Chemical Resources Laboratory, Tokyo Institute of Technology, 4259-R1-18 Nagatsuta-cho, Midori-ku, Yokohama, 226-8503, Japan.
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Biosensors (Basel). 2015 Mar 26;5(2):131-40. doi: 10.3390/bios5020131.
An antibiotic rapamycin is one of the most commonly used immunosuppressive drugs, and also implicated for its anti-cancer activity. Hence, the determination of its blood level after organ transplantation or tumor treatment is of great concern in medicine. Although there are several rapamycin detection methods, many of them have limited sensitivity, and/or need complicated procedures and long assay time. As a novel fluorescent biosensor for rapamycin, here we propose "Q'-body", which works on the fluorescence quench-release principle inspired by the antibody-based quenchbody (Q-body) technology. We constructed rapamycin Q'-bodies by linking the two interacting domains FKBP12 and FRB, whose association is triggered by rapamycin. The fusion proteins were each incorporated position-specifically with one of fluorescence dyes ATTO520, tetramethylrhodamine, or ATTO590 using a cell-free translation system. As a result, rapid rapamycin dose-dependent fluorescence increase derived of Q'-bodies was observed, especially for those with ATTO520 with a lowest detection limit of 0.65 nM, which indicates its utility as a novel fluorescent biosensor for rapamycin.
一种抗生素雷帕霉素是最常用的免疫抑制剂之一，也因其抗癌活性而受到关注。因此，器官移植或肿瘤治疗后其血药浓度的测定在医学上非常重要。尽管有几种雷帕霉素检测方法，但其中许多方法的灵敏度有限，和/或需要复杂的程序和较长的检测时间。作为雷帕霉素的一种新型荧光生物传感器，我们提出了“Q'-body”，它基于基于抗体的淬灭体（Q-body）技术的荧光猝灭-释放原理工作。我们通过连接 FKBP12 和 FRB 两个相互作用的结构域来构建雷帕霉素 Q'-body，其通过雷帕霉素触发结合。融合蛋白分别使用无细胞翻译系统在特定位置与荧光染料 ATTO520、四甲基罗丹明或 ATTO590 之一结合。结果，观察到 Q'-body 快速、剂量依赖性的荧光增强，尤其是与 ATTO520 的荧光增强，其最低检测限为 0.65 nM，表明其可作为雷帕霉素的新型荧光生物传感器。
