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Fluorometric Assay of Hepatitis C Virus NS3 Helicase Activity

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Helicases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 587))

Abstract

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.

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Acknowledgment

This work was supported by the Ministry of Science and Higher Education grant 2P05A 038 29.

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Authors and Affiliations

  1. Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland

    Mariusz Krawczyk & Anna Stankiewicz-DrogoĊ„

  2. Institut Jacques Monod, Paris, France

    Anne-Lise Haenni

  3. Genomed, Warsaw, Poland

    Anna Boguszewska-Chachulska

Authors
  1. Mariusz Krawczyk
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  2. Anna Stankiewicz-DrogoĊ„
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  3. Anne-Lise Haenni
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  4. Anna Boguszewska-Chachulska
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Corresponding author

Correspondence to Mariusz Krawczyk .

Editor information

Editors and Affiliations

  1. Dept. Paediatric Laboratory Medicine, Hospital for Sick Children, University Ave. 555, Toronto, M5G 1X8, Ontario, Canada

    Mohamed M. Abdelhaleem

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Krawczyk, M., Stankiewicz-DrogoĊ„, A., Haenni, AL., Boguszewska-Chachulska, A. (2009). Fluorometric Assay of Hepatitis C Virus NS3 Helicase Activity. In: Abdelhaleem, M. (eds) Helicases. Methods in Molecular Biology, vol 587. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-355-8_15

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  • DOI: https://doi.org/10.1007/978-1-60327-355-8_15

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-354-1

  • Online ISBN: 978-1-60327-355-8

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