecting a change in refractive index, a light scattering signal or a detectable tag.

23. The method of claim 19, wherein the nucleotide, polymerase or both comprise exogenous labels.

24. The method of claim 19, wherein the method further comprises an incorporation step comprising incorporating into the primer a nucleotide that is complementary to the next base.

25. The method of claim 24, wherein the incorporation step comprises using the same type of polymerase that catalyzes the incorporation.

26. A method of sequencing a template nucleic acid molecule, the method comprising providing a template nucleic acid molecule primed with a primer and contacting the primed template nucleic acid molecule with the engineered polymerase of claim 1 and at least one nucleotide molecule and identifying the nucleotide molecule, thereby sequencing the template nucleic acid molecule.

2. An engineered DNA polymerase comprising a variant of SEQ ID NO:

2, the variant being at least 95% identical to SEQ ID NO;

2 and comprising an amino acid substitution at L408, Y409, and P410.

3. An engineered DNA polymerase comprising a variant of SEQ ID NO:

3, the variant being at least 95% identical to SEQ ID NO;

3 and comprising an amino acid substitution at L408, Y409, and P410.