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. © 2009 Humana Press, a part of Springer Science+Business Media, LLC.
CITATION STYLE
Krawczyk, M., Stankiewicz-Drogoń, A., Haenni, A. L., & Boguszewska-Chachulska, A. (2010). Fluorometric assay of hepatitis C virus NS3 helicase activity. Methods in Molecular Biology, 587, 211–221. https://doi.org/10.1007/978-1-60327-355-8_15
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