In vitro selection and characterization of streptomycin-binding RNAs: Recognition discrimination between antibiotics

Abstract

As pathogens continue to evade therapeutical drugs, a better understanding of the mode of action of antibiotics continues to have high importance. A growing body of evidence points to RNA as a crucial target for antibacterial and antiviral drugs. For example, the aminocyclitol antibiotic streptomycin interacts with the 16S ribosomal RNA and, in addition, inhibits group I intron splicing. To understand the mode of binding of streptomycin to RNA, we isolated small, streptomycin-binding RNA aptamers via in vitro selection. In addition, bluensomycin, a streptomycin analogue that does not inhibit splicing, was used in a counter-selection to obtain RNAs that bind streptomycin with high affinity and specificity. Although an RNA from the normal selection (motif 2) bound both antibiotics, an RNA from the counter-selection (motif 1) discriminated between streptomycin and bluensomycin by four orders of magnitude. The binding site of streptomycin on the RNAs was determined via chemical probing with dimethylsulfate and kethoxal. The minimal size required for drug binding was a 46- and a 41-mer RNA for motifs 1 and 2, respectively. Using Pb²⁺ cleavage in the presence and absence of streptomycin, a conformational change spanning the entire mapped sequence length of motif 1 was observed only when both streptomycin and Mg²⁺ were present. Both RNAs require Mg²⁺ for binding streptomycin.