A Genetic Reporter for Visualizing Nitroreductase Activity Using Magnetic Resonance Imaging

The genetic expression of bacterial nitroreductase offers a potent approach for the targeted ablation of eukaryotic cells, holding significant potential for both fundamental research and gene‐directed enzyme‐prodrug therapy. The effectiveness of nitroreductase in these applications critically depends on robust transgene expression, driving a need for imaging probes to visualize nitroreductase expression and activity in gene‐engineered cells. Current probes predominantly rely on optical methods, which are inherently limited by their poor penetration in deep, optically dense tissues. Here, we report the molecular engineering of a genetically encoded reporter capable of detecting nitroreductase activity using magnetic resonance imaging (MRI). This reporter comprises human aquaporin fused to a dihydrofolate reductase (DHFR)‐destabilizing domain. The sensor remains in a degraded “off‐state” until nitroreductase‐mediated cleavage of a caged trimethoprim prodrug releases active trimethoprim, which binds to and stabilizes aquaporin, thereby restoring MRI signals. We optimized signal induction by screening N‐ and C‐terminal fusions and incorporating an evolved DHFR variant. We validated a direct, dose‐dependent correlation between nitroreductase expression and diffusion‐weighted MRI contrast across diverse human cell types. These findings validate the use of destabilized aquaporins as switchable, metal‐free MRI reporters for monitoring nitroreductase activity, facilitating the noninvasive evaluation of nitroreductase‐based genetic tools in intact deep tissues.