An efficient colorimetric assay for RNA synthesis by viral RNA-dependent RNA polymerases, using thermostable pyrophosphatase

作者：Hanh Thi Dieu Nguyen; Chong Youhoon; Oh Deok Kun; Heo Yong Seok; Pham Tan Viet; Kang Lin Woo; Jeon Sung Jong; Kim Dong Eun^*

来源：Analytical Biochemistry, 2013, 434(2): 284-286.

DOI：10.1016/j.ab.2012.11.019

• Colorimetric assay
• Inorganic pyrophosphatase
• RNA-dependent RNA polymerase
• RNA synthesis

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 degrees 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%26apos;s from hepatitis C virus and foot-and-mouth disease virus, we demonstrate that this colorimetric assay facilitates the measurement of RNA polymerase activity.

• 出版日期2013-3-15