Aminoglycoside microarrays to explore interactions of antibiotics with RNAs and proteins.
RNA is an important target for drug discovery efforts. Several clinically used aminoglycoside antibiotics bind to bacterial rRNA and inhibit protein synthesis. Aminoglycosides, however, are losing efficacy due to their inherent toxicity and the increase in antibiotic resistance. Targeting of other RNAs is also becoming more attractive thanks to the discovery of new potential RNA drug targets through genome sequencing and biochemical efforts. Identification of new compounds that target RNA is therefore urgent, and we report here on the development of rapid screening methods to probe binding of low molecular weight ligands to proteins and RNAs. A series of aminoglycosides has been immobilized onto glass microscope slides, and binding to proteins and RNAs has been detected by fluorescence. Construction and analysis of the arrays is completed by standard DNA genechip technology. Binding of immobilized aminoglycosides to proteins that are models for study of aminoglycoside toxicity (DNA polymerase and phospholipase C), small RNA oligonucleotide mimics of aminoglycoside binding sites in the ribosome (rRNA A-site mimics), and a large (approximately 400 nucleotide) group I ribozyme RNA is detected. The ability to screen large RNAs alleviates many complications associated with binding experiments that use isolated truncated regions from larger RNAs. These studies lay the foundation for rapid identification of small organic ligands from combinatorial libraries that exhibit strong and selective RNA binding while displaying decreased affinity to toxicity-causing proteins.
Disney
Matthew D
MD
Laboratory for Organic Chemistry, Swiss Federal Institute of Technology Zürich, ETH Hönggerberg, HCI F315, 8093 Zurich, Switzerland.
Seeberger
Peter H
PH
D000583:Amikacin / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D046228:Microarray Analysis / Q000379:methods*; D018716:Molecular Mimicry; D012335:RNA, Ribosomal / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D012335:RNA, Ribosomal; D000583:Amikacin; D004259:DNA-Directed DNA Polymerase; D010738:Type C Phospholipases