SPECIFICITY OF AMINOGLYCOSIDE BINDING TO RNA CONSTRUCTS DERIVED FROM THE 16S RIBOSOMAL-RNA DECODING REGION AND THE HIV-RRE ACTIVATOR REGION

Aminoglycoside antibiotics can bind to many different types of RNA mol ecules. 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 quantitativ ely measure aminoglycoside affinities to constructs of the HIV-1 RRE t ranscriptional activation region and the prokaryotic rRNA decoding reg ion which is the natural antibacterial target of the aminoglycosides. A fluorescent analog of Rev(34-50) (Fl-Rev(34-50)) was prepared and sh own by fluorescence anisotropy measurements to bind to the HIV-1 RRE r egion 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 bindin g is known to be mimicked by Rev(34-50) The binding is driven by a str ongly negative enthalpic term. Aminoglycosides compete with Fl-Rev(34- 50) binding and competition experiments with semisynthetic aminoglycos ides and neomycin B and tobramycin show binding affinities in the 1-2 mu M range. The binding of aminoglycosides to this construct is thus n ot highly selective. A prokaryotic rRNA construct was also prepared an d shown to bind a fluorescent dye labeled derivative of the antibiotic paromomycin (CRP) stoichiometrically with a dissociation constant of 0.16 mu M. Competition experiments with other aminoglycosides showed b inding in the micromolar range, with limited specificity for aminoglyc oside 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 subnanom olar affinities and specificity of aminoglycoside binding found using in vitro selected RNA molecules (Wang et al., 1996).