Enzymes resistant to photodamage
First Claim
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.
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Accused Products
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.
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Citations
100 Claims
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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.
- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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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.
- 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;
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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. - View Dependent Claims (23)
- a B103 polymerase, a GA-1 polymerase, a PZA polymerase, a Φ
- 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.
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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.
- View Dependent Claims (28)
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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.
- SEQ ID NOs;
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30. A composition comprising a modified recombinant DNA polymerase, which DNA polymerase comprises:
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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.
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31-34. -34. (canceled)
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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.
- glycine, isoleucine, leucine, valine, tyrosine, asparagine, glutamine, serine, threonine, lysine, aspartic acid, and glutamic acid;
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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.
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37-41. -41. (canceled)
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42. A method of increasing an enzyme'"'"'s resistance to photodamage, the method comprising:
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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. - View Dependent Claims (44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59)
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43. A method of increasing an enzyme'"'"'s resistance to photodamage, the methods comprising:
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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.
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60. A composition comprising:
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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.
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61-71. -71. (canceled)
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72. A composition comprising:
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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. - View Dependent Claims (82, 84)
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73-81. -81. (canceled)
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83. (canceled)
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85-86. -86. (canceled)
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87. A method of decreasing photosensitivity, the method comprising:
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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.
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88-99. -99. (canceled)
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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.
Specification