Recombinant polymerases with increased phototolerance

• US 9,758,774 B2
• Filed: 09/08/2016
• Issued: 09/12/2017
• Est. Priority Date: 02/01/2012
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Kamtekar, Satwik, Bibillo, Arek, Lee, Walter, Miller, Erik, Park, Insil
• Primary Examiner(s): Noakes, Suzanne M
• Assistant Examiner(s): Lee, Jae W
• Application Number:
  US15/259,471
• Publication Number:
  US 20170067038A1
• Time in Patent Office: 369 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: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions, and/or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties include increased resistance to photodamage, and can also include enhanced metal ion coordination, reduced exonuclease activity, reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fraction, altered cofactor selectivity, increased yield, increased thermostability, increased accuracy, increased speed, increased readlength, and the like. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.

Claims

1. A composition comprising a φ

29-type (phi29-type) recombinant DNA polymerase, which recombinant polymerase comprises an amino acid sequence that is at least 85% identical to SEQ ID NO;

2, and which recombinant polymerase comprises one or more mutation selected from the group consisting of an amino acid substitution at position S191, an amino acid substitution at position F436, an amino acid substitution at position T438, an amino acid substitution at position A444, an amino acid substitution at position S524, an L139R substitution, and a T520H substitution, wherein identification of positions is relative to SEQ ID NO;

2, and wherein said polymerase exhibits polymerase activity.

2. The composition of claim 1, wherein the recombinant polymerase comprises one or more mutation selected from the group consisting of a T438I substitution, a T438L substitution, and an S524K substitution, wherein identification of positions is relative to SEQ ID NO:

2.

3. The composition of claim 1, wherein the recombinant polymerase comprises one or more mutation selected from the group consisting of an S191A substitution, an S191T substitution, an F436E substitution, an F436A substitution, an F436K substitution, a T438G substitution, a T438V substitution, a T438N substitution, a T438A substitution, an A444L substitution, an A444E substitution, an S524E substitution, and an S524N substitution, wherein identification of positions is relative to SEQ ID NO:

2.

4. The composition of claim 1, wherein the recombinant polymerase comprises one or more mutation or combination of mutations selected from the group consisting of an amino acid substitution at position 250, an amino acid substitution at position 372, an amino acid substitution at position 481, an amino acid substitution at position 509, an amino acid substitution at position 145, an amino acid substitution at position 236, an amino acid substitution at position 247, an amino acid substitution at position 434, an amino acid substitution at position 232, an amino acid substitution at position 512, an amino acid substitution at position 138, an amino acid substitution at position 501, an amino acid substitution at position 505, an amino acid substitution at position 510, an amino acid substitution at position 533, an amino acid substitution at position 536, an amino acid substitution at position 202, an amino acid substitution at position 469, an amino acid substitution at position 434 and an amino acid substitution at position 250, an amino acid substitution at position 505 and an amino acid substitution at position 507, an A434G substitution and an L250H substitution, an A434G substitution and an L250C substitution, a V247A substitution and an L250H substitution, an A434G substitution, a D232E substitution, an E512Q substitution, an E512P substitution, an E512K substitution, a V247A substitution, a V2471 substitution, a Y1451 substitution, an E236G substitution, an L138K substitution, an L139K substitution, an E505K substitution, an E505K substitution and a D507S substitution, a T533Q substitution, a K536Q substitution, a K202E substitution, a K202D substitution, a K202A substitution, a K469A substitution, an E372Y substitution, a K509Y substitution, an A481E substitution, an L250A substitution, an L250C substitution, an L250S substitution, an L250H substitution, and a D507K substitution, wherein identification of positions is relative to SEQ ID NO:

2.

5. The composition of claim 1, wherein the recombinant polymerase comprises E372Y, A481E, and K509Y substitutions, wherein identification of positions is relative to SEQ ID NO:

2.

6. The composition of claim 1, where the recombinant polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

2.

7. The composition of claim 1, wherein the recombinant polymerase comprises one or more exogenous features at the C-terminal and/or N-terminal region of the polymerase.

8. The composition of claim 7, wherein the recombinant polymerase comprises a biotin ligase recognition sequence and a polyhistidine tag.

9. The composition of claim 7, wherein the C-terminal region of the recombinant polymerase comprises a His 10 tag.

10. The composition of claim 1, comprising a phosphate-labeled nucleotide analog.

11. The composition of claim 10, wherein the nucleotide analog comprises a fluorophore.

12. The composition of claim 1, comprising a phosphate-labeled nucleotide analog and a DNA template, wherein the recombinant polymerase incorporates the nucleotide analog into a copy nucleic acid in response to the DNA template.

13. The composition of claim 1, wherein the composition is present in a DNA sequencing system.

14. The composition of claim 13, wherein the sequencing system comprises a zero-mode waveguide.

15. The composition of claim 14, wherein the recombinant polymerase is immobilized on a surface of the zero-mode waveguide in an active form.

16. A method of sequencing a DNA template, the method comprising:

a) providing a reaction mixture comprising;

the DNA template,

a replication initiating moiety that complexes with or is integral to the template,

the recombinant polymerase of claim 1, wherein the polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polymerization reaction, and

one or more nucleotides and/or nucleotide analogs;

b) subjecting the reaction mixture to a polymerization reaction in which the modified recombinant polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogs are incorporated into the resulting DNA; and

c) identifying a time sequence of incorporation of the one or more nucleotides and/or nucleotide analogs into the resulting DNA.

17. The method of claim 16, wherein the subjecting and identifying steps are performed in a zero mode waveguide.

18. A method of making a DNA, the method comprising:

(a) providing a reaction mixture comprising;

a template,

a replication initiating moiety that complexes with or is integral to the template,

the recombinant polymerase of claim 1, which polymerase is capable of replicating at least a portion of the template using the moiety in a template-dependent polymerase reaction, and

one or more nucleotides and/or nucleotide analogs; and

(b) reacting the mixture such that the polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides and/or nucleotide analogs are incorporated into the resulting DNA.

19. The method of claim 18, wherein the mixture is reacted in a zero mode waveguide.

20. The method of claim 18, the method comprising detecting incorporation of at least one of the nucleotides and/or nucleotide analogs.