Figure 1.

Target RNA is cleaved endonucleolytically producing 5′-phosphate and 3′-hydroxyl termini. (A) Preparation of site-specifically labeled substrates and cleavage assay. 5′-³²P-labeled and 3′ aminolinker (L) protected 12-nt oligoribonucleotide was ligated to nonphosphorylated 9-nt oligoribonucleotide using T4 RNA ligase. An aliquot of the ligation product was further 5′-³²P-labeled using T4 polynucleotide kinase. The purified substrates were incubated with affinity-purified RISC programmed with single-stranded guide siRNA. (B) PhosphorImaging of cleavage reactions incubated for 2 h at 30°C, and resolved on a 20% denaturing polyacrylamide gel. 5′-³²P-labeled 9- and 12-nt oligoribonucleotides were loaded as marker in lanes 1 and 2, respectively. The cleavage reactions with single- and double-labeled 21-nt substrate are loaded in lanes 4 and 5, respectively. Lane 3 contains the 12-nt cleavage product isolated from a prior cleavage reaction. (C) Two-dimensional thin layer chromatography analysis of the ribonuclease T2-digested RISC-cleavage product. The oval depicts the unlabeled pAp as detected by UV shadowing. The radioactive signal comigrates with the 5′ ³²pAp released by ribonuclease T2 digestion from the gel-purified 12-nt cleavage product.