Selection of targeting oligos for uniform RNase H cleavage. (A) In general, a surrogate RNA oligonucleotide containing the first 30–50 nt of the target transcript is chemically synthesized with a 5′ FAM group. A series of targeting oligos (TOs) are designed and chemically synthesized. The TOs used in the selection exercise did not require a desthiobiotin group, because unlike the long 3′ cleavage products of in vitro transcripts, the 3′ cleavage products of the surrogate RNA were short and did not interfere with LC-MS analysis. After RNase H cleavage, the fluorescently labeled 5′ cleavage fragments can be analyzed by urea PAGE and LC-MS intact mass analysis. (B) For consistency, the phosphodiester bonds are numbered around the nucleotide hybridized to the 5′ deoxynucleotide of the TO (top panel; a cytidine in this case). Urea PAGE showed that RNase H cleavage is most efficient with FLucTO-25, FLucTO-26, and FLucTO-27. Multiple cleavage products were observed with FLucTO-24, FLucTO-25, and FLucTO-26 (middle panel). LC-MS intact mass analysis of the cleavage products is shown in the lower panel. In the schematics, phosphodiester bonds are shown as “-”. Deoxynucleotides in the TOs are shown as gray bars; ribonucleotides are shown as yellow bars. Numbers represent frequency of cleavage detected at corresponding phosphodiester bonds obtained in triplicated experiments.
