(A) RISC loading and assays using pre-let-7a, let-7a guide, or let-7a duplex RNA are schematically shown. The let-7a-5p target RNA (39 nt) was 5’ 32P-labeled. Ago2 slicing of the target RNA guided by let-7a-5p would produce a 20-nt product (highlighted in red). (B) Pre-let-7a dicing-coupled RISC loading assay: 10 nM Ago2 used. (C) RISC loading of let-7a guide RNA: 5 nM Ago2 used. (D) RISC loading of let-7a duplex RNA: 5 nM Ago2 used. (B, C, D) Sliced target RNA products were fractionated by 15% Urea-PAGE (left panel). A quantitative summary is also shown (right panel). The target RNA slicing efficiency was estimated by the ratio of the radioactivity of the correctly cleaved band over that of the uncut control band. Significant differences were identified by Student’s t-tests: *, p<0.05; **, p<0.01; ***, p<0.001. Error bars, S.E. (n = 3).