Labeled nucleoside polyphosphates
First Claim
1. A composition comprising a terminal phosphate labeled nucleoside polyphosphate of formula wherein P=phosphate (PO3) and derivatives thereof, n is 3 or greater;
- Y is an oxygen or sulfur atom;
B is a nitrogen-containing heterocyclic base;
S is an acyclic moiety, a carbocyclic moiety, or sugar moiety;
L is a fluorescent, a fluorogenic, a chemiluminescent, a colored, a chromogenic, a mass tag, or an electrochemical label with or without a linker containing a hydroxyl group, a sulfhydryl group, an amino group or a haloalkyl group suitable for forming a phosphate ester, a thioester, a phosphoramidate or an alkyl phosphonate linkage at the terminal phosphate of a natural or modified nucleotide.
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Accused Products
Abstract
The present invention describes new compositions of matter in the form of labeled nucleoside polyphosphates with four or more phosphates. In addition compositions of nucleoside polyphosphates with four or more phosphates that are substrates for nucleic acid polymerases with enhanced substrate properties and methods of using these nucleoside polyphosphates for nucleic acid detection, characterization and quantification are described. The compositions provided by this invention include nucleoside polyphosphate, dideoxynucleoside polyphosphate, or deoxynucleoside polyphosphate analogues which have colorimetric, chemiluminescent, or fluorescent moieties, mass tags or an electrochemical tags attached to the terminal-phosphate. When a nucleic acid polymerase uses this analogue as a substrate, an enzyme-activatable label would be present on the inorganic polyphosphate by-product of phosphoryl transfer. Removal of the polyphosphate product of phosphoryl transfer via phosphate or polyphosphate transferring enzyme leads to a detectable change in the label attached thereon. When the polymerase assay is performed in the presence of a phosphatase, there is provided a convenient method for real-time monitoring of DNA or RNA synthesis and detection of a target nucleic acid.
208 Citations
31 Claims
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1. A composition comprising a terminal phosphate labeled nucleoside polyphosphate of formula
wherein P=phosphate (PO3) and derivatives thereof, n is 3 or greater; - Y is an oxygen or sulfur atom;
B is a nitrogen-containing heterocyclic base;
S is an acyclic moiety, a carbocyclic moiety, or sugar moiety;
L is a fluorescent, a fluorogenic, a chemiluminescent, a colored, a chromogenic, a mass tag, or an electrochemical label with or without a linker containing a hydroxyl group, a sulfhydryl group, an amino group or a haloalkyl group suitable for forming a phosphate ester, a thioester, a phosphoramidate or an alkyl phosphonate linkage at the terminal phosphate of a natural or modified nucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 26, 27, 28, 29, 30)
- Y is an oxygen or sulfur atom;
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15. A method of detecting the presence of a DNA sequence comprising the steps of:
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a) conducting a DNA polymerase reaction in the presence of a terminal-phosphate-labeled nucleotide containing at least four phosphates, which reaction results in the production of a labeled polyphosphate; b) permitting said labeled polyphosphate to react with a phosphate or polyphosphate transferring enzyme to produce a detectable species; and c) detecting the presence of said detectable species. - View Dependent Claims (16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 31)
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Specification