Figure 7.

Effect of bulges on RNA cleavage. (A) Schematic representation of guide DNA and target RNAs. (B) TtAgo was pre-incubated with 5′ phosphorylated guide DNA at 55°C for 30 min prior to addition of 5′ radiolabeled RNA substrates, followed by incubation at 75°C for indicated times. Products were resolved on a 15% denaturing polyacrylamide gel. Red arrowheads indicate the cleavage site. The sequence of let-7 is shown on the left to annotate the hydrolysis and cleavage positions. The bulge positions are indicated according to structural studies and inserted nucleotides are shown in blue. H, alkaline hydrolysis ladder of 5′ labeled target RNA. Hydrolysis yields terminal 2′,3′-cyclic phosphate, which further hydrolyze to 2′- and 3′-monophosphate. These distinctly charged products resolve towards the bottom of the gel into double bands and are grouped by brackets. TtAgo cleavage products carry a 3′-OH and therefore migrate slower than the RNA hydrolysis products of the same sequence.