Fig. 1.

N17 random library selection of accessible sites in KRT_366–1067 and KRT_967–1466 transcripts. An N17-RNase-H library selection was performed and the results analyzed on sequencing gels (panel A). Lanes show: 0 = control (no RNase-H); X = experimental; A = limited hydrolysis with RNase-U2, which cleaves at A residues; G = limited hydrolysis with RNase-T1, which cleaves at G residues; and H = limited base hydrolysis, which cleaves randomly at all residues. In the KRT_366–1067 selection (left panel in A), 12 “regions” (R1–12) were identified. Regions are shown as encompassing 17 nt, but the accessible regions may extend in either direction. Regions 1–8 were identified on sequencing gels run for 1.5 h (shown in A); four additional regions, R9–12, were identified on sequencing gels run for 7.5 h (not shown). ASO targeted to the R1–8 regions were then synthesized and tested in specific ASO–RNase-H cleavage reactions. Another N17-RNase-H library selection was performed on KRT_967–1466 (right panel of A), a transcript which also contained the 3′-untranslated region, and seven regions were identified (R1–7). Specific ASO 17mers were synthesized which corresponded to these regions, and tested under the same selection procedure (panel B). Surprisingly, only five of the seven ASOs produced good RNase-H cleavage activity (the ASO targeting R1 and R7 showed very weak activities). When we re-performed and re-examined sequencing gels from the RNase-H-based selections, we found that RNase-H was actually initiating hydrolysis 5–9 nt from the 3′-end of the ASO regions. We re-synthesized ASO targeting regions R1 and R7 which were correspondingly shifted by six nucleotides (designated as Δ1 and Δ7), as well as shifted version for the other regions. When RNase-H protocols were re-performed with the Δ1 and Δ7 ASOs, cleavage was substantially increased for both regions (panel B), yielding results equivalent to those obtain for the other regions. Panel C shows RNAse-H-based cleavage results with three ASOs to regions which did not show any activity in library selection protocols (N1–N3), as well as cleavage of three ASOs matching particular sequences to which three miRs aligned within region 7 (miR-205, miR-150, and miR-211, designated as 7₁–7₃). ASOs targeting the library-selected R6 region is shown for comparison.