Spectroscopic helicase assay

Claim 2

A continuous, kinetic spectroscopic method for detecting helicase activity in a sample, said method comprising the steps of: (a) incubating a mixture comprising a sample, a known initial amount of double-stranded nucleic acid comprising hybridized complementary single-stranded nucleic acid, and a luminescent marker, wherein said marker is bound to said double stranded nucleic acid and present at a concentration whereby said marker is capable of at least 5-fold greater luminescent intensity in the presence of said double-stranded nucleic acid than in the presence of a molar-equivalence of unhybridized said complementary single-stranded nucleic acid, and said concentration is within a concentration range within which the measured activity of one or more control helicases is independent of marker concentration, under conditions under which one or more control helicases would be capable of converting at least a detectable portion of said initial amount of double-stranded nucleic acid into unhybridized said complementary single-stranded nucleic acid; whereby a final amount of said double-stranded nucleic acid is formed; (b) exposing said mixture to light capable of inducing luminescence from said marker; (c) detecting the intensity of said luminescence from said mixture; wherein the intensity of said luminescence from said mixture correlates with said final amount of double-stranded nucleic acid in said mixture and the difference between said initial and said final amount of double-stranded nucleic acid correlates with the helicase activity of said sample.

Claim 3

A continuous, kinetic spectroscopic method for detecting the presence of inhibitors of helicase activity in a sample, said method comprising the steps of: (a) incubating a mixture comprising a sample, a helicase, a known initial amount of double-stranded nucleic acid comprising hybridized complementary single-stranded nucleic acid, and a luminescent marker, wherein said marker is bound to said double stranded nucleic acid and present at a concentration whereby said marker is capable of at least 5-fold greater luminescent intensity in the presence of said double-stranded nucleic acid than in the presence of a molar-equivalence of unhybridized said complementary single-stranded nucleic acid, and said concentration is within a concentration range within which the measured activity of one or more control helicases is independent of marker concentration, under conditions whereby, but for the presence of an inhibitor of said helicase in said sample, said helicase would be capable of converting a control portion of said initial amount of double-stranded nucleic acid into unhybridized said complementary single-stranded nucleic acid, whereby a final amount of said double-stranded nucleic acid is formed; (b) exposing said mixture to light capable of inducing luminescence from said marker; (c) detecting the intensity of said luminescence from said mixture; wherein the intensity of said luminescence from said mixture correlates with said final amount of double-stranded nucleic acid in said mixture and a difference between said initial and said final amount of double-stranded nucleic acid less than said control portion indicates the presence of an inhibitor of helicase activity in said sample.

Claim 4

A continuous, kinetic spectroscopic method for detecting the presence of inhibitors of helicase activity in a sample, said method comprising the steps of: (a) incubating a mixture comprising a sample, a helicase, a known initial amount of double-stranded nucleic acid comprising hybridized complementary single-stranded nucleic acid, and a luminescent marker, wherein said marker is bound to said double stranded nucleic acid and present at a concentration whereby said marker is capable of at least 5-fold greater luminescent intensity in the presence of said double-stranded nucleic acid than in the presence of a molar-equivalence of unhybridized said complementary single-stranded nucleic acid, and said concentration is within a concentration range within which the measured activity of one or more control helicases is independent of marker concentration, under conditions whereby, but for the presence of an inhibitor of said helicase in said sample, said helicase would be capable of converting a control portion of said initial amount of double-stranded nucleic acid into unhybridized said complementary single-stranded nucleic acid, whereby a final amount of said double-stranded nucleic acid is formed; (b) exposing said mixture to light capable of inducing luminescence from said marker; (c) detecting the intensity of said luminescence from said mixture; wherein the intensity of said luminescence from said mixture correlates with said final amount of double-stranded nucleic acid in said mixture and a difference between said initial and said final amount of double-stranded nucleic acid less than said control portion indicates the presence of an inhibitor of helicase activity in said sample.

Claim 5

A method according to claim 1, wherein said at least 5-fold greater luminescent intensity is at least a 10-fold greater luminescent intensity.

Claim 6

A method according to claim 1, wherein said marker is a fluorescent dye.

Claim 7

A method according to claim 1, wherein said marker is a fluorescent dye and said concentration is between 150 and 400 nM.

Claim 8

A method according to claim 1, wherein said marker is Hoechst 33258 (H33258).

Claim 9

A method according to claim 1, wherein said marker is bis-benzimide (DAPI).

Claim 10

A method according to claim 1, wherein said marker is thiazole orange (TO).

Claim 11

A method according to claim 1, wherein said marker is ethidium bromide (EB).

Claim 12

A method according to claim 1, wherein said mixture further comprises a ssDNA specific nuclease to digest unwound strands and prevent renaturation.

Claim 13

A method according to claim 2, wherein said at least 5-fold greater luminescent intensity is at least a 10-fold greater luminescent intensity.

Claim 14

A method according to claim 2, wherein said marker is a fluorescent dye.

Claim 15

A method according to claim 2, wherein said marker is a fluorescent dye and said concentration is between 150 and 400 nM.

Claim 16

A method according to claim 2, wherein said marker is Hoechst 33258 (H33258).

Claim 17

A method according to claim 2, wherein said marker is bis-benzimide (DAPI).

Claim 18

A method according to claim 2, wherein said marker is thiazole orange (TO).

Claim 19

A method according to claim 2, wherein said marker is ethidium bromide (EB).

Claim 20

A method according to claim 2, wherein said mixture further comprises a ssDNA specific nuclease to digest unwound strands and prevent renaturation.