Nature Commun. 6, 6992 (2015)


When it comes to measuring the nanoscale real-time dynamics of single biomolecules non-invasively, the sensitivity of single-molecule fluorescence resonance energy transfer (smFRET) is hard to beat. However, low photon count rates as well as photobleaching and photoblinking issues have limited the technique's temporal resolution to 10 ms. Protein diffusion and conformational dynamics are, however, typically faster. Now, Nam Ki Lee and colleagues demonstrate that the dynamics of a single biomolecule in a buffer solution can be measured by smFRET for tens of milliseconds at submillisecond timescales by simply tethering the molecule to a freely diffusing liposome (through biotin–NeutrAvidin interactions) and using a photoprotection buffer. With this technique, the researchers resolved the 5-ms diffusional rate of a single protein on single-stranded DNA and the faster 1,500 s−1 conformational transition rate of a Holliday junction (a branched nucleic-acid structure). Compared with alternative smFRET approaches, the liposome-tethering variant needs a basic microscopy set-up, and does not require the immobilization of the sample on a glass slide or a high concentration of FRET samples.