FIG 2.

Antisense oligomers direct RNase H cleavage of the s2m element and a conserved single-stranded region (ss3) in vitro. (Top) Design of the six gapmers complementary to the s2m, as well as a nonspecific control gapmer “scr” used in this study (Table 3). The LNA is indicated in orange, s2m-specific phosphorothioate-linked DNA in pink/purple, other phosphorothioate-linked DNA in gray (“scr” and “all”), and phosphodiester-linked DNA in light gray (“some”). RNase H cleavage of the isolated s2m (A and D), 3′ UTR (B and E), the extended 3′ UTR (F), and the predicted single-stranded region ss3 (C). Three target-to-gapmer molar ratios were tested: 1:0.5, 1:1, and 1:2. Incubation of RNA target with RNase H alone does not lead to cleavage (RNase H, last lane) and is not driven by control gapmers with scrambled DNA sequence (“scr”). Incubation of RNA target with gapmer without the addition of RNase H does not lead to degradation either, but it does lead to the appearance of a retarded band that likely corresponds to the target-gapmer duplex.