Evaluation of RNA binding specificity of aminoglycosides with DNA microarrays

We have developed methods for using DNA array technology to probe the entire transcriptome to determine RNA-binding specificity of ligands. Two methods were investigated. In the first, the RNA-binding aminoglycoside antibiotic tobramycin was covalently linked to magnetic beads. The beads were bound to human liver mRNA and washed, and specifically bound RNA was eluted, amplified, and analyzed with DNA array technology. A small number of genes were found to bind specifically to the tobramycin beads. In the second method, the aminoglycoside ligand was added directly to the array hybridization reaction, and the signal compared to a control experiment in the absence of ligand. The aminoglycosides were found to interfere with a small percentage of all hybridization events. These methods differ from traditional DNA array experiments in that the readout is a direct measure of the interaction between mRNA and a ligand, rather than an indirect measure of effect on expression. We expect the results will lead to discovery of new aminoglycoside-binding RNA motifs, and may also have relevance toward understanding and overcoming side effects observed with these antibiotics in the clinic. Keywords: RNA binding analysis 5 samples including controls were analyzed for effect of RNA binding ligands on hybridization of mRNA to DNA microarrays.