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

Mike Filius, Sung Hyun Kim, Ivo Severins, Chirlmin Joo

Research output: Contribution to journalArticlepeer-review

15 Scopus citations


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.

Original languageEnglish
Pages (from-to)3295-3301
Number of pages7
JournalNano Letters
Issue number7
StatePublished - 14 Apr 2021

Bibliographical note

Publisher Copyright:
© 2021 The Authors. Published by American Chemical Society.


  • DNA nanotechnology
  • Single-molecule FRET
  • single-molecule multiplexing
  • structural biology


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