FRET Below and Beyond: New tools for single-molecule fluorescence studies of DNA and Proteins

Single-molecule FRET (smFRET) is a family of techniques within single-molecule fluorescence spectroscopy, which involve measuring the FRET efficiency between a pair of dyes within or between single molecules. As FRET results in a signal that is dependent on the interdye distance, measuring FRET on single molecules allows the experimenter to monitor conformations of biomolecules in a manner free from ensemble averaging. In recent years, advances in smFRET techniques have given access to absolute intramolecular distances for resolving structures, and a wealth of insight into biomolecular kinetics at equilibrium that would not be feasible with ensemble techniques. However, a lack of standardization in technique, and high entry barriers to acquiring instrumentation have made smFRET techniques inaccessible to all but the most specialist of single-molecule fluorescence spectroscopy researchers. Here I present work demonstrating the reproducibility of the accurate FRET technique, alongside many other smFRET labs, on a cost effective and open-source confocal smFRET microscope, as well as a new contact-quenching based method for measuring short distances, which can work in parallel with smFRET experiments, adding to the tool kit of single-molecule fluorescence spectroscopists. Finally, I will present work using these methods to investigate various biomolecular systems, exploring the conformational landscapes of DNA binding proteins, and the flexibility of the damaged substrates they recognise.