Enzymes resistant to photodamage

• US 9,650,672 B2
• Filed: 08/05/2015
• Issued: 05/16/2017
• Est. Priority Date: 03/31/2008
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Bjornson, Keith
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US14/818,728
• Publication Number:
  US 20160017413A1
• Time in Patent Office: 650 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: 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2535/113 Cycle sequencing

Abstract

Provided are compositions comprising modified DNA polymerases that exhibit improved photostability compared to the parental polymerases from which they were derived. Provided are methods for generating enzymes, such as DNA polymerases, with the aforementioned phenotype. Provided are methods of using polymerases with increased resistance to photodamage to make a DNA or to sequence a DNA template.

Claims

1. A composition comprising:

a triplet state quencher, wherein the triplet state quencher is a trivalent lanthanide ion,

a fluorescently labeled or fluorogenic nucleotide analog, and

a recombinant DNA polymerase to which the triplet state quencher is noncovalently bound, wherein the recombinant polymerase comprises an amino acid sequence that is at least 90% identical to SEQ ID NO;

1 or SEQ ID NO;

21,

wherein the nucleotide analog serves as a substrate for the polymerase, wherein the nucleotide analog does not comprise the triplet state quencher, and wherein the triplet state quencher serves as an acceptor for the excited triplet state of the fluorescently labeled analog or of the fluorogenic analog'"'"'s product.

2. The composition of claim 1 wherein the recombinant polymerase comprises one or more mutations relative to a parental polymerase that increase the affinity of the recombinant polymerase'"'"'s exonuclease domain for the trivalent lanthanide ion relative to the parental polymerase while maintaining the polymerase'"'"'s polymerase activity.

3. The composition of claim 1, wherein the lanthanide ion is Eu³⁺ or Tb³⁺.

4. The composition of claim 2, wherein the affinity of the recombinant polymerase'"'"'s exonuclease domain for the trivalent lanthanide ion is increased by at least 100-fold.

5. The composition of claim 1 wherein the recombinant polymerase comprises an EF-hand motif to which the trivalent lanthanide ion binds.

6. The composition of claim 1, wherein the recombinant polymerase comprises a first moiety to which is covalently attached a second moiety that noncovalently binds the triplet state quencher.

7. The composition of claim 6, wherein the first moiety comprises a cysteine residue and the second moiety comprises a chelator to which the trivalent lanthanide ion binds.

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

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

10. The composition of claim 9, wherein the sequencing system comprises a zero mode waveguide.

11. The composition of claim 1, wherein the recombinant polymerase is immobilized on a surface.

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

optionally one or more nucleotides and/or additional nucleotide analogs;

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

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

13. The method of claim 12, wherein the subjecting and identifying steps are performed in a zero mode waveguide.

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

optionally one or more nucleotides and/or additional 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 nucleotides and/or nucleotide analogs are incorporated into the resulting DNA.

15. The method of claim 14, wherein the mixture is reacted in a zero mode waveguide.

16. The method of claim 14, the method comprising detecting incorporation of the fluorescently labeled or fluorogenic nucleotide analog.