Enzymes resistant to photodamage

• US 20100093555A1
• Filed: 03/30/2009
• Published: 04/15/2010
• 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: Park, Insil, Clark, Sonya, Christians, Fred, Bjornson, Keith
• Granted Patent: US 9,127,259 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• 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 modified recombinant DNA polymerase, which modified recombinant polymerase comprises at least a first amino acid substitution relative to a parental polymerase, which first substitution replaces a first phenylalanine, tyrosine, tryptophan, histidine, cysteine, or methionine residue in the parental polymerase with a residue less susceptible to oxidation, wherein the first phenylalanine, tyrosine, tryptophan, histidine, cysteine, or methionine residue in the parental polymerase is within 20 Å

of a fluorophore, which fluorophore is linked to a nucleotide or nucleotide analog occupying the polymerase active site of the polymerase.

2. The composition of claim 1, wherein the first amino acid substitution is other than a Y369R, a Y369H, or a Y369E substitution, wherein numbering of amino acid position is relative to that of SEQ ID NO:

1.

3. The composition of claim 1, wherein the modified recombinant polymerase comprises at least a second amino acid substitution relative to the parental polymerase, which second substitution replaces a second phenylalanine, tyrosine, tryptophan, histidine, cysteine, or methionine residue in the parental polymerase with a residue less susceptible to oxidation, wherein the second amino acid substitution is independent of and at a position in the polymerase different from that of the first amino acid substitution.

4. The composition of claim 1, wherein the at least first amino acid substitution mitigates photodamage of the modified recombinant polymerase as compared to the parental polymerase.

5. The composition of claim 1, wherein the first amino acid substitution is a conservative substitution.

6. The composition of claim 1, wherein the modified recombinant polymerase is a modified recombinant Φ

29-type DNA polymerase.

7. The composition of claim 6, wherein the modified recombinant polymerase is a modified recombinant Φ

29, B103, GA-1, PZA, Φ

15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase.

8. The composition of claim 1, wherein the modified recombinant polymerase comprises at least the first substitution at one or more positions selected from the group consisting of:

position 128, position 137, position 230, position 232, position 246, position 248, position 254, position 300, position 315, position 363, position 367, position 369, position 378, position 385, position 454, position 461, position 482, position 483, position 485, position 489, position 494, position 500, position 505, position 506, position 521, and position 526, wherein numbering of positions is relative to SEQ ID NO;

1.

9. The composition of claim 8, wherein the modified recombinant polymerase comprises at least the first substitution selected from the group consisting of:

M246L, F248L, W367S, Y369V, Y482V, W483S, W483F, W483L, W483V, W483I, W483P, W483Q, H485G, H485N, H485K, H485R, H485A, H485E, H485S, H485I, H485P, H485Q, H485T, H485F, H485G, H485L, Y505V, M506L, Y521V, and F526L.

10. The composition of claim 1, wherein the modified recombinant polymerase is a modified recombinant Φ

29 polymerase comprising at least one amino acid substitution or combination of substitutions, wherein the at least one substitution or combination of substitutions is selected from the group consisting of;

M246L, F248L, W367S, Y369V, Y482V, W483S, W483F, W483L, W483V, W483I, W483P, W483Q, H485G, H485N, H485K, H485R, H485A, H485E, H485S, H485I, H485P, H485Q, H485T, H485F, H485G, H485L, Y505V, M506L, Y521V, F526L, M246L and F248L, W367S and Y369V, Y482V and W483S, Y482V and H485G, W483S and H485G, Y505V and M506L, and Y521V and F526L, wherein numbering of positions is relative to SEQ ID NO;

1.

11. The composition of claim 1, wherein the modified recombinant polymerase is a chimeric polymerase, which chimeric polymerase comprises segments derived from any two or more polymerases selected from the group consisting of:

a B103 polymerase, a GA-1 polymerase, a PZA polymerase, a 015 polymerase, a BS32 polymerase, a M2Y polymerase, an Nf polymerase, a G1 polymerase, a Cp-1 polymerase, a PRD1 polymerase, a PZE polymerase, an SF5 polymerase, a Cp-5 polymerase, a Cp-7 polymerase, a PR4 polymerase, a PR5 polymerase, a PR722 polymerase, an L17 polymerase, and/or a F21 polymerase.

12. The composition of claim 11, wherein the chimeric polymerase further comprises an N62D amino acid substitution, wherein numbering of positions is relative to SEQ ID NO:

1.

13. The composition of claim 12, wherein the chimeric polymerase further comprises one or two amino acid substitutions selected from the group consisting of:

W483F, W483L, W483V, W4831, W483P, W483Q, H485N, H485K, H485A, H485E, H485S, H485I, H485P, H485Q, H485T, H485F, H485G, H485L and H485R.

14. The composition of claim 1, wherein the parental polymerase is a wild type or an exonuclease deficient Φ

29 polymerase.

15. The composition of claim 1, wherein the nucleotide analog comprises a phosphate-labeled nucleotide analog.

16. The composition of claim 15, wherein the phosphate-labeled nucleotide analog comprises from 3 to 7 phosphate groups.

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

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

19. The composition of claim 18, wherein the sequencing system comprises a zero mode waveguide, an optical confinement, or an observation volume.

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

21. A composition comprising a modified recombinant polymerase, wherein the modified recombinant polymerase comprises one or two amino acid substitutions selected from the group consisting of:

W483F, W483L, W483V, W483I, W483P, W483Q, H485N, H485K, H485A, H485E, H485S, H485I, H485P, H485Q, H485T, H485F, H485G, H485L and H485R, wherein numbering of positions is relative to SEQ ID NO;

1.

22. A composition comprising a chimeric polymerase, which chimeric polymerase comprises segments derived from any two or more polymerases selected from the group consisting of:

a B103 polymerase, a GA-1 polymerase, a PZA polymerase, a Φ

15 polymerase, a BS32 polymerase, a M2Y polymerase, an Nf polymerase, a G1 polymerase, a Cp-1 polymerase, a PRD1 polymerase, a PZE polymerase, an SF5 polymerase, a Cp-5 polymerase, a Cp-7 polymerase, a PR4 polymerase, a PR5 polymerase, a PR722 polymerase, an L17 polymerase, and/or a F21 polymerase.

23. The composition of claim 22, wherein the chimeric polymerase further comprises an N62D amino acid substitution, wherein numbering of positions is relative to SEQ ID NO:

1.

24. A composition comprising a modified recombinant DNA polymerase, which modified recombinant polymerase comprises one or more amino acid substitutions relative to a parental polymerase, which one or more substitutions independently replace one or more phenylalanine, tyrosine, tryptophan, histidine, cysteine, or methionine residues in the parental polymerase with a residue or residues less susceptible to oxidation, and which amino acid substitutions mitigate photodamage of the modified recombinant polymerase as compared to the parental polymerase.

25. The composition of claim 24, wherein the one or more amino acid substitutions is other than a Y369R, a Y369H, or a Y369E substitution, wherein numbering of amino acid position is relative to that of SEQ ID NO:

1.

26. The composition of claim 24, wherein the one or more amino acid substitutions is within 20 Å

of the polymerase active site of the polymerase.

27. A composition comprising a modified recombinant polymerase, wherein the modified recombinant polymerase comprises an amino acid sequence selected from the group of amino acid sequences set forth in SEQ ID NOs:

2, 7, 8, 9, 10, 11, and 12, and conservative variations thereof.

28. A polynucleotide encoding the modified recombinant polymerase of claim 27.

29. A composition comprising a polynucleotide that comprises a nucleotide sequence selected from the group of nucleotide sequences set forth in:

SEQ ID NOs;

13, 14, 15, 16, 17, 18, and 19.

30. A composition comprising a modified recombinant DNA polymerase, which DNA polymerase comprises:

a first substituted residue at position 483 and a second substituted residue at position 485, wherein the first and second substituted residues are any amino acid other than tryptophan, tyrosine, methionine, histidine and cytosine, wherein numbering of positions is relative to SEQ ID NO;

1;

a first substituted residue at position 494 and a second substituted residue at position 500, wherein the first and second substituted residues are any amino acid other than tryptophan, tyrosine, methionine, histidine and cytosine, wherein numbering of positions is relative to SEQ ID NO;

1;

a first substituted residue at position 137 and a second substituted residue at position 378, wherein the first and second substituted residues are any amino acid other than tryptophan, tyrosine, methionine, histidine and cytosine, wherein numbering of positions is relative to SEQ ID NO;

1;

a first substituted residue at position 230 and a second substituted residue at position 232, wherein the first and second substituted residues are any amino acid other than tryptophan, tyrosine, methionine, histidine and cytosine, wherein numbering of positions is relative to SEQ ID NO;

1;

or,

a first substituted residue at position 300 and a second substituted residue at position 315, wherein the first and second substituted residues are any amino acid other than tryptophan, tyrosine, methionine, histidine and cytosine, wherein numbering of positions is relative to SEQ ID NO;

1.

31-34. -34. (canceled)

35. A composition comprising a modified recombinant DNA polymerase, which DNA polymerase comprises a first substituted residue at position 505, a second substituted residue at position 506, and a third substituted residue at position and 521,

wherein the first substituted residue is selected from the group consisting of:

glycine, isoleucine, leucine, valine, tyrosine, asparagine, glutamine, serine, threonine, lysine, aspartic acid, and glutamic acid;

wherein the second substituted residue is selected from the group consisting of;

alanine, isoleucine, methionine, valine, glutamine, aspartic acid, and leucine; and

,

wherein the third substituted residue is selected from the group consisting of;

alanine, tyrosine, phenylalanine, isoleucine, leucine, and threonine, wherein numbering of amino acid positions is relative to SEQ ID NO;

1.

36. A composition comprising a modified recombinant DNA polymerase, which modified recombinant polymerase comprises two or more amino acid substitutions relative to a parental polymerase, which substitutions independently replace two or more phenylalanine, tyrosine, tryptophan, histidine, cysteine, or methionine residues in the parental polymerase with a residue or residues less susceptible to oxidation.

37-41. -41. (canceled)

42. A method of increasing an enzyme'"'"'s resistance to photodamage, the method comprising:

identifying at least a first amino acid residue in a parental enzyme as a target for mutation;

mutating the enzyme at the first amino acid residue in the parental enzyme with a residue less susceptible to oxidation, thereby producing a modified recombinant enzyme; and

,

determining whether the resulting modified recombinant enzyme displays increased resistance to photodamage as compared to the parental enzyme in a reaction mixture comprising a photosensitizer.

44. The method of claim 42, wherein the amino acid residue at the first position in the parental enzyme is selected from the group consisting of phenylalanine, tyrosine, tryptophan, histidine, cysteine, and methionine.

45. The method of claim 42 or 43, wherein the photosensitizer is a fluorophore.

46. The method of claim 42 or 43, wherein the photosensitizer comprises a fluorescently labeled substrate or a fluorogenic substrate for the enzyme.

47. The method of claim 42, wherein identifying at least the first amino acid residue in the parental enzyme as a target for mutation comprises structurally modeling the parental enzyme.

48. The method of claim 42, wherein identifying the at least the first amino acid residue in the parental enzyme as a target for mutation comprises structurally modeling the parental enzyme with a substrate in its active site and identifying one or more residues that are within a selected distance from the substrate.

49. The method of claim 42 or 43, wherein the parental enzyme and the recombinant modified enzyme are DNA polymerases.

50. The method of claim 49, wherein the parental enzyme and the recombinant modified enzyme are Φ

29-type polymerases.

51. The method of claim 49, wherein the photosensitizer is a fluorescently labeled or fluorogenic nucleotide analog.

52. The method of claim 49 wherein the first amino acid residue is selected from the group consisting of position 128, position 137, position 230, position 232, position 246, position 248, position 254, position 300, position 315, position 363, position 367, position 369, position 378, position 385, position 454, position 461, position 482, position 483, position 485, position 489, position 494, position 500, position 505, position 506, position 521, and position 526, wherein numbering of positions is relative to SEQ ID NO:

1.

53. The method of claim 52, wherein the residues are within 20 Å

of an active site.

54. The method of claim 52, wherein the resulting modified recombinant polymerase comprises at least a first amino acid substitution selected from the group consisting of:

M246L, F248L, W367S, Y369V, Y482V, W483S, W483F, W483L, W483V, W483I, W483P, W483Q, H485G, H485N, H485K, H485R, H485A, H485E, H485S, H485I, H485P, H485Q, H485T, H485F, H485G, H485L, Y505V, M506L, Y521V, and F526L.

55. The method of claim 42, wherein the resulting modified recombinant enzyme is a modified recombinant Φ

29 polymerase comprising at least one amino acid substitution or combination of substitutions selected from the group consisting of;

M246L, F248L, W367S, Y369V, Y482V, W483S, W483F, W483L, W483V, W483I, W483P, W483Q, H485G, H485N, H485K, H485R, H485A, H485E, H485S, H485I, H485P, H485Q, H485T, H485F, H485G, H485L, Y505V, M506L, Y521V, F526L, M246L and F248L, W367S and Y369V, Y482V and W483S, Y482V and H485G, W483S and H485G, Y505V and M506L, and Y521V and F526L.

56. The method of claim 42 or 43, wherein the determining step is performed in a zero-mode waveguide.

57. The method of claim 42, wherein identifying at least the first amino acid residue in a parental enzyme as a target for mutation comprises identifying two or more amino acid residues in the parental enzyme as targets for mutation;

and wherein mutating the enzyme at least the first position comprises mutating the enzyme at the two or more amino acid residues to independently replace the amino acid residues in the parental enzyme with a residue or residues less susceptible to oxidation.

58. The method of claim 42, comprising making a library of modified recombinant enzymes, a plurality of members of the library comprising one or more mutations at one or more positions.

59. The method of claim 58, comprising screening the library to identify at least one member exhibiting increased resistance to photodamage as compared to the parental enzyme in a reaction mixture comprising a photosensitizer.

43. A method of increasing an enzyme'"'"'s resistance to photodamage, the methods comprising:

identifying at least two parental enzymes;

generating a chimeric enzyme from the at least two parental enzymes; and

,

determining whether the chimeric enzyme displays increased resistance to photodamage as compared to the parental enzymes in a reaction mixture comprising a photosensitizer.

60. A composition comprising:

a triplet state quencher,

a fluorophore other than an amino acid residue, and

a polypeptide with which the triplet state quencher and the fluorophore are covalently or noncovalently associated,

wherein the triplet state quencher serves as an acceptor for the excited triplet state of the fluorophore.

61-71. -71. (canceled)

72. A composition comprising:

a triplet state quencher,

a fluorescently labeled or fluorogenic nucleotide analog, and

a modified recombinant DNA polymerase to which the triplet state quencher is covalently or noncovalently bound,

wherein the nucleotide analog serves as a substrate for the polymerase 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.

82. 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 72, wherein the modified 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 modified 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.

84. 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 72, wherein the modified 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.

73-81. -81. (canceled)

83. (canceled)

85-86. -86. (canceled)

87. A method of decreasing photosensitivity, the method comprising:

introducing into a parental polypeptide a binding site for a triplet state quencher, thereby producing a modified polypeptide;

binding the triplet state quencher to the modified polypeptide; and

,

determining whether, in a mixture comprising a fluorophore and the modified polypeptide with bound quencher, the fluorophore and/or the modified polypeptide display increased resistance to photodamage, as compared to the fluorophore and/or the parental polypeptide in a mixture comprising the fluorophore and the parental polypeptide.

88-99. -99. (canceled)

100. A composition comprising a modified recombinant DNA polymerase comprising a first substitution consisting of N62D, a second substitution consisting of T368F, a third substitution consisting of E375Y, a fourth substitution consisting of K512Y, a fifth substitution at position 483 and a sixth substitution at position 485, wherein numbering of positions is relative to SEQ ID NO:

1.