Oligonucleotide-directed misfolding of RNAs

This work describes a general method for inhibiting RNA function, O&barbelow;ligonucleotide D&barbelow;irected Misfolding of R&barbelow;NA (ODMiR). ODMiR uses shorter oligonucleotides to direct the folding of a RNA during transcription by trapping it in an inactive fold. The ODMIR method was demonstrated with the Candida albicans group I intron. Oligonucleotides were designed from secondary structure prediction or screened as complements to consecutive 12-mer regions of the intron's sequence. One oligonucleotide 8-mer designed to bind to a predicted misfolded structure of the intron has an IC₅₀ of 150 nM. This misfolded structure is only 2.2 kcal/mol less favorable than the predicted lowest free energy structure. The 8-mer, L(TACCTTTC), where L represents a Locked Nucleic Acid (LNA) residue, binds to nucleotides 252-259, which are part of the slow folding P3 pseudoknot. The TLCT LACLGALCGLGCL C oligonucleotide inhibited self-splicing the most efficiently, with an IC₅₀ of about 30 nM. This oligonucleotide binds to nucleotides 235–246. Nucleotides 245 and 246 are part of the slower folding P7/P3 region while 240–243 participate in tertiary interactions. Both of these oligonucleotides induce structural rearrangements in P2.1 and P3.; The ODMiR method was extended to Escherichia coli RNase P. The most efficient inhibitor of RNase P function from a screen of oligonucleotides complementary to 12-mer regions of RNase P (M1) RNA was m(CAGCCUACCCGG). This 12-mer inhibits 50% of pre-tRNA processing at about 200 nM and induces structural rearrangements in pseudoknot P4, despite binding elsewhere. It is likely that the best inhibitors of RNA function will disrupt the formation of long-range interactions, such as the pseudoknots in both the C. albicans group I intron and RNase P RNA.; The binding of oligonucleotides to one or both sides of an RNA internal loop was investigated by gel retardation. Surprisingly, the binding affinities of oligonucleotides for the internal loop studied can be predicted well by the Individual Nearest Neighbor-Hydrogen Bond (INN-HB) model. Oligonucleotide complements tethered with non-nucleotide linkers are able to bind tightly to their RNA complement with sequence specificity. This suggests that RNA internal loops can be target with oligonucleotides that are shorter than those currently used to target linear sequence.