RNA molecules that specifically and stoichiometrically bind aminoglycoside antibiotics with high affinities
RNA molecules that specifically and stoichiometrically bind aminoglycoside antibiotics with high affinities
复制标题
DOI：
10.1021/bi960878w
复制
发表时间：
1996-09-24
期刊：
影响因子：
2.9
通讯作者：
Rando, RR
中科院分区：
文献类型：
--
作者：
Wang, Y;Killian, J;Rando, RR
以前已经选择了能够与氨基糖苷类抗生素妥布霉素高亲和力结合的RNA适配子(Wang&Rando，1995)。在各种构建体中都发现了共同的序列，这些序列通过Mold二级结构预测被映射到茎环区域。一种基于妥布霉素的亲和切割试剂在它们的共识区域特异性地切割适配子。发展了一种荧光去偏振方法，以准确地测量氨基糖苷与RNA结构物结合的亲和力和化学计量比。选择与氨基糖苷类抗生素妥布霉素结合的RNA适配子(J6RNA)，亲和力为0.77 nm，化学计量比为1：1。荧光标记的5-羧基四甲基罗丹明妥布霉素(CRT)被用作荧光去偏振研究中的配体。J6RNA结合是妥布霉素的特异性结合，与结构相关的氨基糖苷类化合物的亲和力为10(3)-10(4)，比妥布霉素低10(3)-10(4)。这种类型的氨基糖苷结合适配子对于揭示RNA-氨基糖苷识别的规律是有用的。
RNA aptamers had previously been selected which were able to bind to the aminoglycoside antibiotic tobramycin with high affinity (Wang & Rando, 1995). Consensus sequences are found in a variety of constructs, and these sequences were mapped to stem-loop regions by Mfold secondary structure prediction. A tobramycin-based affinity cleavage reagent specifically cleaves the aptamers in their consensus reg ions. A fluorescence depolarization method is developed to accurately measure the affinity and stoichiometry of aminoglycoside binding to RNA constructs. An RNA aptamer (J6RNA) selected to bind to the aminoglycoside antibiotic tobramycin is shown to do so with an affinity of 0.77 nM and a stoichiometry of 1:1. (Fluorescently labeled) 5-carboxytetramethylrhodamine tobramycin (CRT) is used as a ligand in the fluorescence depolarization studies. J6RNA binding is quite specific for tobramycin, and weakly binds structurally related aminoglycosides with affinities 10(3)-10(4)-fold lower than that for tobramycin. Specific aminoglycoside binding aptamers of this type should be useful for revealing the rules of RNA-aminoglycoside recognition.