Halo Tag

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Introduction

Crystal structure of psHaloTag labeled with JF635-HTL in the dark state

Drag the structure with the mouse to rotate

The [Photoswitchable Halo Tag] (psHaloTag) is a unique chemigenetic system designed to provide reversible, light-controlled fluorescence tunable through both genetic and synthetic modifications, with promising applications for dynamic imaging in biological microscopy ^[1]. psHaloTag addresses a major gap in the availability of reliable, reversible systems suitable for live-cell imaging. Many current systems are limited in tracking long-term, cyclical processes or in refreshing the pool of observable molecules. psHaloTag combines the genetically encoded HaloTag protein with the light-sensing sAsLOV2 domain. When illuminated with 450 nm light, psHaloTag undergoes a reversible change that activates a bound rhodamine dye ligand, resulting in a significant increase in fluorescence. This strong, multiple-cycle reporter overcomes the limitations of irreversible systems. It is an important tool for achieving precise spatiotemporal control in demanding applications such as live-cell Super-Resolution Microscopy (SMLM), where many established photosensitive probes cannot be reactivated.

1 Features
2 Mechanism
3 Structural highlights
4 Relevance

Features

Photoswitching | Reversible ON/OFF fluorescence control using 450 nm light.
Chemigenetic | Genetically encoded protein plus high-performance synthetic dye.
Live-Cell Ready | Robust, visible-light activation across various targets.

Mechanism

The psHaloTag works as an allosteric photoswitch.

Protein Scaffold: An engineered HaloTag protein with the light-sensing sAsLOV2 domain.
Ligand: Binds to a fluorogenic rhodamine dye (JF635-HTL).

Light ON (450 nm): Light activates the sAsLOV2 domain using Flavin Mononucleotide (FMN)

cofactor, causing a conformational change (Jα helix unfolding).

Fluorescence ON: This change forces the bound dye into its fluorescent state.

Light OFF (Dark): The protein structure relaxes back to its original shape, shifting

the dye back to its non-fluorescent state.

Fig.1. General principle of photoswitchable HaloTag (psHaloTag) and open-closed equilibrium of JF635-HaloTag ligand (HTL). L1 and L2 denote linkers.

The psHaloTag variants show a strong, reversible, deep-red fluorescence turn-on when activated by light. This provides a well-controlled tool for improved imaging.

Structural highlights

The protein is made by inserting the light-sensing sAsLOV2 domain into the HaloTag protein. An extended helix connects the domains. This connection makes sure the light-induced structural change from the LOV domain is effectively transmitted to the HaloTag region, where the rhodamine dye is bound. Performance-boosting mutations are found near the headgroup of the rhodamine dye. These mutations likely stabilize the interaction to enhance the fluorescence ON/OFF ratio.

Relevance

Although irreversibly photoactivatable fluorophores are well established, reversible reporters that can be reactivated multiple times remain scarce, and only a few have been applied in living cells using generalizable protein labelling methods. This system improves techniques like SMLM by allowing precise control of single-molecule emitter density over time by achieving sub-diffraction resolution in living cells. Also, it exploits high-quality synthetic dyes for bright colours and stability in light whose performance does not rely on oxygen and is less affected by pH changes compared to traditional fluorescent proteins (FPs).