Exploiting polymerase promiscuity: A simple colorimetric RNA polymerase assay.
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.
Vassiliou
W
W
Department of Biology, Indiana University, Bloomington, Indiana 47405, USA.
Epp
J B
JB
Wang
B B
BB
Del Vecchio
A M
AM
Widlanski
T
T
Kao
C C
CC
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000378:metabolism; D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D017795:Bromovirus / Q000201:enzymology; D003124:Colorimetry / Q000379:methods*; D004396:Coloring Agents; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D001908:Diarrhea Viruses, Bovine Viral / Q000201:enzymology; D004353:Drug Evaluation, Preclinical / Q000379:methods; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000097:blood / Q000378:metabolism; D001467:Hordeum / Q000821:virology; D006801:Humans; D007700:Kinetics; D009596:Nitrophenols / Q000097:blood / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D012313:RNA / Q000096:biosynthesis / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D013053:Spectrophotometry; D013379:Substrate Specificity; D013698:Templates, Genetic; D014764:Viral Proteins; D014780:Viruses / Q000201:enzymology*
D004396:Coloring Agents; D009596:Nitrophenols; D014764:Viral Proteins; D012313:RNA; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; C070852:RNA polymerase SP6; C062210:bacteriophage T7 RNA polymerase; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases; D000469:Alkaline Phosphatase; C024836:4-nitrophenol