Monitoring helicase activity with molecular beacons

Craig A. Belon, David N. Frick

Research output: Contribution to journalArticle

47 Scopus citations

Abstract

A high-throughput, fluorescence-based helicase assay using molecular beacons is described. The assay is tested using the NS3 helicase encoded by the hepatitis C virus (HCV) and is shown to accurately monitor helicase action on both DNA and RNA. In the assay, a ssDNA oligonucleotide molecular beacon, featuring a fluorescent moiety attached to one end and a quencher attached to the other, is annealed to a second longer DNA or RNA oligonucleotide. Upon strand separation by a helicase and ATP, the beacon strand forms an intramolecular hairpin that brings the tethered fluorescent and quencher molecules into juxtaposition, quenching fluorescence. Unlike currently available real-time helicase assays, the molecular beacon - based helicase assay is irreversible. As such, it does not require the addition of extra DNA strands to prevent products from re-annealing. Several variants of the new assay are described and experimentally verified using both Cy3 and Cy5 beacons, including one based on a sequence from the HCV genome. The HCV genome - based molecular beacon helicase assay is used to demonstrate how such an assay can be used in high-throughput screens and to analyze HCV helicase inhibitors.

Original languageEnglish (US)
Pages (from-to)433-442
Number of pages10
JournalBioTechniques
Volume45
Issue number4
DOIs
StatePublished - Oct 1 2008
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biotechnology

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