Single-molecule FRET is a versatile tool to study nucleic acids and proteins at the nanometer scale. However, currently, only a couple of FRET pairs can be reliably measured on a single object, which makes it difficult to apply single-molecule FRET for structural analysis of biomolecules. Here, we present an approach that allows for the determination of multiple distances between FRET pairs in a single object. We use programmable, transient binding between short DNA strands to resolve the FRET efficiency of multiple fluorophore pairs. By allowing only a single FRET pair to be formed at a time, we can determine the pair distance with subnanometer precision. The distance between other pairs are determined by sequentially exchanging DNA strands. We name this multiplexing approach FRET X for FRET via DNA eXchange. Our FRET X technology will be a tool for the high-resolution analysis of biomolecules and nanostructures.
Bibliographical noteFunding Information:
We thank Viktorija Globyte and Raman van Wee for critical reading and feedback. We thank Misha Klein for help on the analysis software. C.J. was supported by Vrije Programma (SMPS) of the Foundation for Fundamental Research on Matter and Human Frontier Science Program (RGP0026/2019).
© 2021 The Authors. Published by American Chemical Society.
- DNA nanotechnology
- Single-molecule FRET
- single-molecule multiplexing
- structural biology