Synthetic multicolor antigen-stabilizable nanobody platform for intersectional labelling and functional imaging

We present a synthetic toolkit of antigen-stabilizable fluorescent nanobodies (VIS-Fbs) spanning the entire visible spectrum from 450 nm to 660 nm. By engineering over twenty fluorescent proteins (FPs) and biosensors into eight nanobodies, we established a generalizable design of VIS-Fbs, which fluoresce brightly only upon binding to cognate antigens. Our synthetic approach includes constitutive, photoactivatable and photoswitchable FPs, and intensiometric FP-based biosensors. VIS-Fbs carrying biosensors enable simultaneous monitoring of two metabolites at confined locations, while FP-based VIS-Fbs targeting biosensors allow ratiometric functional imaging in the mouse brain. We further used VIS-Fbs to track endogenous β-catenin dynamics in zebrafish embryos during normal development and under Wnt/β-catenin signaling modulation. VIS-Fbs provide background-free visualization of intracellular proteins, multicolor detection of multiple antigens, and selective targeting of defined cell populations and compartments. This synthetic biology-driven platform enables precise studies of protein dynamics, cellular processes, and complex biological systems with high specificity and minimal background.