Sequential Activation of DNA Sensor Enables Correlated Imaging of Dual-Enzyme Activities in Living Cells

The DNA repair system relies on the coordinated action of multiple enzymes to maintain genomic stability, with apurinic/apyrimidinic endonuclease 1 (APE1) and flap endonuclease 1 (FEN1) playing pivotal roles in the long-patch base excision repair (LP-BER) pathway. Elevated levels of APE1 and FEN1 have been associated with tumor progression and resistance to therapy, making them key biomarkers for cancer diagnosis and treatment monitoring. Here, we present a sequentially activated AND-logic DNA sensor (D-AF) for the correlated imaging of APE1 and FEN1 in living cells. The sensor operates through a sequential process: APE1 first recognizes and cleaves an apurinic site, initiating structural changes that enable FEN1 to cleave a 5' flap, resulting in restored fluorescence. We demonstrate the use of the D-AF-based nanosensor for <i>in situ</i> imaging of APE1 and FEN1 activities in cancer cells and for monitoring of enzyme dynamics during chemotherapy. This platform offers a valuable tool for investigating DNA repair mechanisms and their roles in cancer diagnosis and treatment.