Fluorometric assay of hepatitis C virus NS3 helicase activity.
The development of techniques based on fluorescence has made it possible to create new types of assays that represent an advantageous alternative to old tests relying on radioactivity. Such a novel approach has been applied to develop a high-throughput assay to measure the helicase activity of the hepatitis C virus (HCV) NS3 protein and the inhibitory potential of several classes of compounds. The NS3 helicase is one of the most promising targets of anti-HCV-oriented screening of compounds due to the urgent need for more effective and tolerable drugs. The 96- or 384-well microplate assay that we developed is based on the use of a quenched double-stranded DNA substrate labeled with a fluorophore (Cy3 or FAM) and with a Black Hole Quencher 1 or 2. It allows for direct (real-time) measurements of substrate unwinding and inhibition of unwinding by anti-helicase compounds. After a few modifications of buffers and assay conditions this method can be applied to various variants of HCV helicase and other proteins with helicase activities.
Krawczyk
Mariusz
M
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
Stankiewicz-Drogoń
Anna
A
Haenni
Anne-Lise
AL
Boguszewska-Chachulska
Anna
A
D001681:Biological Assay / Q000295:instrumentation / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005470:Fluorometry / Q000295:instrumentation / Q000379:methods*; D016679:Genome, Viral; D009690:Nucleic Acid Conformation; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D004247:DNA; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases