A continuous nonradioactive assay for RNA-dependent RNA polymerase activity

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• 作者：Lahser ; Frederick C. ; Malcolm ; Bruce A.
• 关键词：RNA-dependent RNA polymerase ; RdRp ; Coupled-enzyme assay ; Luciferase ; ATP sulfurylase ; Hepatitis C virus ; Poliovirus
• 刊名：Analytical Biochemistry
• 出版年：2004
• 出版时间：February 15, 2004
• 年：2004
• 卷：325
• 期：2
• 页码：247-254
• 全文大小：193 K

文摘

Current assays for the activity of viral RNA-dependent RNA polymerases (RdRps) are inherently end-point measurements, often requiring the use of radiolabeled or chemically modified nucleotides to detect reaction products. In an effort to improve the characterization of polymerases that are essential to the life cycle of RNA viruses and develop antiviral therapies that target these enzymes, a continuous nonradioactive assay was developed to monitor the activity of RdRps by measuring the release of pyrophosphate (PP_i) generated during nascent strand synthesis. A coupled-enzyme assay method based on the chemiluminescent detection of PP_i, using ATP sulfurylase and firefly luciferase, was adapted to monitor poliovirus 3D polymerase (3D^pol) and the hepatitis C virus nonstructural protein 5B (NS5B) RdRp reactions. Light production was dependent on RdRp and sensitive to the concentration of oligonucleotide primer directing RNA synthesis. The assay system was found to be amenable to sensitive kinetic studies of RdRps, requiring only 6nM 3D^pol to obtain a reliable estimate of the initial velocity in as little as 4 min. The assay can immediately accommodate the use of both homopolymer and heteropolymer RNA templates lacking uridylates and can be adapted to RNA templates containing uridine by substituting α-thio ATP for ATP. The low background signal produced by other NTPs can be corrected from no enzyme (RdRp) controls. The effect of RdRp/RNA template preincubation was assessed using NS5B and a homopolymer RNA template and a time-dependent increase of RdRp activity was observed. Progress curves for a chain terminator (3^′-deoxyguanosine 5^′-triphosphate) and an allosteric NS5B inhibitor demonstrated the predicted time- and dose-dependent reductions in signal. This assay should facilitate detailed kinetic studies of RdRps and their potential inhibitors using either standard or single-nucleotide approaches.