Specificity of aminoglycoside binding to RNA constructs derived from the 16S rRNA decoding region and the HIV-RRE activator region

Aminoglycoside antibiotics can bind to many different types of RNA molecules. It was of interest to determine the nature of the selectivity of binding of aminoglycosides to important, biologically relevant RNA targets. Fluorescence anisotropy methods were developed to quantitatively measure aminoglycoside affinities to constructs of the HIV-1 RRE transcriptional activation region and the prokaryotic rRNA decoding region which is the natural antibacterial target of the aminoglycosides. A fluorescent analog of Rev34-50 (Fl-Rev34-50) was prepared and shown by fluorescence anisotropy measurements to bind to the HIV-1 RRE region with a stoichiometry of 1 and a dissociation constant of 7.6 nM. RRE RNA is a target for the arginine rich Rev protein, and the binding is known to be mimicked by Rev34-50. The binding is driven by a strongly negative enthalpic term. Aminoglycosides compete with Fl-Rev34-50 binding and competition experiments with semisynthetic aminoglycosides and neomycin B and tobramycin show binding affinities in the 1-2 microM range. The binding of aminoglycosides to this construct is thus not highly selective. A prokaryotic rRNA construct was also prepared and shown to bind a fluorescent dye labeled derivative of the antibiotic paromomycin (CRP) stoichiometrically with a dissociation constant of 0.16 microM. Competition experiments with other aminoglycosides showed binding in the micromolar range, with limited specificity for aminoglycoside type, suggesting that much of the aminoglycoside molecule is not involved in binding. The relatively modest specificity in the binding of aminoglycoside described above is to be contrasted to the subnanomolar affinities and specificity of aminoglycoside binding found using in vitro selected RNA molecules (Wang et al., 1996).