Kristina Ganzinger - DNA-PAINT single-particle tracking - Imaging ONEWORLD

Описание: This week features - DNA-PAINT single-particle tracking (DNA-PAINT-SPT) enables extended single-molecule studies of membrane protein interactions by Dr Kristina Ganzinger.

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 commonly-used 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-labelled oligonucleotides transiently binding to a complementary DNA strand attached to the molecule of interest. Several fluorescently-labelled oligonucleotides can be accommodated on the complementary DNA strand simultaneously, and enable tracking of individual molecules for unprecedented observation periods. I will show that DNA-PAINT based single-particle tracking (DNA-PAINT-SPT) can be readily combined with existing protein tagging methods, such as SNAP tags or nanobodies. I will report on leveraging this technique from a proof-of-principle implementation to a useful tool for the SPT community by introducing simultaneous live cell dual-colour DNA-PAINT-SPT for quantifying protein dimerization and tracking proteins in living cell membranes, demonstrating its improved performance over single-dye SPT.