Figure 6.

Coumestans inhibit NS5B-RNA binary complex formation. Binding of NS5B to RNA as a function of individual coumestan derivative was evaluated by UV-mediated cross-linking. NS5BCΔ21 (1.5 μg) was incubated with 30 nM [5′-³²P] labeled rA₂₀/U₁₂ template/primer (200K Cerenkov c.p.m.) in a standard irradiation mixture in the absence or presence of increasing concentrations of the indicated coumestan compound and exposed to UV radiation. The cross-linked species were resolved by SDS-PAGE and visualized on a phosphorimager. The extent of NS5B-RNA cross-linked species formed was quantified using ImageQuant software (Molecular Dynamics). Lane 1 in each set represents the control reaction carried out in the absence of the inhibitor. Lanes 2–9 represent indicated compounds at concentrations of 1, 5, 10, 15, 30, 50, 100 and 200 μM, respectively.