RNA molecules are the functional targets for aminoglycosides. In order to approach an understanding of the rules which underlie aminoglycoside-RNA recognition, high-affinity RNA aptamers have been prepared which discriminate among various aminoglycosides [Wang et al. (1996) Biochemistry 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 103-104 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 constant of approximately 5 nM. This construct can strongly discriminate between similar aminoglycosides with respect to binding. Elimination of either the three or the single nucleotide bulge eliminates specific aminoglycoside binding. The structure of the loop region is also critical. These studies demonstrate that simplified RNA molecules can be generated which bind aminoglycosides specifically and with high affinities.
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