High-Resolution Single-Molecule FRET via DNA eXchange (FRET X)

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.

Automated summary

The research team introduced a new method that allows for determining multiple distances between FRET pairs in a single object by resolving the FRET efficiency of multiple fluorophore pairs through transient binding of short DNA strands. This FRET X technology is expected to be a tool for high-resolution analysis of biomolecules and nanostructures.