THE BINDING-SITE OF A SPECIFIC AMINOGLYCOSIDE BINDING RNA MOLECULE

A small (40 nucleotides) stem-loop derivative (J6f1) of a specific ami noglycoside-binding RNA aptamer, containing a 3 nt and a 1 nt bulge, h as previously been shown to stoichiometrically bind tobramycin with a dissociation constant of approximately 5 nM [Hamasaki, K., Killian, J. , Cho, J. and Rando, R. R. (1997) Biochemistry 36, 1367-1371]. This co nstruct can strongly discriminate among similar aminoglycosides with r espect to binding. A combination of chemical interference studies, che mical modification studies, and mutational studies are performed to de fine the aminoglycoside binding site of J6f1. Recognition of the amino glycoside by J6f1 involves contacts with nucleotide bases, rather than with the phosphate backbone. The binding site 1 comprised of part of the stem-loop region. The two bulges are also essential for high affin ity and stoichiometric binding of tobramycin. These bulges are probabl y important for prying open the double helical region, thereby allowin g the aminoglycoside access to the nucleotide bases.