Post-transcriptional Modifications Modulate rRNA Structure and Ligand Interactions.

Post-transcriptional modifications play important roles in modulating the functions of RNA species. The presence of modifications in RNA may directly alter its interactions with binding partners or cause structural changes that indirectly affect ligand recognition. Given the rapidly growing list of modifications identified in noncoding and mRNAs associated with human disease, as well as the dynamic control over modifications involved in various physiological processes, it is imperative to understand RNA structural modulation by these modifications. Among the RNA species, rRNAs provide numerous examples of modification types located in differing sequence and structural contexts. In addition, the modified rRNA motifs participate in a wide variety of ligand interactions, including those with RNA, protein, and small molecules. In fact, several classes of antibiotics exert their effects on protein synthesis by binding to functionally important and highly modified regions of the rRNAs. These RNA regions often display conservation in sequence, secondary structure, tertiary interactions, and modifications, trademarks of ideal drug-targeting sites. Furthermore, ligand interactions with such regions often favor certain modification-induced conformational states of the RNA. Our laboratory has employed a combination of biophysical methods such as nuclear magnetic resonance spectroscopy (NMR), circular dichroism, and UV melting to study rRNA modifications in functionally important motifs, including helix 31 (h31) and helix h44 (h44) of the small subunit rRNA and helix 69 (H69) of the large subunit rRNA. The modified RNA oligonucleotides used in these studies were generated by solid-phase synthesis with a variety of phosphoramidite chemistries. The natural modifications were shown to impact thermal stability, dynamic behavior, and tertiary structures of the RNAs, with additive or cooperative effects occurring with multiple, clustered modifications. Taking advantage of the structural diversity offered by specific modifications in the chosen rRNA motifs, phage display was used to select peptides that bind with moderate (low micromolar) affinity and selectivity to modified h31, h44, and H69. Interactions between peptide ligands and RNAs were monitored by biophysical methods, including electrospray ionization mass spectrometry (ESI-MS), NMR, and surface plasmon resonance (SPR). The peptides compare well with natural compounds such as aminoglycosides in their binding affinities to the modified rRNA constructs. Some candidates were shown to exhibit specificity toward different modification states of the rRNA motifs. The selected peptides may be further optimized for improved RNA targeting or used in screening assays for new drug candidates. In this Account, we hope to stimulate interest in bioorganic and biophysical approaches, which may be used to deepen our understanding of other functionally important, naturally modified RNAs beyond the rRNAs.