Figure 5. Circular let-7a does not support Argonaute2-catalysed cleavage of mRNA.

(A) Generation of circular let-7a. Let-7a RNA was circularized using T4 RNA ligase and gel purified. Linear let-7a RNA was prepared in an identical fashion and gel purified from a reaction lacking T4 RNA ligase. Linear and circularized ³²P-end-labelled let-7a prior to gel purification. Samples were analysed by 10%polyacrylamide gel under denaturing conditions. (B) Both linear and circular let-7aRNAcan anneal to the mRNA target. Reactions (20 µl) containing 50 mM Tris, pH 7.5, 200 mM NaCl, 1 mM MgCl₂, radiolabelled target mRNA (1 nM) and circular or linear let-7a (0.5, 2.5 or 5 nM) were incubated for 30 min. Samples were analysed by 10% polyacrylamide gel under native conditions. (C) Circular let-7a RNA does not support mRNA cleavage. Reactions (20 µl) containing 50 mM Tris, pH 7.5, 50 mM KCl, 1 mM MgCl₂ and 200 nM GST–Argonaute2 were preincubated for 30 min with linear or circularized let-7a RNA (as indicated). 5′-end-labelled target mRNA (1 nM) was added to each reaction followed by a 15 min incubation. Formamide loading buffer was added and reactions were analysed by 12%polyacrylamide gel run under denaturing conditions. Quantification of the cleavage activity is shown in the right-hand.