Monitoring helicase activity with molecular beacons

Craig A. Belon, David N. Frick

Research output: Contribution to journalArticle

45 Citations (Scopus)

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

Fingerprint

Hepacivirus
Assays
Monitoring
Viruses
Oligonucleotides
DNA
Fluorescence
Genome
RNA
Genes
Adenosine Triphosphate
Throughput
Quenching
Annealing
Molecules

ASJC Scopus subject areas

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

Cite this

Monitoring helicase activity with molecular beacons. / Belon, Craig A.; Frick, David N.

In: BioTechniques, Vol. 45, No. 4, 01.10.2008, p. 433-442.

Research output: Contribution to journalArticle

Belon, Craig A. ; Frick, David N. / Monitoring helicase activity with molecular beacons. In: BioTechniques. 2008 ; Vol. 45, No. 4. pp. 433-442.
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