Reagents for monitoring nucleic acid amplification and methods of using same

A method of detecting nucleic acid amplification comprising the steps of:

• (a) contacting a polymerase with a primer, a template, nucleobase triphosphates and one or more molecules of the formula (1) shown below in a medium in which said polymerase is active:
• wherein:
• Nuc represents a nucleobase selected from the group consisting of adenine, cytosine, guanine, thymidine, uracil and analogs thereof;
• N represents a number from 1 through 8 indicating the number of phosphate moieties present in said molecule;
• X1 and X2 are independently selected from the group H, OH, NH2, O-alkyl, N-alkyl, S-Alkyl, P-alkyl, a halogen, an alkyl, a cyclo-alkyl, and an aromatic containing group, wherein the alkyl and cycloalkyl possess varying degrees of unsaturation and are optionally substituted;
• and
• F and Q comprise a pair of indicators, wherein cleavage of the molecule results in separation of the indicators which, in turn, yields a change in a measurable signal;
• and
• (b) detecting a change a measurable signal.

A method of detecting a polymerase comprising the steps of:

• (a) contacting said polymerase with a primer, a template, nucleobase triphosphates and one or more molecules of the formula (1) shown below in a medium in which said polymerase is active:
• wherein:
• Nuc represents a nucleobase selected from the group consisting of adenine, cytosine, guanine, thymidine, uracil and analogs thereof;
• N represents a number from 1 through 8 indicating the number of phosphate moieties present in said molecule;
• X1 and X2 are independently selected from the group H, OH, NH2, O-alkyl, N-alkyl, S-Alkyl, P-alkyl, a halogen, an alkyl, a cyclo-alkyl, and an aromatic containing group, wherein the alkyl and cycloalkyl possess varying degrees of unsaturation and are optionally substituted;
• and
• F and Q comprise a pair of indicators, wherein cleavage of the molecule results in separation of the indicators which, in turn, yields a change in a measurable signal;
• and
• (b) detecting a change said measurable signal.

A method of detecting a phosphodiesterase comprising the steps of:

• (a) contacting one and one or more molecules of the formula (1) shown below with a phosphodiesterase in a medium in which said phosphodiesterase is enzymatically active:
• wherein:
• Nuc represents a nucleobase selected from the group consisting of adenine, cytosine, guanine, thymidine, uracil and analogs thereof;
• N represents a number from 1 through 8 indicating the number of phosphate moieties present in said molecule;
• X1 and X2 are independently selected from the group H, OH, NH2, O-alkyl, N-alkyl, S-Alkyl, P-alkyl, a halogen, an alkyl, a cyclo-alkyl, and an aromatic containing group, wherein the alkyl and cycloalkyl possess varying degrees of unsaturation and are optionally substituted;
• and
• F and Q comprise a pair of indicators, wherein cleavage of the molecule results in separation of the indicators which, in turn, yields a change in a measurable signal;
• and
• (b) detecting a change in said measurable signal.

A method of detecting nucleobase cyclase activity comprising the steps of:

• (a) contacting one or more molecules of the formula (1) shown below with said cyclase in a medium in which said cyclase is enzymatically active
• wherein:
• Nuc represents a nucleobase selected from the group consisting of adenine, cytosine, guanine, thymidine, uracil and analogs thereof;
• N represents a number from 1 through 8 indicating the number of phosphate moieties present in said molecule;
• X1 and X2 are independently selected from the group H, OH, NH2, O-alkyl, N-alkyl, S-Alkyl, P-alkyl, a halogen, an alkyl, a cyclo-alkyl, and an aromatic containing group, wherein the alkyl and cycloalkyl possess varying degrees of unsaturation and are optionally substituted;
• and
• F and Q comprise a pair of indicators, wherein cleavage of the molecule results in separation of the indicators which, in turn, yields a change in a measurable signal;
• and
• (b) detecting a change in said measurable signal.

An assay for detecting the presence of an active viral polymerase in a sample comprising the following components:

• a polymerase with a primer, a template, nucleobase triphosphates and one or more molecules of the formula (1) shown below:
• wherein:
• Nuc represents a nucleobase selected from the group consisting of adenine, cytosine, guanine, thymidine, uracil and analogs thereof;
• N represents a number from 1 through 8 indicating the number of phosphate moieties present in said molecule;
• X1 and X2 are independently selected from the group H, OH, NH2, O-alkyl, N-alkyl, S-Alkyl, P-alkyl, a halogen, an alkyl a cyclo-alkyl, and an aromatic containing group, wherein the alkyl and cycloalkyl possess varying degrees of unsaturation and are optionally substituted;
• and
• F and Q comprise a pair of indicators, wherein cleavage of the molecule results in separation of the indicators which, in turn, yields a change in a measurable signal.

A method of labeling a nucleic acid comprising:

• (a) contacting a polymerase with a primer, a nucleic acid template, nucleoside triphosphates and one or more compounds of the formula (1) shown below in a medium in which said polymerase is active:
• wherein:
• Nuc represents a nucleobase selected from the group consisting of adenine, cytosine, guanine, thymidine, uracil and analogs thereof;
• N represents a number from 1 through 8 indicating the number of phosphate moieties present in said molecule;
• X1 and X2 are independently selected from the group H, OH, NH2, O-alkyl, N-alkyl, S-Alkyl, P-alkyl, a halogen, an alkyl, a cyclo-alkyl, and an aromatic containing group, wherein the alkyl and cycloalkyl possess varying degrees of unsaturation and are optionally substituted;
• and
• F and Q comprise a pair of indicators, wherein cleavage of the molecule results in separation of the indicators which, in turn, yields a change in a measurable signal;
• and
• (b) allowing the one or more compounds of formula (1) to be cleaved and incorporated into a nucleic acid synthesized by said polymerase, thereby resulting in a labeled nucleic acid that emits a measurable signal.