Multiplexed no-wash cellular imaging using BenzoTag, an evolved self-labeling protein.

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Self-labeling proteins like HaloTag7 are crucial for localizing synthetic molecules, such as fluorescent dyes, within cells.
• HaloTag7's utility is limited by slow labeling kinetics with most dyes, hindering live-cell applications.
• Fast labeling rates are essential for real-time cellular assays.

Purpose of the Study:

• To engineer a novel HaloTag variant, BenzoTag, with enhanced performance using a fluorogenic benzothiadiazole dye.
• To improve conjugation kinetics and signal amplification for robust live-cell labeling.
• To enable multiplexed labeling assays by assessing orthogonality with existing systems.

Main Methods:

• Yeast surface display was employed for molecular evolution of HaloTag variants.
• The performance of the BenzoTag-benzothiadiazole system was evaluated against the HaloTag7-silicon rhodamine system.
• Labeling kinetics, signal intensity, and dye concentration requirements were assessed in live cells.

Main Results:

• The BenzoTag-benzothiadiazole system demonstrated significantly improved conjugation kinetics compared to HaloTag7-silicon rhodamine.
• Robust, no-wash fluorescence labeling was achieved in live cells with rapid enzyme saturation (<100 seconds) and low dye concentrations (as low as 7 nM).
• BenzoTag exhibited orthogonality to HaloTag7, enabling multiplexed labeling experiments.

Conclusions:

• The BenzoTag-benzothiadiazole system offers superior performance for live-cell fluorescence labeling compared to current HaloTag7-based methods.
• This new system provides robust, sensitive, and time-efficient labeling for various cell-based assays.
• BenzoTag facilitates real-time monitoring of biological processes and drug actions in live cells.