Directed RNA cleavage with RNase H. (A) Previously reported RNase H cleavage sites (17–19). (B) Synthetic RNA oligonucleotide containing the first 33 nt of an artificial FLuc transcript (synFLuc-AC) and targeting oligos FLucTO-25 designed based on A. Inverted triangles represent RNase H cleavage sites reported in the literature (A) and in this study (B). Deoxynucleotides in TOs are colored in blue; ribonucleotides are colored in green. The predicted loop region of the synthetic RNA oligonucleotides based on an RNA folding algorithm (RNAFold, University of Vienna) is indicated. (C) RNA cleavage using E. coli or Thermus thermophilus RNase H at 37°C. The cleavage efficiency of both enzymes was similar but Tth RNase H generates more uniform cuts than the E. coli enzyme.
