A fluorescence-based assay for multisubunit DNA-dependent RNA polymerases.
The properties of DNA-dependent RNA polymerases have been studied since the 1960s, but considerable interest in probing RNA polymerase structure/function relationships, the roles of different classes of RNA polymerases in cellular processes, and the feasibility of using RNA polymerases as drug targets still exists. Historically, RNA polymerase activity has been measured by the incorporation into RNA of radioisotopically labeled nucleotides. We report the development of an assay for RNA polymerase activity that uses the dye RiboGreen to detect transcripts by fluorescence and is thus free of the expense, short shelf life, and high handling costs of radioisotopes. The method is relatively quick and can be performed entirely in microplate format, allowing for the processing of dozens to hundreds of samples in parallel. It should thus be well-suited to use in drug screening and analysis of chromatographic fractions. As RiboGreen fluorescence is enhanced by binding to either RNA or DNA, template DNA must be removed by DNase digestion and ultrafiltration between the transcription and the detection phases of the assay procedure. Although RiboGreen fluorescence is sensitive to changes in solvent environment, solvent exchange in the ultrafiltration step allows comparison of transcription levels even under extremes of salt, pH, etc.
Kuhlman
Peter
P
Department of Chemistry and Biochemistry, Denison University, Granville, OH 43023, USA. kuhlman@denison.edu
Duff
Heidi L
HL
Galant
Ashley
A
D012321:DNA-Directed RNA Polymerases / Q000032:analysis* / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes; D007700:Kinetics; D008722:Methods; D012997:Solvents / Q000494:pharmacology; D014158:Transcription, Genetic
D005456:Fluorescent Dyes; D012997:Solvents; D012321:DNA-Directed RNA Polymerases