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.