Exchangeable HaloTag Ligands for Super-Resolution Fluorescence Microscopy

The specific and
covalent labeling of the protein HaloTag with
fluorescent probes in living cells makes it a powerful tool for bioimaging.
However, the irreversible attachment of the probe to HaloTag precludes
imaging applications that require transient binding of the probe and
comes with the risk of irreversible photobleaching. Here, we introduce
exchangeable ligands for fluorescence labeling of HaloTag (xHTLs)
that reversibly bind to HaloTag and that can be coupled to rhodamines
of different colors. In stimulated emission depletion (STED) microscopy,
probe exchange of xHTLs allows imaging with reduced photobleaching
as compared to covalent HaloTag labeling. Transient binding of fluorogenic
xHTLs to HaloTag fusion proteins enables points accumulation for imaging
in nanoscale topography (PAINT) and MINFLUX microscopy. We furthermore
introduce pairs of xHTLs and HaloTag mutants for dual-color PAINT
and STED microscopy. xHTLs thus open up new possibilities in imaging
across microscopy platforms for a widely used labeling approach.