Fig. 2.

Ligand binding to the HCV IRES. (A) Docking of the benzimidazole 1 at the C58-G110 base pair in the crystal structure of the HCV subdomain IIa target complex in comparison with the geometry of a C-G○G triple. (B) Titrations of Cy3/Cy5-labeled HCV subdomain IIa RNA with benzimidazole 1 and guanine 3. Curves show normalized FRET signal for 1 (●) and guanine (▼), as well as the normalized fluorescence signals of the donor Cy3 (■) and acceptor Cy5 (♦) for guanine. Fitting of a single-site binding curve to the benzimidazole 1 FRET response gave an EC₅₀ value for ligand binding of 3.4 ± 0.3 μM. Because the FRET signal did not reach saturation in the guanine titration, affinity was estimated by fitting to the Cy3 emission, which resulted in an EC₅₀ value for ligand binding of 1,050 ± 69 μM (guanine). (C) Effect of guanine on IRES-driven translation, as measured in an in vitro translation assay. Error bars in B and C represent ±1 SD calculated from triplicate experiments.