Single-molecule studies of membrane protein interactions via continuous DNA-mediated fluorophore exchange

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.