guanosine-triphosphate and 4-nitrophenol

We developed a convenient colorimetric assay for monitoring RNA synthesis from DNA-dependent RNA polymerases (DdRp) and viral RNA-dependent RNA polymerases (RdRp). ATP and GTP with a p-nitrophenyl moiety attached to the gamma-phosphate were synthesized (PNP-NTPs). These PNP-NTPs can be used for RNA synthesis by several RNA polymerases, including the RdRps from brome mosaic virus and bovine viral diarrhea virus and the DdRps from bacteriophage T7 and SP6. When the polymerase reactions were performed in the presence of alkaline phosphatase, which digests the p-nitrophenylpyrophosphate side-product of phosphoryl transfer to the chromogenic p-nitrophenylate, an increase in absorbence at 405 nm was observed. These nucleotide analogues were used in continuous colorimetric monitoring of polymerase activity. Furthermore, the PNP-NTPs were found to be stable and utilized by RNA polymerases in the presence of human plasma. This simple colorimetric polymerase assay can be performed in a standard laboratory spectrophotometer and will be useful in screens for inhibitors of viral RNA synthesis.

Topics: Adenosine Triphosphate; Alkaline Phosphatase; Bacteriophage T7; Base Sequence; Bromovirus; Colorimetry; Coloring Agents; Diarrhea Viruses, Bovine Viral; DNA-Directed RNA Polymerases; Drug Evaluation, Preclinical; Guanosine Triphosphate; Hordeum; Humans; Kinetics; Nitrophenols; Plasmids; RNA; RNA-Dependent RNA Polymerase; Spectrophotometry; Substrate Specificity; Templates, Genetic; Viral Proteins; Viruses