Development of a microscopic platform for real-time monitoring of biomolecular interactions
Development of a microscopic platform for real-time monitoring of biomolecular interactions
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DOI：
10.1101/gr.4235806
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发表时间：
2006-01-01
期刊：
影响因子：
7
通讯作者：
Harada, Y
中科院分区：
文献类型：
--
作者：
Sasuga, Y;Tani, T;Harada, Y
我们开发了一种新的显微平台，通过结合微珠标记技术与全内反射荧光显微镜（TIRFM）的分子相互作用的实时分析。探针微珠的光学操作，然后在固体表面上进行光固定，使得LIS能够产生具有极高密度的阵列（在25个药丸X 25个药丸区域中> 100个微珠），并且TIRFM使得可以监测荧光标记的靶在探针微珠上的结合反应而不去除游离靶。我们通过分析抗原和抗体之间以及小化合物和蛋白质之间的相互作用证明了该平台的高性能。然后，大肠杆菌的总细胞裂解物中的重组蛋白水平进行了定量的关联动力学使用抗体固定的微珠阵列，作为一个模型的蛋白质谱阵列。此外，结合体外合成偶联蛋白标记，我们还可以从动力学角度分析核因子κ B（p50）与DNA的相互作用。这些结果表明，该平台使Lis能够：（1）实时监测荧光标记的靶与多个探针的结合过程而不去除游离靶，（2）仅从所有早期阶段的缔合动力学确定游离靶的浓度，以及（3）大大减少用于分子相互作用分析的靶溶液的所需体积，原则上至亚纳升。这种基于微珠的微阵列系统的独特功能开辟了在极小体积的靶溶液（如单细胞提取物）中探索具有广泛亲和力的分子相互作用的途径。
We developed a new microscopic platform for the real-time analysis of molecular interactions by combining microbead-tagging techniques with total internal reflection fluorescent microscopy (TIRFM). The optical manipulation of probe microbeads, followed by photo immobilization on a solid surface, enabled LIS to generate arrays with extremely high density (> 100 microbeads in a 25 pill x 25 pill area), and TIRFM made it possible to monitor the binding reactions Of fluorescently labeled targets onto probe microbeads without removal of free targets. We demonstrated the high performance of this platform through analyses of interactions between antigen and antibody and between small compounds and proteins. Then, recombinant protein levels in total cellular lysates of Escherichia coli were quantified from the association kinetics using antibody-immobilized microbead arrays, which served as a model for a protein-profiling array. Furthermore, in combination with in vitro synthesis-coupled protein labeling, we could kinematically analyze the interaction Of nuclear factor kappa B (p50) with DNA. These results demonstrated that this platform enabled Lis to: (1) monitor binding processes of fluorescently labeled targets to multiple probes in real-time without removal of free targets, (2) determine concentrations of free targets only from the association kinetics at ail early phase, and (3) greatly reduce the required volume of the target solution, in principle to subnanoliter, for molecular interaction analysis. The unique features of this microbead-based microarray system open the way to explore molecular interactions with a wide range of affinities in extremely small volumes of target solutions, such as extracts from single cells.