Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
ACS Chem Biol. 2012 Feb 17;7(2):403-10. doi: 10.1021/cb200253h. Epub 2011 Nov 11.
Argonaute proteins are the core components of the microRNP/RISC. The biogenesis and function of microRNAs and endo- and exo- siRNAs are regulated by Ago2, an Argonaute protein with RNA binding and nuclease activities. Currently, there are no in vitro assays suitable for large-scale screening of microRNP/RISC loading modulators. We describe a novel in vitro assay that is based on fluorescence polarization of TAMRA-labeled RNAs loaded to human Ago2. Using this assay, we identified potent small-molecule inhibitors of RISC loading, including aurintricarboxylic acid (IC(50) = 0.47 μM), suramin (IC(50) = 0.69 μM), and oxidopamine HCL (IC(50) = 1.61 μM). Small molecules identified by this biochemical screening assay also inhibited siRNA loading to endogenous Ago2 in cultured cells.
Argonaute 蛋白是 microRNP/RISC 的核心成分。microRNA 的生物发生和功能以及内源性和外源性 siRNA 受到 Argonaute 蛋白（一种具有 RNA 结合和核酸酶活性的 Argonaute 蛋白）的调节。目前，还没有适合大规模筛选 microRNP/RISC 加载调节剂的体外测定法。我们描述了一种新的基于 TAMRA 标记的 RNA 加载到人 Ago2 的荧光偏振的体外测定法。使用该测定法，我们鉴定出有效的 RISC 加载小分子抑制剂，包括金顶菌素（IC（50）= 0.47 μM）、苏拉明（IC（50）= 0.69 μM）和氧化多巴胺盐酸盐（IC（50）= 1.61 μM）。通过这种生化筛选测定法鉴定的小分子也抑制了培养细胞中内源性 Ago2 的 siRNA 加载。
