Fig. 3.

MicroInspector-based alignment of miRs for the MGB_1–502 transcript and analysis of RNase-H-based cleavage. Using the web-based MicroInspector tool, the miR database was aligned to MGB_1–502 and compared with the N17-RNase-H-based library selection protocol (panel A shows library selection results, which identified eight accessible regions, designated as R1–8, indicated to the left). Lanes 0, X, A, G, and H were as described in the legend of Figure 1. Panel B shows alignment results using the MicroInspector tool; library-selected regions R1–8 are shown in blue at the top. As is evident, most of the identified miRs (>80% of 36 miRs) overlapped (≥5 nt) the library-selected accessible sites. Also of interest is the fact that no miRs overlap R4and especially R5 regions, so not all accessible regions are necessarily targeted. ASOs targeting the identified accessible sites all showed good activity in RNase-H-based assays (panel C). Three specific miRs (circled) targeting R2, R3, or R6 were selected based on their match scores, and 17mer ASOs were synthesized which matched a portion of the region the miRs targeted (designated as S1–3; panel B). Panel D shows the sequence and target alignment of ASO S1. The ASO corresponding to R6 identified by library selection was synthesized and tested using the RNase-H-based protocol, as were the S1–S3 ASOs (panel E). Results showed that all of the S1–S3 ASOs, which aligned with accessible sites in the target RNA, produced RNase-H-based cleavage equivalent to the library-selected R6 region. Randomly choosing ASOs not in library-selected sites nearly always results in little or no activity (data not shown). Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]