We developed a convenient colorimetric assay for monitoring RNA synthesis f
rom DNA-dependent RNA polymerases (DdRp) and viral RNA-dependent RNA polyme
rases (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 al
kaline phosphatase, which digests the p-nitrophenylpyrophosphate side-produ
ct 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. (C) 2000 Academic Press.