单分子荧光共振能量转移 X.

Single-Molecule FRET X.

机构信息

Department of BioNanoScience, Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands.

出版信息

Methods Mol Biol. 2024;2694:203-213. doi: 10.1007/978-1-0716-3377-9_10.

Abstract

Fluorescence resonance energy transfer (FRET) is a photophysical phenomenon that has been repurposed as a biophysical tool to measure nanometer distances. With FRET by DNA eXchange, or FRET X, many points of interest (POIs) in a single object can be probed, overcoming a major limitation of conventional single-molecule FRET. In FRET X, short fluorescently labeled DNA imager strands specifically and transiently bind their complementary docking strands on a target molecule, such that at most a single FRET pair is formed at each point in time and multiple POIs on a single molecule can be readily probed. Here, we describe the sample preparation, image acquisition, and data analysis for structural analysis of DNA nanostructures with FRET X.

摘要

荧光共振能量转移（FRET）是一种光物理现象，现已被重新用作生物物理工具来测量纳米距离。通过 DNA 交换的荧光共振能量转移（FRET by DNA Exchange，或 FRET X），可以探测单个物体中的多个感兴趣点（POI），克服了传统单分子 FRET 的一个主要限制。在 FRET X 中，短的荧光标记 DNA 成像链特异性且短暂地与其在靶分子上的互补对接链结合，使得在每个时间点最多形成一个 FRET 对，并且可以很容易地探测单个分子上的多个 POI。在这里，我们描述了使用 FRET X 进行 DNA 纳米结构的结构分析的样品制备、图像采集和数据分析。