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Direct fluorometric measurement of hepatitis C virus helicase activity
Author(s) -
Boguszewska-Chachulska Anna M.,
Krawczyk Mariusz,
Stankiewicz Anna,
Gozdek Agnieszka,
Haenni Anne-Lise,
Strokovskaya Ludmila
Publication year - 2004
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2004.04.072
Subject(s) - helicase , rna helicase a , chemistry , ns3 , dna , enzyme , förster resonance energy transfer , rna , protease , biochemistry , fluorescence , microbiology and biotechnology , biophysics , biology , physics , gene , quantum mechanics
The non‐structural protein 3 (NS3) of hepatitis C virus (HCV) is a highly promising target for anti‐HCV therapy because of its multiple enzymatic activities, such as RNA‐stimulated nucleoside triphosphatase, RNA helicase and serine protease. The helicase domain of NS3 as well as domain 2 of the helicase were expressed in a baculovirus system to obtain in high yield active proteins for prospective studies of complexes of the helicase with its inhibitors. A novel direct fluorometric test of helicase activity with a quenched DNA substrate, 3 ′ labeled with a Cy3 dye and 5 ′ labeled with a Black Hole Quencher, was developed and optimal reaction conditions established. This test based on fluorescence resonance energy transfer is simple and fast. It allows for direct measurements of enzyme activity, circumventing laborious and complicated radioactive techniques that are poorly reproducible. The results obtained encourage us to propose this new fluorescent assay as a method enabling high throughput screening of anti‐helicase compounds.