Ribonuclease H cleavage assay

The reaction mixture (5 μl) contained 3′-FITC-tRNA^Phe (1 μM), one of the bulge-inducing conjugates (at 40 μM concentration), 50 mM Tris–HCl pH 7.0, 0.2 M KCl and 1 mM EDTA. In parallel, the control experiment was carried out under identical conditions, but with 3′-FITC-tRNA^Phe alone. Considerable excess of the conjugate (BC2, BC3, BC4 or BC5) was used over 3′-FITC-tRNA^Phe (40:1) to ensure efficient hybridization prior to treatment with ribonuclease H. The mixtures were incubated at 37°C for 30 min. RNase H (1U) was added to BC:RNA complexes and incubated at 37°C for 15 min. The reactions were quenched by RNA precipitation with 2% (w/v) lithium perchlorate in acetone (75 μl). RNA pellet was collected by centrifugation and dissolved in loading buffer (8 M urea, 0.025% bromophenol blue, 0.025% xylene cyanol). RNA cleavage products were resolved in 12% polyacrylamide/8 M urea gel electrophoresis using Tris/Borate/EDTA (TBE) as running buffer. To identify cleavage sites, an imidazole ladder and an RNase T1-ladder produced by partial tRNA^Phe cleavage with 2 M imidazole buffer (pH 7.0) and RNase T1, respectively, were run in parallel. The gel was analyzed using ChemiDoc-MP (Bio-Rad).