Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University, 7-1-20 Minatojimaminamimachi, Chuo-ku, Kobe 650-0047, Japan.
Bioconjug Chem. 2009 Dec;20(12):2242-6. doi: 10.1021/bc9002184.
Bioactive small molecules such as metabolites and drugs play important roles in regulating biological functions. A technique for visualizing such small molecules is very useful to understand their molecular mechanisms. In this study, an RNA-protein conjugate, which consists of an RRE-RNA sensor protein (EYFP-Rev-ECFP) and an altered RRE-RNA, was constructed to detect bioactive small molecules by fluorescent resonance energy transfer (FRET). We designed a theophylline-aptamer-inserted RRE-RNA (Theo-RRE) to detect theophylline as a model target molecule. Theo-RRE formed an RNA-protein conjugate with EYFP-Rev-ECFP in the presence of theophylline. As a result, theophylline was specifically detected down to 10 microM by the FRET increase in distinction from theophylline analogue, caffeine, in cell lysates.
生物活性小分子，如代谢物和药物，在调节生物功能方面发挥着重要作用。一种可视化这些小分子的技术对于理解它们的分子机制非常有用。在这项研究中，构建了一种 RNA-蛋白质缀合物，由 RRE-RNA 传感器蛋白（EYFP-Rev-ECFP）和改变的 RRE-RNA 组成，通过荧光共振能量转移（FRET）来检测生物活性小分子。我们设计了一种茶碱适体插入的 RRE-RNA（Theo-RRE）来检测茶碱作为模型靶分子。在茶碱存在的情况下，Theo-RRE 与 EYFP-Rev-ECFP 形成 RNA-蛋白质缀合物。结果，与茶碱类似物咖啡因相比，在细胞裂解物中通过 FRET 的增加可以特异性检测到低至 10μM 的茶碱。
