A Dual-Channel Self-Calibrated Fluorescent Probe for the Correlation of Nitroreductase Activity and DNA Damage

The therapeutic efficacy of hypoxia-activated prodrugs relies on nitroreductase (NTR)-mediated activation and the subsequent DNA damage caused by cytotoxic metabolites. Current methods cannot monitor NTR activity and DNA damage simultaneously, limiting the ability to assess their correlation. Here, we present a dual-channel ratiometric fluorescent probe, Me-NBP, for simultaneous monitoring of NTR activity and nucleic acid status within a single molecular system. The probe combines a 4-nitro-sulfonyl methylene unit as the NTR-responsive trigger, enhancing green fluorescence upon enzymatic reduction, with a positively charged pyridinium moiety that binds nucleic acids and generates a red fluorescence signal. The two emission channels are well separated (∼100 nm), allowing ratiometric self-calibration and quantitative detection with minimal spectral crosstalk. Using Me-NBP, we demonstrate the correlation between NTR activity and DNA damage in living cells, evaluate prodrug activation in tumor-bearing mice, and distinguish malignant from adjacent normal tissues in human breast cancer samples. This work provides a robust analytical tool for studying enzyme-mediated prodrug activation and its downstream effects in biological systems.