Studying Interactions Of Aminoglycosides With RNAs And Proteins Via Microarray

Aminoglycosides are important drugs for infectious diseases, especially for diseases caused by gram-negative bacteria. However, due to the increasing resistance of bacteria and their inherent toxicity, aminoglycosides are losing efficacy in clinical application. Thus, developing new aminoglycosides attracts more interests. Several clinically used aminoglycosides are mainly exerted by inhibition of protein synthesis through binding to bacterial 16 S rRNA-A site, which is the subunit for the 30 S rRNA in ribosome. In this article, carbohydrate microarray technology, a kind of high-throughput method, was used to explore the interactions of aminoglycosides with RNAs and proteins. Six kinds of aminoglycosides were immobilized onto the chemical modified glass slides. Then they were hybridized with fluorescently labeled RNAs and proteins, analyzing the interaction of aminoglycosides with RNAs and proteins by detecting the fluorescence intensity after hybridization. The main results are as follows:(1) Tetraethylene glycol disuccinimidyl disuccinate(TGDD) and tetraethylene glycol phthaloyl phthalate(TGPP) were synthesized and used to modify the slides. We constructed the carbohydrate microarray by the covalent immobilized way, which were used to explore the interactions of aminoglycosides with RNAs and proteins;(2) The interactions of aminoglycosides with 16 S rRNA Asite mimic and 18 S rRNA Asite mimic were studied. All immobilized aminoglycosides bind to the rRNA mimics, but the fluorescence intensity were different. The fluorescence intensities for the 16 S rRNA Asite mimic are obviously higher than for the 18 S rRNA Asite mimic, which illustrated that the microarray method can be utilized as a screen not only for tight binding to RNA but also specific binding to bacterial rRNA mimic, which could rapidly predicted the effects for aminoglycosides;(3) The interactions between aminoglycosides and group I ribozyme RNA(~400 nucleotide) were studied. Group I ribozyme is one of the model for study of new RNA targets. The results show that immobilized aminoglycosides can derectly bind to the group I ribozyme, illustrating that carbohydrate microarray could accelerated the discovery of new RNA therapeutic targets;(4) The interactions of aminoglycosides with proteins(DNA polymerase and phospholipase C) were studied. Binding of immobilized aminoglycosides to proteins that are models for study of aminoglycoside toxicity. The results showed that streptomycin has the strongest fluorescent intensity, consistent with its own side effects. The fluorescent intensity for amikacin, netilmicin and sisomicin weaker than streptomycin, indicating that side-effects of amikacin, netilmicin and sisomicin were lower than streptomycin.These studies lay the foundation for rapid identification of new RNA-binding ligands with strong binding affinity for their desired targets but weak affinity to toxicity-causing proteins.