title: An efficient colorimetric assay for RNA synthesis by viral RNA-dependent RNA polymerases, using thermostable pyrophosphatase.
abstract: RNA-dependent RNA polymerase (RdRp) is essential for the replication of RNA genome-containing positive-strand RNA viruses. We developed a simple colorimetric assay to quantify the RNA synthesis activity of RdRp by measuring the pyrophosphates released during nascent RNA synthesis. RNA polymerase reaction was quenched by heating at 70 °C for 5 min, during which thermostable inorganic pyrophosphatase converted the accumulated pyrophosphates into inorganic phosphates. Subsequently, the amount of inorganic phosphate was measured using a color-developing reagent. Using RdRp's from hepatitis C virus and foot-and-mouth disease virus, we demonstrate that this colorimetric assay facilitates the measurement of RNA polymerase activity.
authors.affiliation: Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, Republic of Korea.
authors.forename: Hanh Thi Dieu
authors.initials: HT
authors.lastname: Nguyen
authors.forename: Youhoon
authors.initials: Y
authors.lastname: Chong
authors.forename: Deok-Kun
authors.initials: DK
authors.lastname: Oh
authors.forename: Yong-Seok
authors.initials: YS
authors.lastname: Heo
authors.forename: Pham Tan
authors.initials: PT
authors.lastname: Viet
authors.forename: Lin-Woo
authors.initials: LW
authors.lastname: Kang
authors.forename: Sung-Jong
authors.initials: SJ
authors.lastname: Jeon
authors.forename: Dong-Eun
authors.initials: DE
authors.lastname: Kim
mesh_terms: D003124:Colorimetry; D004355:Drug Stability; D011755:Pyrophosphatases / Q000378:metabolism*; D012313:RNA / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D013696:Temperature; D014780:Viruses / Q000201:enzymology*
chemical_list: D012313:RNA; D012324:RNA-Dependent RNA Polymerase; D011755:Pyrophosphatases