Single-molecule FRET and tracking of transfected biomolecules in living cells

Proteins and DNA in cells exhibit different conformational states, which are influenced by dynamic interactions with other biomolecules. All these interactions are affected by the molecules' localization within the cell, i.e., their compartmentalization. Such, in cellula, compartment-specific dynamics is difficult to measure, because of limitations in instrumentation, autofluorescence of cells, and the necessity to track diffusing molecules. Here, we present a bottom-up engineering approach, which allows us to track transfected proteins in cellula and to analyze time-resolved single-molecule FRET efficiencies. This has been achieved by alternating laser excitation-based dual-channel (donor and FRET, acceptor) tracking with a HILO microscope. We validate our strategy by characterizing long-term static FRET traces of customized DNA with known dye positions. We utilize two different transfection strategies, namely microinjection (physical) and a transfection mediated by the toxin Streptolysin-O (biological). By comparing in vitro and in cellula measurements we show that the cellular environment in this case changes the FRET efficiency by about 25%. In addition, we evaluate single-molecule FRET traces for the heat shock protein Hsp90 in cellula. The obtained FRET efficiency distribution is largely consistent with known Hsp90 structures and in vitro distributions, but also shows some clear differences. Altogether, we show that FRET-TTB opens the path to study protein state changes of transfected biomolecules in living cells, including their time-resolved cellular localization.