# Photoclickable Halotag ligands for spatiotemporal multiplexed protein labeling on living cells

## TL;DR

A new light-controlled labeling method allows precise, multicolor tracking of proteins on living cells using HaloTag and photoclickable ligands.

## Contribution

A naphthyl-substituted DIO ligand enables fast, selective, and multiplexed spatiotemporal labeling of HaloTagged proteins on living cells.

## Key findings

- A naphthyl-substituted DIO ligand shows superior photoswitching and photoclick efficiency for HaloTag labeling.
- Two- and three-color labeling of cell surface regions is achieved with high spatial and temporal precision.
- The system supports combinatorial labeling and is compatible with long-term tracking and cellular barcoding.

## Abstract

Precise spatiotemporal control over fluorescence labeling is a powerful approach for selective marking and tracking of proteins of interest within living systems. Here, we report a photoclickable labeling platform based on the 2,3-diaryl-indanone epoxide (DIO) photoswitch scaffold and the self-labeling protein HaloTag. Upon illumination, the protein-bound DIO undergoes reversible photoisomerization to form a metastable oxidopyrylium ylide (PY) that reacts with ring-strained dipolarophiles via [5 + 2] cycloaddition, enabling covalent spatiotemporal labeling. We synthesize and characterize a library of DIO-HaloTag and DIO-SNAP-tag ligands, systematically examining the effects of linker architecture and scaffold substitution on the photoswitching and photoclick reactivity in vitro and on living cells. We identify a naphthyl-substituted DIO ligand exhibiting superior photoswitching and photoclick efficiency, allowing fast, selective labeling of HaloTagged proteins on the surface of living cells using visible light activation (405 nm). Using this system, we achieve two- and three-color labeling of defined cell surface regions with excellent spatial and temporal precision, additionally allowing combinatorial labeling. Together, this work establishes a versatile framework for multiplexed, light-directed protein labeling compatible with living systems, with promising future applications including multiplexed long-term tracking and cellular barcoding.

A novel photoclickable HaloTag ligand enables multicolor spatiotemporal labeling of proteins on the surface of living cells.