Cardo Lucia, Karunatilaka Krishanthi S, Rueda David, Sigel Roland K O
Institute of Inorganic Chemistry, University of Zurich, Zurich, Switzerland.
Methods Mol Biol. 2012;848:227-51. doi: 10.1007/978-1-61779-545-9_15.
A procedure to investigate the folding of group II intron by single molecule Fluorescence Resonance Energy Transfer (smFRET) using total internal reflection fluorescence microscopy (TIRFM) is described in this chapter. Using our previous studies on the folding and dynamics of a large ribozyme in the presence of metal ions (i.e., Mg(2+) and Ca(2+)) and/or the DEAD-box protein Mss116 as an example, we here describe step-by-step procedures to perform experiments. smFRET allows the investigation of individual molecules, thus, providing kinetic and mechanistic information hidden in ensemble averaged experiments.
本章介绍了一种使用全内反射荧光显微镜（TIRFM）通过单分子荧光共振能量转移（smFRET）研究II组内含子折叠的方法。以我们之前关于在金属离子（即Mg(2+)和Ca(2+)）存在下以及/或者DEAD-box蛋白Mss116存在时大型核酶的折叠和动力学的研究为例，我们在此描述进行实验的逐步程序。smFRET能够对单个分子进行研究，因此可以提供隐藏在总体平均实验中的动力学和机制信息。
