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 the RNA-binding specificity of ligands. Two methods were investigated. In the first method, 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 was compared with 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 that the results will lead to the discovery of new aminoglycoside-binding RNA motifs and may also have relevance toward understanding and overcoming the side effects observed with these antibiotics in the clinic.
Liang
Fu-Sen
FS
Department of Chemistry and The Skaggs Institute for Chemical Biology and Array Core Facility, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Greenberg
William A
WA
Hammond
Jennifer A
JA
Hoffmann
Julia
J
Head
Steven R
SR
Wong
Chi-Huey
CH
D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008099:Liver / Q000502:physiology; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods; D012313:RNA / Q000378:metabolism*; D014031:Tobramycin / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D008024:Ligands; D012313:RNA; D014031:Tobramycin