IN-VITRO SELECTION AND CHARACTERIZATION OF STREPTOMYCIN-BINDING RNAS - RECOGNITION DISCRIMINATION BETWEEN ANTIBIOTICS

As pathogens continue to evade therapeutical drugs, a better understan ding of the mode of action of antibiotics continues to have high impor tance. A growing body of evidence points as RNA as a crucial target fo r antibacterial and antiviral drugs. For example, the aminocyclitol an tibiotic streptomycin interacts with the 16S ribosomal RNA and, in add ition, inhibits group I intron splicing. To understand the mode of bin ding of streptomycin to RNA, we isolated small, streptomycin-binding R NA aptamers via in vitro selection. In addition, bluensomycin, a strep tomycin analogue that does not inhibit splicing, was used in a counter -selection to obtain RNAs that bind streptomycin with high affinity an d specificity. Although an RNA from the normal selection (motif 2) bou nd both antibiotics, an RNA from the counter-selection (motif 1) discr iminated between streptomycin and bluensomycin by four orders of magni tude. The binding site of streptomycin on the RNAs was determined via chemical probing with dimethylsulfate and kethoxal. The minimal size r equired 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 str eptomycin, a conformational change spanning the entire mapped sequence length of motif I was observed only when both streptomycin and Mg2+ w ere Present. Both RNAs require Mg2+ for binding streptomycin.