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...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2006-08, Vol.103 (33), p.12311-12316
Main Authors: Liang, Fu-Sen, Greenberg, William A., Hammond, Jennifer A., Hoffmann, Julia, Head, Steven R., Wong, Chi-Huey
Format: Article
Language:English
Subjects:
Aminoglycosides
Animals
Anti-Bacterial Agents - metabolism
Antibiotics
Atoms & subatomic particles
Binding sites
Biological Sciences
Complementary DNA
Deoxyribonucleic acid
DNA
DNA probes
Fluorescence
Humans
Ligands
Liver - physiology
Messenger RNA
Molecular Sequence Data
Molecular Structure
Molecules
Nucleic Acid Hybridization
Nucleotide sequences
Oligonucleotide Array Sequence Analysis - instrumentation
Oligonucleotide Array Sequence Analysis - methods
Physical Sciences
Ribonucleic acid
RNA
RNA - metabolism
Studies
Tobramycin - metabolism
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Summary: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 aminoglycosidebinding RNA motifs and may also have relevance toward understanding and overcoming the side effects observed with these antibiotics in the clinic.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0605264103