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利用糖芯片技术检测氨基糖苷类抗生素与RNAs和蛋白质之间的相互作用

王雪玉1 王晓丽1 张蓓蕾1 胡静2 尹健1

(1.江南大学生物工程学院糖化学与生物技术教育部重点实验室, 江苏无锡 214122)
(2.江南大学无锡医学院, 江苏无锡 214122)

【摘要】氨基糖苷类抗生素是一类广谱型抗细菌感染药物,其不断增加的细菌耐药性很大程度上限制了它的临床应用,研究和开发新型氨基糖苷类抗生素具有重要意义。将氨基糖苷类抗生素固定到玻璃片基上,制成糖芯片,再分别与荧光标记的RNAs和蛋白质杂交,通过分析杂交后的荧光信号强度检测它们之间的相互作用。结果显示,氨基糖苷类抗生素芯片可以特异性地与r RNA的A位点模拟物、I型核酶和蛋白酶结合。因此糖芯片技术不仅可以检测氨基糖苷类抗生素与r RNAs的特异性结合,而且可以应用于寻找新型RNA结合配体的研究,为快速鉴定和筛选可紧密结合RNA靶标且毒性较低的新型氨基糖苷类抗生素奠定了一定的基础。

【关键词】 氨基糖苷类抗生素芯片;rRNA的A位点模拟物;I型核酶;Klenow DNA聚合酶;磷脂酶C;

【DOI】

【基金资助】 国家自然科学基金(No.21502071); 江苏省自然科学基金(Nos.BK20140154,BK20150140); 江南大学公共卫生研究中心项目(No.JUPH201502)资助;

Interactions of aminoglycosides with RNAs and proteins via carbohydrate microarray

Xueyu Wang1 Xiaoli Wang1 Beilei Zhang1 Jing Hu2 Jian Yin1

(1.Key Laboratory of Carbohydrate Chemistry and Biotechnology Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, Jiangsu, China 214122)
(2.Wuxi Medical School, Jiangnan University, Wuxi, Jiangsu, China 214122)

【Abstract】Aminoglycosides are broad-spectrum antibacterials to treat bacterial infections, especially gram-negative bacteria infections. However, aminoglycosides are losing efficacy because of the increase in antibiotic resistance and their inherent toxicity, attracting more interests in developing new aminoglycosides. Several clinically used aminoglycosides are mainly exerted by inhibition of protein synthesis through binding to bacterial rRNA. The bacterial ribosome RNA is the most currently exploited RNA drug target. Identification of new compounds that target RNAs is indispensable to fight with the growing threat that bacteria pose to human safety. In this work, we used carbohydrate microarrays to probe interactions of low molecular weight ligands with RNAs and proteins. Carbohydrate microarrays, comprising hundreds to thousands of different glycan structures on surfaces in a spatially discrete pattern, are sensitive and versatile tools to study the interactions between biological macromolecules. Herein, aminoglycosides have been immobilized onto the modified glass microscope slides and their interactions with RNAs and proteins are then measured through the labeled fluorescence. The results displayed that microarray can be used to detect the binding of aminoglycosides with three types of target molecules, including the small RNA oligonucleotide mimics of aminoglycoside binding sites in the ribosome (rRNA A-site mimics), the large group Ⅰ ribozyme RNA (approximately 400 nucleotide) and certain proteins (toxicity-causing enzymes, such as DNA polymerase and phospholipase C). For rRNA A-site mimics, the fluorescence intensities of 16 S rRNA is stronger than that of 18 S rRNA, illustrating that as a screen technique, the microarray method can not only determine the binding affinity to RNA but also detect the specific binding to bacterial rRNA mimic. The ability to screen group Ⅰ ribozyme RNA can be helpful to the discovery of new RNA therapeutic targets. Binding of immobilized aminoglycosides to toxicity-causing proteins (DNA polymerase and phospholipase C) is a new method to study of aminoglycoside toxicity. These studies lay the foundation for rapid identification of new RNA-binding ligands with strong and specific binding affinity for their desired targets.

【Keywords】 aminoglycoside microarrays; rRNA A-site mimics; group Ⅰ ribozyme; klenow DNA polymerase; phospholipase C;

【DOI】

【Funds】 National Natural Science Foundation of China (No. 21502071); Natural Science Foundation of Jiangsu Province, China (Nos. BK20140154, BK20150140); Fund of the Public Health Research Center at Jiangnan University (No. JUPH201502);

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This Article

ISSN:1000-3061

CN: 11-1998/Q

Vol 32, No. 10, Pages 1362-1371

October 2016

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Abstract

  • 1 Materials and methods
  • 2 Results and analysis
  • 3 Conclusions
  • References