FIGURE 6.

Complexes containing PACT but not TRBP enhance miRNA strand selection. (A) Sequence and thermodynamic analysis of miRNA-1, miRNA-2, and miRNA-3. Thermodynamics were calculated for the four terminal base pairs and 2-nt 3′ overhangs at each end of the duplex. (B) Ago2 alone cleaves target RNAs strand-selectively using miRNA-1. This strand selection is not enhanced by the presence of Dicer and TRBP but is strongly enhanced by the presence of Dicer and PACT. Lanes labeled “G” contain a target corresponding to the guide strand, and lanes labeled “P” contain a target that is complementary to the passenger strand. (C) Quantification of the ratio of target cleavage mediated by the guide strand compared to that mediated by the passenger strand. Data shown are means (±SD) from three separate experimental replicates. (D) Ago2 alone exhibits low levels of strand-selectivity using miRNA-2. Complexes containing Dicer and either TRBP or PACT enhance this strand-selectivity. (E) Quantification of the ratio of target cleavage mediated by the guide strand compared to that mediated by the passenger strand. Data shown are means (±SD) from three separate experimental replicates. (F) Ago2 alone exhibits strong strand-selectivity using miRNA-3. This strand selection is not enhanced by the presence of Dicer and TRBP but is strongly enhanced by the presence of Dicer and PACT. (G) Quantification of the ratio of target cleavage mediated by the guide strand compared to that mediated by the passenger strand. Data shown are means (±SD) from three separate experimental replicates.