Specific binding of aminoglycoside antibiotics to RNA.
BACKGROUND
Aminoglycoside antibiotics interfere with ribosomal protein synthesis and with intron splicing. Various lines of evidence suggest that RNA is the molecular target for aminoglycosides, but little is known about the recognition process. Is recognition of a particular aminoglycoside specific for certain RNA structures? If so, what are the rules for recognition? We have begun to investigate this problem by in vitro selection of RNA molecules that can specifically bind to the aminoglycoside antibiotic tobramycin.


RESULTS
An RNA diversity library was used to select for sequences capable of binding to the aminoglycoside antibiotic tobramycin. After six cycles of selection, 82% of the RNA bound to tobramycin specifically. The selected RNA was reverse-transcribed into DNA, which was then cloned. At low selection stringency, an extremely large number of clones, on the order of 10(7), produced RNAs capable of binding tobramycin with Kds in the microM range (values similar to that observed for the binding of tobramycin to Escherichia coli ribosomes). Sequencing of 18 of the clones revealed no obvious consensus sequence. At higher selection stringencies (Kds in the nM range) only two consensus sequences for binding were observed.


CONCLUSIONS
We have shown that RNA molecules can be readily selected that bind the aminoglycoside tobramycin. The RNAs that bind tobramycin with high affinity contain consensus binding regions that may be confined to predicted stem-loop structures. These studies open the way for understanding the basis of RNA-aminoglycoside recognition.
Wang
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Y
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Rando
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D000900:Anti-Bacterial Agents / Q000378:metabolism* / Q000494:pharmacology; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012313:RNA / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014031:Tobramycin / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D007202:Indicators and Reagents; D012313:RNA; D004247:DNA; D014031:Tobramycin