Development and Characterization of a Red Fluorescent Protein-Based Sensor RZnP1 for the Detection of Cytosolic Zn²

Fluorescent sensors have been developed to record Zn²⁺ dynamics and measure Zn²⁺ concentrations within the cell. Most previous efforts on developing single-wavelength sensors are focused on green sensors. Here, we engineer a genetically encoded, single red fluorescent protein-based Zn²⁺ sensor, Red Zinc Probe (RZnP1), which can detect intracellular concentrations of Zn²⁺. RZnP1 demonstrates a sensitive response to cytosolic Zn²⁺ (K_d = 438 pM), decent brightness (quantum yield (QY) = 0.15), good in situ dynamic range (F_max/F_min = 4.0), and specificity for Zn²⁺ over other biologically relevant metal cations. RZnP1 offers a way to image Zn²⁺ with multiple intracellular ions in tandem. We demonstrate the simultaneous recording of Zn²⁺ and Ca²⁺ using RZnP1 alongside the Ca²⁺ sensor GCaMP5G in HeLa cells. We also use RZnP1 with mito-GZnP2, a green fluorescent protein (GFP)-based mitochondrial Zn²⁺ sensor, to track Zn²⁺ dynamics in the cytosol and mitochondria concurrently in rat primary neuron culture. Our work not only expands the toolbox of Zn²⁺ sensors but also demonstrates techniques for imaging Zn²⁺ dynamics along with other cations and between multiple subcellular compartments simultaneously.