Inhibition of Escherichia coli RNase P by oligonucleotide directed misfolding of RNA

O ligonucleotide d irected mi sfolding of R NA (ODMiR) uses short oligonucleotides to inhibit RNA function by exploiting the ability of RNA to fold into different structures with similar free energies. It is shown that the 2′- O -methyl oligonucleotide, m(CAGCCUACCCGG), can trap Escherichia coli RNase P RNA (M1 RNA) in a nonfunctional structure in a transcription mixture containing RNase P protein (C5 protein). At about 200 nM, the 12-mer thus inhibits 50% of pre-tRNA processing by RNase P. Roughly 10-fold more 12-mer is required to inhibit RNase P containing full-length, renatured RNase P RNA. Diethyl pyrocarbonate modification in the presence of 12-mer reveals increased modification of sites in and interacting with P4, suggesting a structural rearrangement of a large pseudoknot important for catalytic activity. Thus, the ODMiR method can be applied to RNAs even when folding is facilitated by a cognate protein.