Liang Shuang, Connell Gregory J
Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, USA.
RNA. 2009 Oct;15(10):1929-38. doi: 10.1261/rna.1720209. Epub 2009 Aug 20.
An RNA editing reaction that is both essential and specific to the trypanosomatid parasites is an attractive target for new drug development. Although high-throughput screening of chemical libraries is a powerful strategy often used to identify new drugs, the available in vitro editing assays do not have the necessary sensitivity and format for this approach to be feasible. A ruthenium labeled reporter RNA is described here that overcomes these limitations as it can both detect edited product in the low femtomole range and is ideal for high-throughput format. The reporter RNA consists of an RNA editing substrate linked to a streptavidin-binding aptamer that is initially held within an inactive conformation. An in vitro selection strategy optimized the linkage so that the streptavidin-binding aptamer is only activated by an editing-induced conformational change. An electrochemiluminescent signal results from the ruthenium label when the reporter is bound to the bottom of a streptavidin-coated microtiter plate where it can be stimulated by a carbon electrode. Chemical probing, mutagenesis, and binding affinity measurements were used to characterize the reporter. The highly sensitive assay could be adapted to a broad range of RNA processing reactions.
对于锥虫寄生虫而言既至关重要又具有特异性的RNA编辑反应，是新药开发的一个有吸引力的靶点。尽管对化学文库进行高通量筛选是常用于鉴定新药的一种强大策略，但现有的体外编辑检测方法对于这种方法可行来说并不具备必要的灵敏度和形式。本文描述了一种钌标记的报告RNA，它克服了这些局限性，因为它既能在低飞摩尔范围内检测编辑产物，又非常适合高通量形式。该报告RNA由与链霉亲和素结合适体相连的RNA编辑底物组成，该适体最初处于无活性构象。一种体外筛选策略优化了这种连接，使得链霉亲和素结合适体仅通过编辑诱导的构象变化而被激活。当报告分子与链霉亲和素包被的微量滴定板底部结合时，钌标记会产生电化学发光信号，在那里它可以被碳电极激发。化学探针、诱变和结合亲和力测量被用于表征该报告分子。这种高灵敏度检测方法可适用于广泛的RNA加工反应。
