A novel single molecule fluorescence quenching technique for measuring distances below 3 nm

Single-molecule FRET (smFRET) is a powerful biophysical tool for measuring intramolecular distances in biomolecules, however FRET is only effective over a 3-10nm scale. In this work we advance a quenching based method which uses Cy3B and Atto647N, a common dye pair used for smFRET. The quenching effect arising from this dye pair is sensitive to distance changes below 3nm, which is significantly lower than its R0 of 6.2nm. It has previously been used to measure binary on/off transitions, such as local DNA melting in polymerase complexes.