Figure 5.

Competition assays. (A and B) dsRNA competes with and inhibits the RNase III-specific cleavage of HCV RNA. Cleavage by RNase III of S₁ transcript labeled at (A) 5′ end; (B) 3′ end, in the presence of increasing concentrations of competitor dsRNA. (A) Lanes 1 and 2 correspond to 5′ end-labeled RNA alone (incubated on ice) and incubated with buffer, respectively; lane 3, standard reaction with RNase III; lanes 2–9, cleavage reactions using a constant concentration of S₁ (10 ng) and increasing concentrations of dsRNA (20, 40, 60, 80, 100 and 120 ng, respectively). (B) Identical to (A) but using 3′ end-labeled S₁. (C) Reverse competition of RNase III cleavage of T7 R1.1 RNA with HCV RNA. A constant amount of a 60 nt fragment of T7 R1.1 RNA (internally labeled) was cleaved in the presence of increasing concentrations of HCV RNA S₁ without label. Lane 1 and 2, RNA T7 R1.1 alone or incubated with buffer, respectively; lane 3, standard RNase reaction, but with final concentration of RNase III of 0.0001 U/µl; lane 4, standard reaction with RNase III (0.0005 U/µl); lanes 5–9, RNase III cleavage of a constant concentration of T7 R1.1 RNA (1.8 nM) and increasing concentrations of S₁ RNA (0.45, 0.9, 1.8, 3.6 and 7.2 nM respectively); lane 9, inhibition of T7 R1.1 RNA cleavage with 300 ng of Penicillium chrysogenum dsRNA (positive control).