A high-throughput fluorescence screen to monitor the specific binding of antagonists to RNA targets.
Since RNA molecules can form intricate three-dimensional structures, it should be possible to design specific, high-affinity antagonists directed against these structures. To begin to explore the validity of this possibility, high-throughput screening methods are required to assay for RNA antagonists. A fluorescence quenching technique is described here in a 96-well plate format which is capable of screening chemical diversity libraries. A pyrene-containing aminoglycoside analog is used to accurately monitor antagonist binding to a prokaryotic 16S rRNA A-site decoding region construct. This rRNA region comprises the natural target for aminoglycoside antibiotics. The fluorescence technique reported here should be generally adaptable to monitor the binding of structurally novel antagonists to any selected RNA target.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Avenue, Boston, Massachusetts, 02115, USA.
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D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000378:metabolism; D005456:Fluorescent Dyes; D007700:Kinetics; D012336:RNA, Ribosomal, 16S / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D005456:Fluorescent Dyes; D012336:RNA, Ribosomal, 16S