MINIMAL RNA CONSTRUCTS THAT SPECIFICALLY BIND AMINOGLYCOSIDE ANTIBIOTICS WITH HIGH AFFINITIES

RNA molecules are the functional targets for aminoglycosides. In order to approach an understanding of the rules which underlie aminoglycosi de-RNA recognition, high-affinity RNA aptamers have been prepared whic h discriminate among various aminoglycosides [Wang et al. (1996) Bioch emistry 35, 12338-12346]. One of these aptamers, J6, which is 109 nts in length, binds the aminoglycoside tobramycin stoichiometrically with a dissociation constant of 0.77 +/- 0.03 nM. Aminoglycosides, similar in structure to tobramycin, bind with affinities diminished by 10(3)- 10(4) compared to tobramycin. Experiments are reported here which are designed to reveal the nature of the tobramycin binding domain of J6. A small (40 nts) stem-loop derivative of J6, containing a 3 nt and a 1 nt bulge, stoichiometrically binds tobramycin with a dissociation con stant of approximately 5 nM. This construct can strongly discriminate between similar aminoglycosides with respect to binding. Elimination o f either the three or the single nucleotide bulge eliminates specific aminoglycoside binding. The structure of the loop region is also criti cal. These studies demonstrate that simplified RNA molecules can be ge nerated which bind aminoglycosides specifically and with high affiniti es.