Multiplexed No-wash Cellular Imaging Using BenzoTag, an Evolved Self-labeling Protein

Overview

Journal Chem Sci

Publisher Royal Society of Chemistry

Specialty Chemistry

Date 2024 Oct 21

PMID 39430930

Authors

Bryan J Lampkin

Benjamin J Goldberg

Joshua A Kritzer

Affiliations

Soon will be listed here.

Abstract

Self-labeling proteins are powerful tools for exploring biology as they enable the precise cellular localization of a synthetic molecule, often a fluorescent dye. HaloTag7 is the most popular self-labeling protein due to its broad utility, its bio-orthogonality, and the simplicity of its chloroalkane ligand. However, reaction rates of HaloTag7 with different chloroalkane-containing substrates are highly variable and rates are only very fast for rhodamine-based dyes. This is a major limitation for the HaloTag system because fast labeling rates are critical for live-cell assays. Here, we use yeast surface display to produce a HaloTag variant, BenzoTag, with improved performance with a fluorogenic benzothiadiazole dye. Molecular evolution improved conjugation kinetics and increased the signal from the dye-protein complex, allowing for robust, no-wash fluorescence labeling in live cells. The new BenzoTag-benzothiadiazole system has improved performance compared to the best existing HaloTag7-silicon rhodamine system, including saturation of intracellular enzyme in under 100 seconds and robust labeling at dye concentrations as low as 7 nM. The BenzoTag system was also found to be sufficiently orthogonal to the HaloTag7-silicon rhodamine system to enable multiplexed no-wash labeling in live cells. The BenzoTag system will be immediately useful for a large variety of cell-based assays monitoring biological processes and drug action in real time.

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