Real-Time DynamicTracking of Multiple Base ExcisionRepair Enzymes in Living Cells

Simultaneous in situ monitoring of base excision repair (BER) correlated enzymes like apurinic/apyrimidinic endonuclease 1 (APE1) and flap endonuclease 1 (FEN1) in living cells offers valuable insights into their roles in disease development and cytotoxicity caused by pollutants, but comprehensive analysis is currently hindered by diverse enzyme functions and limited methods. In this study, we developed a dual-activatable DNA fluorescent probe (AP-FLAP) to simultaneously visualize APE1 and FEN1 activities, revealing the BER-related DNA damage caused by various environmental pollutants within living cells. The AP-FLAP probe was designed by ingeniously integrating a dumbbell structure containing a 5′ flap and a hairpin structure containing AP sites into a single oligonucleotide probe. APE1 specifically hydrolyzed the AP sites, releasing a 5-carboxy-X-rhodamine (ROX) signal, while FEN1 recognized and cleaved the 5′ flap, releasing a 6-carboxyfluorescein (FAM) signal. The probe allowed for independent determination of APE1 and FEN1 activities with good specificity and sensitivity. Subsequently, we applied the AP-FLAP probe to investigate base damage induced by 1-methylphenanthrene (1-MP) and 6-chlorobenzo[a]pyrene (6-Cl-BaP) in human umbilical vein endothelial cells (HUVECs). Significant base damage by 1-MP and 6-Cl-BaP exposure was revealed, with a positive correlation of damage degree with different exposure concentrations from 0.1 to 100 μM. Notably, 6-Cl-BaP caused significant damage even at 0.1 μM, in a concentration-dependent manner. Our work provides a powerful tool for elucidating BER molecular mechanisms and DNA damage repair under environmental exposure and opens new avenues for developing multifunctional nucleic acid probes for a wide range of applications in chemical biology and biomedical research.