Monitoring helicase activity with molecular beacons.
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.
Belon
Craig A
CA
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA.
Frick
David N
DN
D001483:Base Sequence; D002232:Carbocyanines / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D016679:Genome, Viral; D016174:Hepacivirus / Q000201:enzymology / Q000235:genetics; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D002232:Carbocyanines; D004247:DNA; D012313:RNA; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C089187:cyanine dye 3; C085321:cyanine dye 5