National Cancer Institute, NIH, Bethesda, MD 20892-4256, USA.
Methods. 2010 Jun;51(2):220-32. doi: 10.1016/j.ymeth.2010.01.022. Epub 2010 Jan 22.
The application of FRET-based molecular biosensors provided confirmation of the central model of Ran GTPase function and led to important new insights into its physiological role. In many fields of cell biology, methods employing FRET are a standard approach that is becoming increasingly accessible due to advances in instrumentation and available fluorophores. However, the optimal design of a FRET sensor remains to be the cornerstone of any successful FRET application. Utilizing the recent literature on FRET applications and our studies on Ran, we outline the basic considerations involved in designing molecular FRET sensors. We point to several broadly applicable principles that were used in many different FRET sensors that can detect a wide range of molecular events. Using the FRET sensors for Ran that we created as examples, we then focus on the practical aspects of FRET assays. We describe the preparation of a bipartite FRET sensor consisting of ECFP-Ran and EYFP-importin beta and its validation as a reporter for FRET-based high throughput screening in small molecule libraries. Finally, we review the design and optimization of monomolecular FRET sensors that monitor the RanGTP-RanBP1 interaction, and of sensors detecting the RanGTP-regulated importin beta cargo release.
基于荧光共振能量转移（FRET）的分子生物传感器的应用为 Ran GTPase 功能的中心模型提供了证实，并为其生理作用带来了重要的新见解。在细胞生物学的许多领域中，采用 FRET 的方法是一种标准方法，由于仪器和可用荧光团的进步，这种方法变得越来越容易获得。然而，FRET 传感器的最佳设计仍然是任何成功的 FRET 应用的基石。利用关于 FRET 应用的最新文献和我们在 Ran 上的研究，我们概述了设计分子 FRET 传感器所涉及的基本考虑因素。我们指出了在许多不同的 FRET 传感器中广泛应用的几个普遍适用的原则，这些传感器可以检测广泛的分子事件。我们使用我们创建的用于 Ran 的 FRET 传感器作为示例，然后重点介绍 FRET 测定的实际方面。我们描述了由 ECFP-Ran 和 EYFP-importin beta 组成的二分 FRET 传感器的制备，并将其验证为小分子文库中基于 FRET 的高通量筛选的报告物。最后，我们回顾了监测 RanGTP-RanBP1 相互作用的单分子 FRET 传感器的设计和优化，以及检测 RanGTP 调节的 importin beta 货物释放的传感器的设计和优化。
