RNA molecules that specifically and stoichiometrically bind aminoglycoside antibiotics with high affinities.
RNA aptamers had previously been selected which were able to bind to the aminoglycoside antibiotic tobramycin with high affinity (Wang & Rando, 1995). Consensus sequences are found in a variety of constructs, and these sequences were mapped to stem-loop regions by Mfold secondary structure prediction. A tobramycin-based affinity cleavage reagent specifically cleaves the aptamers in their consensus regions. A fluorescence depolarization method is developed to accurately measure the affinity and stoichiometry of aminoglycoside binding to RNA constructs. An RNA aptamer (J6RNA) selected to bind to the aminoglycoside antibiotic tobramycin is shown to do so with an affinity of 0.77 nM and a stoichiometry of 1:1. (Fluorescently labeled) 5-carboxytetramethylrhodamine tobramycin (CRT) is used as a ligand in the fluorescence depolarization studies. J6RNA binding is quite specific for tobramycin, and weakly binds structurally related aminoglycosides with affinities 10(3)-10(4)-fold lower than that for tobramycin. Specific aminoglycoside binding aptamers of this type should be useful for revealing the rules of RNA-aminoglycoside recognition.
Wang
Y
Y
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Killian
J
J
Hamasaki
K
K
Rando
R R
RR
D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001483:Base Sequence; D001667:Binding, Competitive; D002240:Carbohydrate Sequence; D004492:Edetic Acid / Q000378:metabolism; D004917:Erythromycin / Q000378:metabolism; D005296:Ferrous Compounds / Q000494:pharmacology; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000378:metabolism; D005601:Framycetin / Q000378:metabolism; D005839:Gentamicins / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D009843:Oligoribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D012235:Rhodamines; D014031:Tobramycin / Q000031:analogs & derivatives / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D005296:Ferrous Compounds; D005456:Fluorescent Dyes; D005839:Gentamicins; D009843:Oligoribonucleotides; D012235:Rhodamines; D005601:Framycetin; D012313:RNA; D004917:Erythromycin; D004492:Edetic Acid; D014031:Tobramycin