| Assay Method Information | |
| | Inhibition Assay |
| Description: | Measurement of HCV NS5B polymerization activity was performed by evaluating the amount of radiolabeled GTP incorporated by the enzyme in a newly synthesized RNA using heteropolymeric RNA template/primer. The RdRp assay was carried out in 384-well plates using 50 nM of purified NS5B enzyme, which was incubated with 300 nM 5'-biotinylated oligo(rG13)/poly(rC) or oligo (rU15)/poly(rA) primer-template, 600 nM of GTP, and 0.1 μCi of [3H]GTP or [3H]UTP in 25 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 25 mM KCl, 17 mM NaCl and 3 mM of DTT. The 30 μL reaction mixture was incubated at room temperature for 2 h before stopping the reaction by adding 30 μL of streptavidin coated SPA-beads (GE Healthcare, Uppsala, Sweden) in 0.5 M EDTA. The 30 μL reaction was terminated after 2 hours at 25° C. upon addition of 30 μl streptavidin-coated SPA beads (GE Healthcare, Uppsala, Sweden 5 mg/ml in 0.5 M EDTA). After incubation at 25° C. for 30 min, the plate was counted using a Packard TopCount microplate reader (30 sec/well, 1 min count delay) and IC50 values were calculated (Table 1: IC50 1bJ4). |
| Affinity data for this assay | |
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