In vitro selection analysis of neomycin binding RNAs with a mutagenized pool of variants of the 16S rRNA decoding region

An in vitro selection for neomycin B binding was carried out with an RNA pool containing a 47-nucleotide domain of the decoding region of 16S ribosomal RNA, mutated at 30% per base position. The degenerate region was comprised of an oligonucleotide analogue (''motif A'') of the decoding region in 30S subunits which has previously been shown to interact with the aminoglycoside antibiotic neomycin B and tRNA ligands. After five cycles of selection/amplification, RNA sequences were isolated which specifically bound to neomycin B. Cloning and sequencing showed that none of the isolated clones shared primary sequence or secondary structure homology with the decoding region of 16S RNA. Instead, a new set of sequences was isolated which could be folded into a defined hairpin structure designated as motif B. We investigated the affinity of motif A, motif B, the unselected pool RNA, and the corresponding unmutagenized ''parent'' RNA to neomycin B at different Mg2+ concentrations. Under buffer conditions of low ionic strength all RNAs tested bound nonspecifically to neomycin B. However, motif B bound to neomycin B at Mg2+ concentrations at which binding of the other RNAs tested was significantly lower or not detectable. This is consistent with motif B exhibiting a higher affinity for neomycin B than motif A under these conditions. Motif B has previously been isolated from an in vitro selection to identify RNA sequences with affinity to neomycin B using a completely randomized RNA pool which shared no relationship to motif A. Our results indicate that motif B might represent a highly optimized RNA sequence for neomycin B binding; conversely, the A-site motif in 16S rRNA (motif A) might not be an optimal target for neomycin B recognition.