Chemigenetic Encoding of Fluorescent Dyes Enables High‐Fidelity and Wash‐Free Imaging of Proteins in Live Cells

Fluorescent dyes are indispensable chemical tools for protein labeling, yet their utility in live-cell imaging remains constrained by background fluorescence from off-target interactions. A chemigenetic strategy is presented that integrates synthetic dye chemistry with genetically encoded fluorescence to achieve high-fidelity, wash-free imaging of target proteins. By leveraging Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) and Halo-tag dyes, a system is engineered where fluorescence emission depends on FPs donor excitation, ensuring only dyes bound to target protein are fluorescent, while nonspecifically bound dyes remain dark, enhancing the signal-to-noise ratio (SNR). This approach is implemented by fusing Halo-tag with FPs (sfGFP, mCherry) to create FRET pairs with Halo dyes (O-Rho, Si-Rho), yielding chemigenetic fluorophores (GLH-O, CLH-Si) with improved SNR even at low expression levels. By optimizing FRET efficiency, the developed probes CH-Si are combined with standard FPs and commercial dyes, and achieve four-color structured illumination microscopy (SIM) imaging of target proteins, and track the mitochondria interactions with endoplasmic reticulum, and lysosomes. Furthermore, by incorporating the ultra-stable mStayGold as donor, a photostable fluorophore (SLH-O) capable of long-term super-resolution imaging is developed. This versatile strategy combines chemical and genetic tools, offering a generalizable platform for high-precision studies of protein and cellular processes.