Figure 4.

Analysis of TA RNA duplex formation by RNA band-shift assay and in vitro processing by RNase III. (A) 5′-radiolabeled RCd9 RNA was incubated with increasing concentrations of CD2907.1 under two different conditions referred as N (Native) and F (Full RNA duplex) conditions, respectively. Native RCd9:CD2907.1 complexes were formed at 37°C for 5 min in TMN buffer, and full duplexes were obtained after a denaturation-annealing treatment in TE Buffer (2 min 90°C, 30 min 37°C). The complexes were immediately loaded on native polyacrylamide gels to control for hybridization efficiency or submitted to in vitro processing by RNase III (B). RNase III digestion of free or complexed RCd9 was performed at 37°C in TMN buffer containing 1 μg tRNA from 1 min to 15 min with 0.05 units of RNase III per sample. Samples were analyzed on 8% polyacrylamide-Urea gels.