Engineering a highly selective FRET-based genetically encoded Zn2+ sensor for real-time live-cell zinc imaging.

Genetically encoded Zn2+ probes (GEZPs) provide powerful tools for visualizing intracellular zinc dynamics with spatial and temporal resolution in living cells. Recently developed single-module ZnM-based scaffolds are derived from CaM-based metal-binding motifs and can serve as effective sensing elements. ZnM preferentially binds Zn2+, but their selectivity can be partially compromised by competing ions such as Ni2+, which share similar coordination properties. Here, we developed TV-ZnM1, a ratiometric FRET-based GEZP constructed through a stepwise engineering strategy centered on the ZnM sensing module. First, we optimized the linker architecture guided by AlphaFold3 structural predictions to improve donor-acceptor coupling. Next, we refined the metal-binding site through targeted residue substitutions, achieving high Zn2+ selectivity and reduced interference from competing divalent ions. The probe exhibits submicromolar Zn2+ affinity (Kd = 4.63 × 10-7 M by fluorescence titration and 1.17 × 10-7 M by PAR assay) and a robust ratiometric response with a 1.71-fold change in living cells. TV-ZnM1 enables quantitative, real-time imaging of intracellular Zn2+ dynamics during pyroptosis, allowing the first continuous observation of zinc redistribution throughout the entire pyroptotic process. This modular strategy offers a versatile framework for designing FRET-based sensors applicable to diverse intracellular targets, expanding the toolkit for live-cell imaging applications.