Figure 4.

Cleavage of 5’-[³²P]-labeled miR-21 by miR-21-miRNase and/or RNase H. Autoradiographs of 18% polyacrylamide/8 M urea gel, showing the patterns of cleavage of miR-21 at its 2- (A), 5- (C), and 10-fold (E) molar excess over miR-21-miRNase or h-ODN. Autoradiographs I, IV, and VII represent cleavage of miR-21 by miR-21-miRNase alone, whereas autoradiographs II, V, and VIII show cleavage of miR-21 by a mixture of miR-21-miRNase and RNase H. Autoradiographs III, VI, and IX demonstrate cleavage of the complex formed between miR-21 and h-ODN by RNase H alone (100 U/ml). Duplexes formed by 5′-[³²P]-miR-21 (10, 25, and 50 µM) and h-ODN or miRNase (5 µM) were incubated at 37°C for 24–72 h. Lanes Im and T1: imidazole ladder and partial RNA digestion with RNase T1, respectively; “control”: RNA was incubated in the absence of oligonucleotide or conjugate and in the presence of RNase H (100 U/ml). Diagrams (B), (D), and (F) show time dependency of cleavage of miR-21 by miR-21-miRNase alone, by a synergetic action of miR-21-miRNase and RNase H, or by RNase H (100 U/ml) alone when miR-21 was in a complex with h-ODN at 2-, 5- and 10-fold molar excess of miR-21 over miR-21-miRNase or h-ODN, respectively. (G) Positions of miR-21 cleavage induced by miR-21-miRNase (red arrows), RNase H in the duplex with h-ODN (black arrows), and by combination of miR-21-miRNase and RNase H. (H) The hypothetical representation of miR-21 cleavage by a combination of miRNase and RNase H.