Poster abstracts

Poster number 34 submitted by Danielle Dremann

Development of Heptameric Peptides to Target H69 of the Bacterial Ribosome

Danielle N. Dremann (Department of Chemistry, Wayne State University), Christine S. Chow (Department of Chemistry, Wayne State University)

Abstract:
The lack of new antibiotics is a growing concern due to an increase in bacterial resistance across the different classes of known drugs. One limitation of the current antibiotics is their overlapping binding sites on the target ribosome; therefore, novel sites are essential for the success of new antibiotics. Helix 69 (H69) is a 19-nucleotide region of the 50S ribosomal subunit. Although the exact function of H69 is currently unknown, it has been implicated to have important roles in ribosomal processes such as subunit association, ribosomal recycling, and tRNA selection. Conserved across phylogeny, bacterial H69 also contains three pseudouridine residues. To determine the drug potential and functional applications of the H69 motif, phage display was used to reveal a heptameric peptide sequence that binds to H69 with moderate (low micromolar) affinity and selectivity. Although this technique is useful in identifying moderate binders, tight-binding sequences may not be found due to the target's implicated role in various translational processes. To surpass this limitation, chemical modifications or mutations of the parent sequence were made by using solid-phase peptide synthesis. Relative dissociation constants for the RNA-peptide complexes were determined by using electrospray ionization mass spectrometry. Modified peptides with greater binding affinity than the parent were further characterized by using a variety of biophysical techniques. A visual assay was developed to further screen peptide libraries to choose novel ligands that can be further characterized and tested for binding and selectivity to H69 RNA. The work described supports that natural, specific ligands can be identified to target oligonucleotide targets and improved binding can be achieved with the use of chemical modifications. Furthermore, H69 functionality can be elucidated by using these peptide ligands combined with biological and biophysical techniques making this experimentation applicable to other oligonucleotide constructs implicated in essential biological processes.

Keywords: Ribosome, Helix 69, Peptide Ligands