Quantifying protein-protein interactions in cell membranes is key to understand many cellular processes, including a cell’s communication with its surroundings via cell surface receptors. While single-molecule tracking is a valuable tool to measure receptor-ligand interaction kinetics in cell membranes, photobleaching limits the observation times of individual molecules to only a few seconds with commonlyused fluorescent proteins or dyes. In practice, this makes it often hard to reliably distinguish real interactions from random colocalization events. Here, we show that this limitation can be overcome by a new labelling method based on DNA-PAINT, with fluorescently-labeled oligonucleotides transiently binding to a complementary DNA strand attached to the molecule of interest. Several fluorescentlylabeled oligonucleotides can be accommodated on the complementary DNA strand simultaneously, and enable tracking of individual molecules for unprecedented observation periods. We present this new method that can be readily combined with existing protein tagging methods, such as SNAP or CLIP tags. Furthermore, we show its implementation invitro and in live cells by studying the dimerization of FKBP proteins that were reconstituted on a supported lipid bilayer or expressed at the cell membrane.

Springer Science
Eur. Biophys. J.
Physics Cellular Interactions

Niederauer, C, Wang-Henderson, M, Stein, J, Stehr, F, Bauer, J, Jungmann, R, … Ganzinger, K.A. (2021). Single-molecule studies of membrane protein interactions via continuous DNA-mediated fluorophore exchange. In Eur. Biophys. J. (Vol. 50, pp. 54–54). Springer Science. doi:10.1007/s00249-021-01558-w