Department of Chemistry and The Center of Excellence in Bioinformatics and Life Sciences, University at Buffalo, The State University of New York, 657 Natural Sciences Complex, Buffalo, New York 14260, USA.
Biochemistry. 2010 Mar 9;49(9):1833-42. doi: 10.1021/bi901998m.
RNAs have diverse structures that are important for biological function. These structures include bulges and internal loops that can form tertiary contacts or serve as ligand binding sites. The most commonly exploited RNA drug target for small molecule intervention is the bacterial ribosome, more specifically the rRNA aminoacyl-tRNA site (rRNA A-site) which is a major target for the aminoglycoside class of antibiotics. The bacterial A-site is composed of a 1 x 1 nucleotide all-U internal loop and a 2 x 1 nucleotide all-A internal loop separated by a single GC base pair. Therefore, we probed the molecular recognition of a small library of four aminoglycosides for binding a 16384-member bacterial rRNA A-site-like internal loop library using two-dimensional combinatorial screening (2DCS). 2DCS is a microarray-based method that probes RNA and chemical spaces simultaneously. These studies sought to determine if aminoglycosides select their therapeutic target if given a choice of binding all possible internal loops derived from an A-site-like library. Results show that the bacterial rRNA A-site was not selected by any aminoglycoside. Analyses of selected sequences using the RNA Privileged Space Predictor (RNA-PSP) program show that each aminoglycoside preferentially binds different types of internal loops. For three of the aminoglycosides, 6''-azido-kanamycin A, 5-O-(2-azidoethyl)-neamine, and 6''-azido-tobramycin, the selected internal loops bind with approximately 10-fold higher affinity than the bacterial rRNA A-site. The internal loops selected to bind 5''-azido-neomycin B bind with an affinity similar to that of the therapeutic target. Selected internal loops that are unique for each aminoglycoside have dissociation constants ranging from 25 to 270 nM and are specific for the aminoglycoside they was selected to bind compared to the other arrayed aminoglycosides. These studies further establish a database of RNA motifs that are recognized by small molecules that could be used to enable the rational and modular design of small molecules targeting RNA.
RNA 具有多种结构，这些结构对于生物功能很重要。这些结构包括突环和内部环，可以形成三级接触或作为配体结合位点。小分子干预的最常用的 RNA 药物靶点是细菌核糖体，更具体地说是 rRNA 氨酰-tRNA 位点（rRNA A 位），这是氨基糖苷类抗生素的主要靶点。细菌 A 位由一个 1 x 1 核苷酸全 U 内部环和一个 2 x 1 核苷酸全 A 内部环组成，由一个 GC 碱基对隔开。因此，我们使用二维组合筛选（2DCS）探测了一小部分氨基糖苷类药物与 16384 个成员的细菌 rRNA A 位样内部环文库结合的分子识别。2DCS 是一种基于微阵列的方法，可同时探测 RNA 和化学空间。这些研究旨在确定如果给与来自 A 位样文库的所有可能内部环的选择，氨基糖苷类药物是否会选择它们的治疗靶标。结果表明，细菌 rRNA A 位未被任何氨基糖苷类药物选择。使用 RNA 特权空间预测器（RNA-PSP）程序对选定序列进行分析表明，每种氨基糖苷类药物都优先结合不同类型的内部环。对于三种氨基糖苷类药物，6''-叠氮卡那霉素 A、5-O-（2-叠氮乙基）-新霉素和 6''-叠氮妥布霉素，所选内部环的结合亲和力比细菌 rRNA A 位高约 10 倍。与治疗靶标结合的 5''-叠氮新霉素 B 的内部环具有相似的亲和力。每种氨基糖苷类药物特有的选定内部环的解离常数范围为 25 至 270 nM，与其他排列的氨基糖苷类药物相比，它们对所选结合的氨基糖苷类药物具有特异性。这些研究进一步建立了一个小分子识别 RNA 基序的数据库，可用于实现针对 RNA 的小分子的合理和模块化设计。
