Department of Chemistry, University of Zurich, Zurich, Switzerland.
Laserinstitut Hochschule Mittweida, University of Applied Sciences Mittweida, Mittweida, Germany.
Methods Mol Biol. 2020;2113:1-16. doi: 10.1007/978-1-0716-0278-2_1.
Imaging fluorescently labeled biomolecules on a single-molecule level is a well-established technique to follow intra- and intermolecular processes in time, usually hidden in the ensemble average. The classical approach comprises surface immobilization of the molecule of interest, which increases the risk of restricting the natural behavior due to surface interactions. Encapsulation of such biomolecules into surface-tethered phospholipid vesicles enables to follow one molecule at a time, freely diffusing and without disturbing surface interactions. Further, the encapsulation allows to keep reaction partners (reactants and products) in close proximity and enables higher temperatures otherwise leading to desorption of the direct immobilized biomolecules.Here, we describe a detailed protocol for the encapsulation of a catalytically active RNA starting from surface passivation over RNA encapsulation to data evaluation of single-molecule FRET experiments in TIRF microscopy. We present an optimized procedure that preserves RNA functionality and applies to investigations of, e.g., large ribozymes and RNAs, where direct immobilization is structurally not possible.
在单分子水平上对荧光标记的生物分子进行成像，是一种能够实时追踪分子内和分子间过程的成熟技术，而这些过程通常隐藏在整体平均水平中。经典的方法包括将感兴趣的分子固定在表面上，这会增加由于表面相互作用而限制其自然行为的风险。将这些生物分子包封在表面连接的磷脂囊泡中，可以实现逐个分子的追踪，使分子自由扩散，并且不会干扰表面相互作用。此外，包封可以使反应伙伴（反应物和产物）保持近距离，并允许在其他情况下会导致直接固定化生物分子解吸的更高温度。在这里，我们描述了一种从表面钝化到 RNA 包封再到 TIRF 显微镜中单分子 FRET 实验数据评估的详细方案，用于从表面钝化到 RNA 包封再到 TIRF 显微镜中单分子 FRET 实验数据评估的详细方案。我们提出了一种优化的程序，该程序可以保持 RNA 的功能，并且适用于例如大核酶和 RNA 的研究，在这些研究中，直接固定在结构上是不可能的。
