SNAP-tag2 for faster and brighter protein labeling
Stefanie Kühn, Veselin Nasufović, Jonas Wilhelm, Julian Kompa, Eline M.F. de Lange, Yin‐Hsi Lin, Cornelia Egoldt, Jonas S. Fischer, Artem Lennoi, Mirosław Tarnawski, Jochen Reinstein, Rifka Vlijm, Julien Hiblot, Kai Johnsson
Abstract
Abstract SNAP-tag is a powerful tool for labeling proteins with synthetic fluorophores in bioimaging. However, its utility in live-cell applications can be constrained by its relatively slow labeling kinetics and the limited cell permeability of its substrates. Here, we introduce improved labeling substrates and an engineered SNAP-tag for faster labeling in vitro and in live cells. SNAP-tag2 presents a second-order rate constant with rhodamine substrates that approaches 10 7 s −1 M −1 , a 100-fold improvement over the corresponding SNAP-tag–substrate pairs. When labeled with highly fluorogenic dyes, SNAP-tag2 also shows a fivefold increase in fluorescence brightness relative to currently used SNAP-tag. The increased labeling kinetics and brightness of SNAP-tag2 translate into greatly improved performance in various live-cell (super-resolution) imaging applications.