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A continuous nonradioactive assay for RNA-dependent RNA polymerase activity
Frederick C Lahser1, Bruce A Malcolm
1Department of Antiviral Therapeutics, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA.
Analytical Biochemistry
|January 31, 2004
Summary
A new continuous, nonradioactive assay measures pyrophosphate release to monitor viral RNA-dependent RNA polymerase (RdRp) activity. This method enables sensitive kinetic studies and inhibitor screening for developing antiviral therapies.
Area of Science:
- Biochemistry
- Virology
- Enzymology
Background:
- Current viral RNA-dependent RNA polymerase (RdRp) assays are end-point measurements using labeled nucleotides.
- Characterizing RdRps is crucial for understanding RNA virus replication and developing antiviral drugs.
Purpose of the Study:
- Develop a continuous, nonradioactive assay to monitor RdRp activity.
- Enable sensitive kinetic studies and inhibitor screening for RdRp enzymes.
Main Methods:
- Adapted a coupled-enzyme assay using chemiluminescent detection of pyrophosphate (PP(i)) release.
- Utilized ATP sulfurylase and firefly luciferase to monitor poliovirus 3D polymerase (3D(pol)) and hepatitis C virus NS5B RdRp reactions.
- Assessed primer concentration, template compatibility, and inhibitor effects.
Main Results:
- Established a light-dependent assay sensitive to RdRp concentration and primer levels.
- Demonstrated suitability for kinetic studies, requiring minimal enzyme (6nM 3D(pol)) for rapid velocity estimation.
- Successfully monitored inhibitor effects (chain terminator and allosteric inhibitor) on NS5B activity.
- Observed time-dependent increases in RdRp activity upon preincubation with RNA template.
Conclusions:
- The developed continuous, nonradioactive assay facilitates detailed kinetic studies of RdRps.
- This assay is valuable for screening RdRp inhibitors and advancing antiviral drug discovery.
- The method is adaptable to various RNA templates and nucleotide analogs.