A modified fluorescent intercalator displacement assay for RNA ligand discovery.
A modified fluorescent intercalator displacement assay for RNA ligand discovery.
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DOI：
10.1016/j.ab.2010.09.020
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发表时间：
2011-01-15
影响因子：
2.9
通讯作者：
Chow CS
中科院分区：
文献类型：
--
作者：
Asare-Okai PN;Chow CS
荧光嵌入剂置换（FID）是一种方便实用的识别新的核酸结合配体的工具。FID的成功是基于这样一个事实，即它可以被塑造成一个多功能的筛选测定，用于评估化合物的相对结合亲和力的核酸。FID是一种无标签的方法;研究中的靶RNA和配体或小分子不必为了进行检查而进行修饰。本研究以3-甲基-2-（（1-（3-（三甲基铵基）丙基）-4-亚喹啉基）甲基）苯并噻唑（TO-PRO）为荧光指示剂，建立了一种改进的用于筛选RNA结合配体的氢火焰离子化检测方法。电喷雾电离质谱（ESI-MS）结果提供了直接证据，证明FID测定中观察到的荧光强度降低与染料分子从RNA中的置换相关。该方法成功地应用于筛选各种RNA结合配体与一组小发夹RNA。鉴定了以中等亲和力结合所选RNA构建体（A位点、TAR、h31和H69）的配体。
Fluorescent intercalator displacement (FID) is a convenient and practical tool for identifying new nucleic-acid-binding ligands. The success of FID is based on the fact that it can be fashioned into a versatile screening assay for assessing the relative binding affinities of compounds to nucleic acids. FID is a tagless approach; the target RNAs and the ligands or small molecules under investigation do not have to be modified in order to be examined. In this study, a modified FID assay for screening RNA-binding ligands was established using 3-methyl-2-((1-(3-(trimethylammonio)propyl)-4-quinolinylidene)methyl)benzothiazolium (TO-PRO) as the fluorescent indicator. Electrospray ionization mass spectrometry (ESI-MS) results provide direct evidence that correlates the reduction in fluorescence intensity observed in the FID assay with displacement of the dye molecule from RNA. The assay was successfully applied to screen a variety of RNA-binding ligands with a set of small hairpin RNAs. Ligands that bind with moderate affinity to the chosen RNA constructs (A-site, TAR, h31, and H69) were identified.
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