Ago2 can cleave preannealed siRNA–mRNA duplexes in vitro. (A) Efficiency of preannealing. In vitro transcribed, radiolabeled firefly mRNA with one perfect CXCR4 site (FL1P) was heated with (+) or without (−) the antisense strand of CXCR4 and allowed to anneal by cooling to room temperature. FL1P mRNA was resolved on 8% native urea-PAGE gel. Preannealed CXCR4–FL1P is indicated by an arrow. (B) Ago2 can cleave preannealed siRNA–mRNA duplexes. Recombinant wild-type Ago1–4 or mutant Ago1 (Ago1-DDH) and Ago2 (Ago2-DDR) with swapped RNase III catalytic triads were incubated either with the CXCR4 (dsCXCR4) containing radiolabeled passenger/sense (sCXCR4) strand or with radiolabeled FL1P with (+) or without (−) preannealed CXCR4 (PA asCXCR4). The products were resolved on 8% denaturing urea-PAGE gels. Cleavage products are indicated by arrows. (C) Efficiency of covalent BrdU-mediated UV crosslinking. In vitro transcribed, radiolabeled FL1P mRNA was incubated with (+) or without (−) BrdU-modified antisense strand of the CXCR4 (CL asCXCR4) and crosslinked by UV exposure. FL1P mRNA was resolved on 8% denaturing urea-PAGE gel. Crosslinked CXCR4–FL1P is indicated by an arrow. (D) Ago2 and Ago1-DDH can cleave crosslinked FL1P–CXCR4 duplexes in vitro. Recombinant wild-type Ago1–4 or mutant Ago1-DDH and Ago2-DDR were incubated either with radiolabeled FL1P crosslinked to CXCR4 (CL asCXCR4) or with radiolabeled FL1P and CXCR4 (+asCXCR4). The products were resolved on 8% denaturing urea-PAGE gels. Cleavage of the crosslinked FL1P produced specific cleavage product of >48 nt (arrow on the left) due to covalent crosslink between the 3′ end CXCR4 and the cleavage product, while cleavage of the uncrosslinked FL1P produced specific cleavage product of 48 nt as expected (arrow on the right). Partial RNaseT1 digestion of FL1P was used as the ladder.
