A tetracycline-binding RNA aptamer

Abstract

Aptamers are perfect tools to study the interaction of small ligands with RNA. To study the mode of interaction of tetracycline with RNA, we isolated aptamers with high affinity to this antibiotic via in vitro selection. One of the selected aptamers, cb28, which has a comparable affinity to tetracycline as the small ribosomal subunit, was characterised in more detail. Cb28 binds only to typical tetracyclines, while atypical tetracyclines are not recognised. The hydroxyl group at position 6 is an essential determinant for recognition, while modifications at positions 4, 5 and 7 do not interfere with RNA binding. Binding of tetracycline to cb28 is magnesium dependent. The secondary structure of cb28 was determined by lead cleavage and DMS modification. Upon tetracycline binding, nucleotides in J2/3 and the P5 stem-loop are protected from cleavage by lead, indicating a conformational change in the RNA. This conformational change was confirmed by tetracycline dependent changes in the DMS modification pattern. Photo-induced affinity incorporation of tetracycline into cb28 resulted in a crosslink to position G76, a residue in L5. The mode of binding of tetracycline to the cb28 aptamer resembles its interaction with the primary binding site on the small ribosomal subunit.