Nature Commun. 6, 6992 (2015)

Credit: NATURE PUBLISHING GROUP

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.