Real-time Monitoring of RNA Helicase Activity Using Fluorescence Resonance Energy Transfer in Vitro

Biochem Biophys Res Commun. 2010 Feb 26;393(1):131-6. doi: 10.1016/j.bbrc.2010.01.100. Epub 2010 Feb 1.

Abstract

We have developed a continuous fluorescence assay based on fluorescence resonance energy transfer (FRET) for the monitoring of RNA helicase activity in vitro. The assay is tested using the hepatitis C virus (HCV) NS3 helicase as a model. We prepared a double-stranded RNA (dsRNA) substrate with a 5' fluorophore-labeled strand hybridized to a 3' quencher-labeled strand. When the dsRNA is unwound by helicase, the fluorescence of the fluorophore is emitted following the separation of the strands. Unlike in conventional gel-based assays, this new assay eliminates the complex and time-consuming steps, and can be used to simply measure the real-time kinetics in a single helicase reaction. Our results demonstrate that Alexa Fluor 488 and BHQ1 are an effective fluorophore-quencher pair, and this assay is suitable for the quantitative measurement of the RNA helicase activity of HCV NS3. Moreover, we found that several extracts of marine organisms exhibited different inhibitory effects on the RNA and DNA helicase activities of HCV NS3. We propose that this assay will be useful for monitoring the detailed kinetics of RNA unwinding mechanisms and screening RNA helicase inhibitors at high throughput.

MeSH terms

  • Antiviral Agents / chemistry
  • Antiviral Agents / isolation & purification*
  • Antiviral Agents / pharmacology
  • Drug Evaluation, Preclinical
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / isolation & purification
  • Enzyme Inhibitors / pharmacology
  • Fluorescence Resonance Energy Transfer / methods*
  • Hepacivirus / enzymology*
  • RNA Helicases / antagonists & inhibitors*
  • RNA Helicases / chemistry
  • RNA Helicases / metabolism
  • RNA, Double-Stranded / chemistry
  • RNA, Double-Stranded / metabolism
  • Substrate Specificity
  • Time Factors
  • Tissue Extracts / chemistry
  • Tissue Extracts / pharmacology
  • Viral Nonstructural Proteins / antagonists & inhibitors*
  • Viral Nonstructural Proteins / chemistry
  • Viral Nonstructural Proteins / metabolism

Substances

  • Antiviral Agents
  • Enzyme Inhibitors
  • NS3 protein, hepatitis C virus
  • RNA, Double-Stranded
  • Tissue Extracts
  • Viral Nonstructural Proteins
  • RNA Helicases