Exchangeable HaloTag Ligands for Super-Resolution Fluorescence Microscopy

Abstract

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.

Reversible Live‐Cell Labeling with Retro‐engineered HaloTags Enables Long‐Term High‐ and Super‐Resolution Imaging

“…With these features, reHaloTag‐based labeling nicely complements the recently developed non‐covalent HaloTag ligands (xHTLs) [19] . These xHTLs bind substantially more transiently, with complex lifetimes in the milliseconds to second's regime, compared to the 15–200 s observed for the reHaloTag system.…”

“…With these features, reHaloTag-based labeling nicely complements the recently developed non-covalent HaloTag ligands (xHTLs). [19] These xHTLs bind substantially more transiently, with complex lifetimes in the milliseconds to second's regime, compared to the 15-200 s observed for the reHaloTag system. The longer lifetimes and resulting higher affinities of reHaloTag variants compared to the xHTL system can potentially ensure higher signal-to-background in ensemble measurements as well as higher tracking fidelity in single molecule imaging approaches.…”

De Novo Designed Self-Assembling Rhodamine Probe for Real-Time, Long-Term and Quantitative Live-Cell Nanoscopy

“…). The strategy is also compatible with the recently reported exchangeable HaloTag labeling method, which will further reduce photobleaching and enable long-term super-resolution imaging. , We envision our strategy will inspire the creation of more innovative self-assembling fluorogenic probes for live-cell nanoscopy.…”

Single-molecule tracking (SMT): a window into live-cell transcription biochemistry

“…The development of genetically encoded, self-labeling tags like Halo and SNAP, together with cellpermeable fluorophores like JF dyes, has combined the best of both worlds, making it possible to label target proteins with bright, photostable synthetic fluorophores in live cells [109][110][111][112][113][114]. These tools continue to be improved [115,116].…”