STRUCTURAL PROBING AND DAMAGE SELECTION OF CITRULLINE-SPECIFIC AND ARGININE-SPECIFIC RNA APTAMERS IDENTIFY BASE POSITIONS REQUIRED FOR BINDING

In a recent study, an RNA aptamer for the specific recognition of the amino acid L-arginine was evolved from an in vitro selected L-citrulli ne binding parent sequence [M. Famulok (1994) J. Am. Chem. Soc. 116, 1 698-1706]. We have now carried out a structural analysis of these apta mers by using chemical modification experiments. Footprinting experime nts and a damage selection approach were performed to identify those p ositions protected from modification in the presence of the amino acid s and modifications that interfere with the binding of the ligand. It is shown that of the two bulged regions present in both aptamers one c an be modified without loss of binding activity whereas in the other b ulge nearly every position is shown to be involved in the recognition of the ligands. This might be indicative for non-canonical base pairin g to occur within the non-Watson-Crick paired regions which might be s tabilized by the complexed amino acid. Binding to the cognate amino ac id significantly enhances the conformational stability of the RNA. We also tested the sensitivity of both aptamers towards lead (II) ion ind uced cleavage and identified a hypersensitive cleavage site within the invariant bulged region. Lead cleavage is inhibited by the complexed amino acid, indicating a conformational change of the aptamer upon lig and binding. NMR titration data obtained with both aptamers and their cognate ligands confirm the proposed conformational changes and indica te the formation of a 1:1 complex of RNA:amino acid.