Evaluation of RNA-binding specificity of aminoglycosides with DNA microarrays

Abstract

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. aminoglycoside-RNA interactionsA minoglycosides are among the most commonly used broadspectrum antibiotics in the clinic. Since the initial discovery of streptomycin in 1944, a series of aminoglycoside antibiotics, including kanamycin, neomycin B, gentamicin, and tobramycin have been successfully introduced as treatments for serious systemic infections and have been used for a variety of topical applications.The origin of the bactericidal activity of aminoglycosides has been extensively investigated and is due to binding to the bacterial ribosome (1). Aminoglycosides bind specifically to the 16S rRNA, near the mRNA decoding site. This binding decreases the fidelity of protein translation by lowering the energetic cost of a conformational transition in the ribosome that is required for the discrimination between near-cognate and cognate tRNAs (2-4). Interference with translation causes the production of erroneous proteins, and accumulation of mutant membrane proteins compromises the integrity of the bacterial envelope, which then allows the uptake of more aminoglycosides, resulting in further misreading and, eventually, cell lysis (5). Other aminoglycosides, such as apramycin and spectinomycin, act in a different mode by inhibiting the translocation process (6, 7).Under physiological conditions, aminoglycosides are cationic. The importance of electrostatic interactions in specific binding between aminoglycosides and their anionic RNA targets has been well established (8,9). Binding of aminoglycosides to nonribosomal RNAs may contribute to their potential therapeutic applications but also to the side effects that are observed for this class of compounds. Serious side effects, including ototoxicity and nephrotoxicity, have been reported and have limited the clinical use of aminoglycosides.Methods, such as surface plasm...

A fluorescence-based screen for ribosome binding antibiotics

“…Previous probes have been incorporated into the bases of a model A-site, allowing changes in the fluorescence to be measured as changes to the RNA occur on drug binding [13,41,42]. In other techniques, fluorescence resonance energy transfer (FRET) analysis is used by labeling the RNA with a donor molecule and labeling an aminoglycoside with an acceptor dye [43,44].…”

New trends in the use of aminoglycosides

“…AGs have long been known to exert their antibacterial functions by binding to the bacterial ribosome and interfering with protein translation. To further probe their mechanism of action, different approaches have been developed and employed in the past decades, including uorescence-based assays, 3,53-56 computational simulations, 57,58 microarray assays, [59][60][61][62][63] and X-ray crystallography. 4,64,65 As a result, AGs have been demonstrated to bind not only to the ribosomal decoding A-site on the 16S rRNA, causing miscoding in the nascent polypeptide, but also to the helix 69 (h69) in the large 50S ribosomal subunit, which is critical in the processes of mRNA/ tRNA translocation and ribosome recycling.…”

Approaches to Validate and Manipulate RNA Targets with Small Molecules in Cells

“…This approach has been used by the Wong group (89) to gain insight into cellular RNAs that are bound by aminoglycosides, thereby representing potential off-targets. Binding was determined by applying the RNAs pulled down by aminoglycosides to DNA microarrays (89).…”

“…This approach has been used by the Wong group (89) to gain insight into cellular RNAs that are bound by aminoglycosides, thereby representing potential off-targets. Binding was determined by applying the RNAs pulled down by aminoglycosides to DNA microarrays (89). In an analogous approach, we appended biotin to a bioactive compound and created an affinity matrix using streptavidin beads (Figure 7) (18).…”