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.
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