Tracking Single Particles for Hours via Continuous DNA-mediated Fluorophore Exchange

Fluorophores are commonly used to covalently label biomolecules for monitoring their motion in single particle tracking experiments. However, photobleaching is still a major bottleneck in these experiments, as the fluorophores finite photon budget typically limits observation times to merely a few seconds. Here, we overcome this inherent constraint via continuous fluorophore exchange based on DNA-PAINT, whereby fluorescently-labeled oligonucleotides bind to a 54 bp single-stranded DNA handle attached to the molecule of interest. When we assayed our approach in vitro by tracking single DNA origami, first surface-immobilized and subsequently diffusing on supported lipid bilayers, we were able to observe these origami for up to hours without losing their fluorescence signals. Our versatile and easily implemented labeling approach allows monitoring single-molecule motion and interactions over unprecedented observation periods, opening the doors to advanced quantitative studies.