Helicases promote the unwinding of double-stranded nucleic acids and are involved in virus maintenance and DNA replication which makes them a target for antiviral and anticancer drugs. A novel assay allows for screening helicase inhibitors. It uses a double-stranded DNA substrate with one of the strands bound to the fluorophore Cy5 and the quencher lAbRQ at opposite ends. If the double-strand is unwound by a helicase, the labeled strand is released and forms a hairpin. This brings both ends of the strand together and the signal of Cy5 is quenched.
The unwinding capacity of HCV NS3 helicase and the impact of different concentrations of a known inhibitor were analyzed on a BMG LABTECH microplate reader. With increasing inhibitor concentrations the fluorescence value decreased, as it was expected due to quenching of the fluorophore in the hairpin structure. Furthermore, IC50 values of helicase inhibiting compounds were easily determined using the MARS Data Analysis Software.