Efficient Colorimetric Assay of RNA Polymerase Activity Using Inorganic Pyrophosphatase and Ammonium Molybdate

3Therefore, continuous hydrolysis of pyrophosphate by pyrophophatases can enhance the yield of RNA or DNA synthesis during the nucleic acid polymerization. We have set out the experiments to monitor the activity of nucleic acids polymerase using the PPase, which can be applicable to a high-throughput screening of antiviral drugs against various viral RNA polymerases. Currently, activity assay of RNA polymerases is mainly based on the direct measurement of RNA polymerase’s incorporation of radioactive ribonucleotides in the synthesizing nucleic acids. Such radioactive assays cannot be easily adapted to high throughput screening. In our studies, the amounts of inorganic phosphate are to be quantitatively measured with color-developing reagent after the RNA polymerase reaction, which is generated from hydrolysis of pyrophosphates by the PPase. This colorimetric coupledenzyme system will facilitate measurement of RNA polymerase activity without need of cumbersome radioactivity assay.