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

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.