Development of antibiotics that target new regions of functionality is a possible way to overcome antibiotic resistance. In this study, the interactions of aminoglycoside antibiotics with helix 69 of the E. coli 23S rRNA in the context of complete 70S ribosomes or the isolated 50S subunit were investigated by using chemical probing and footprinting analysis. Helix 69 is a dynamic RNA motif that plays major roles in bacterial ribosome activity. Neomycin, paromomycin, and gentamicin interact with the stem region of helix 69 in complete 70S ribosomes, but have diminished binding to the isolated 50S subunit. Pseudouridine modifications in helix 69 were shown to impact the aminoglycoside interactions. These results suggest a requirement for a specific conformational state of helix 69 for efficient aminoglycoside binding, and imply that this motif may be a suitable target for mechanism-based therapeutics.

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.

Antibiotic resistance prevents successful treatment of common bacterial infections, making it clear that new target locations and drugs are required to resolve this ongoing challenge. The bacterial ribosome is a common target for antibacterials due to its essential contribution to cell viability. The focus of this work is a region of the ribosome called helix 69 (H69), which was recently identified as a secondary target site for aminoglycoside antibiotics. H69 has key roles in essential ribosomal processes such as subunit association, ribosome recycling, and tRNA selection. Conserved across phylogeny, bacterial H69 also contains two pseudouridines and one 3-methylpseudouridine. Phage display revealed a heptameric peptide sequence that targeted H69. Using solid-phase synthesis, peptide variants with higher affinity and improved selectivity to modified H69 were generated. Electrospray ionization mass spectrometry was used to determine relative apparent dissociation constants of the RNA–peptide complexes.

•Pseudouridine (Ψ) modulates pH-dependent structural changes of helix 69 (H69) RNAs.•Mutant A1912G requires Ψs for pH-dependent structural changes in H69 loop region.•A1919G mutation leads to formation of a U/Ψ1911•G1919 wobble pair.•G1919 and Ψ modifications work synergistically to modulate H69 structural changes.•U1917C mutation leads to pH-dependent changes in the H69 loop and stem regions.Centrally located at the ribosomal subunit interface and mRNA tunnel, helix 69 (H69) from 23S rRNA participates in key steps of translation. Ribosome activity is influenced by three pseudouridine modifications, which modulate the structure and conformational behavior of H69. To understand how H69 is affected by the presence of pseudouridine in combination with sequence changes, the biophysical properties of wild-type H69 and representative mutants (A1912G, U1917C, and A1919G) were examined. Results from NMR and circular dichroism spectroscopy indicate that pH-dependent structural changes of wild-type H69 and the chosen mutants are modulated by pseudouridine and loop sequence. The effects of the mutations on global stability of H69 are negligible; however, pseudouridine stabilizes H69 at low pH conditions. Alterations to induced conformational changes of H69 likely result in compromised function, as indicated by previous biological studies.

Cisplatin can be modified with various ligands to alter the size and charge distribution of the complex. Several amino acid-linked platinum(II) complexes were synthesized, and a reactivity study with 16S ribosomal RNA was carried out. The amino acid-linked analogues show altered specificity compared to the parental compound cisplatin.

Helix 69 of Escherichia coli 23S rRNA has important roles in specific steps of translation, such as subunit association, translocation, and ribosome recycling. An M13 phage library was used to identify peptide ligands with affinity for helix 69. One selected sequence, NQVANHQ, was shown through a bead assay to interact with helix 69. Electrospray ionization mass spectroscopy revealed an apparent dissociation constant for the amidated peptide and helix 69 in the low micromolar range. This value is comparable to that of aminoglycoside antibiotics binding to the A site of 16S rRNA or helix 69. Helix 69 variants (human) and unrelated RNAs (helix 31 or A site of 16S rRNA) showed two- to fourfold lower affinity for NQVANHQ-NH2. These results suggest that the peptide has desirable features for development as a lead compound for novel antimicrobials.

Cisplatin can be modified with various ligands to alter the size and charge distribution of the complex. Several amino acid-linked platinum(II) complexes were synthesized, and a reactivity study with 16S ribosomal RNA was carried out. The amino acid-linked analogues show altered specificity compared to the parental compound cisplatin.