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<p>We present a synthetic tool kit of antigen-stabilizable fluorescent nanobodies (VIS–Fbs) spanning the entire visible spectrum from 450 nm to 660 nm. By engineering over 20 fluorescent proteins (FPs) and biosensors into 8 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, as well as 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 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.</p>

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

  • 期刊论文

  • 发表日期：

    2026年4月22日

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  • Natalia V. Barykina,
  • Erin M. Carey,
  • Olena S. Oliinyk,
  • Juliana M. Mendonça-Gomes,
  • Sofia de Oliveira,
  • Axel Nimmerjahn,
  • Vladislav V. Verkhusha

摘要

We present a synthetic tool kit of antigen-stabilizable fluorescent nanobodies (VIS–Fbs) spanning the entire visible spectrum from 450 nm to 660 nm. By engineering over 20 fluorescent proteins (FPs) and biosensors into 8 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, as well as 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 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.