Poster abstracts

Poster number 120 submitted by Jacob Sieg

Development of a fluorescence assay for monitoring ligand binding to regulatory RNA thermometers

Jacob Sieg (Department of Chemistry & Biochemistry Ohio University, Athens Ohio), Chunxi Zeng (Department of Chemistry & Biochemistry, Molecular & Cellular Biology Program Ohio University, Athens Ohio), Erin R. Murphy (Department of Chemistry & Biochemistry, Molecular & Cellular Biology Program, Department of Biomedical Sciences Ohio University, Athens Ohio), Jennifer V. Hines (Department of Chemistry & Biochemistry, Molecular & Cellular Biology Program Ohio University, Athens Ohio)

Abstract:
The ompA RNA thermometer regulates translation of virulence essential ompA proteins in Gram-negative bacteria via temperature dependent seclusion of the Shine-Dalgarno sequence. In this study, a molecular beacon style approach was used to develop a moderate throughput assay that screens for ligands that bind the ompA RNA thermometer. Eight aminoglycoside antibiotics known to bind other RNAs were screened using this assay to test its sensitivity. Then aminoglycoside binding was characterized by follow-up EC50 determinations and thermodenaturation experiments. All aminoglycosides in this study were shown to bind the ompA thermometer. Binding was cooperative indicating likely binding at multiple sites. Furthermore, binding was primarily related to aminoglycoside charge, indicating that binding is driven by electrostatics. This information can be used to inform future drug discovery studies of RNA thermometers.

Keywords: thermometer, fluorescence, aminoglycoside