Fluorescence-Based Detection of FEN1 Nuclease Activity and Screening of Small-Molecule Inhibitors

FEN1 (Flap Endonuclease 1) is a key DNA nuclease involved in DNA replication and damage repair, playing a crucial role in maintaining genomic stability. Elevated levels of FEN1 activity are closely associated with the development of various cancers, making FEN1 a recognized biomarker and drug target for lung, breast, and prostate cancers. Conventional radioisotope tagging methods for detecting FEN1 nuclease activity present challenges in terms of convenience and safety. In this study, a DNA fragment substrate with a flap structure was synthesized in vitro. This substrate is labeled with a fluorophore at the 5' end of the flap, allowing the FEN1 nuclease to specifically recognize and cleave the designated site. FEN1 activity was then determined by detecting the fluorophore signal after gel electrophoresis. This method enables rapid and effective assessment of FEN1 nuclease activity and the screening of its inhibitors. It provides a sensitive and specific approach for analyzing FEN1 activity and evaluating the efficacy of small-molecule inhibitors. Furthermore, it contributes significantly to research on DNA damage repair and cancer development, offering a novel strategy for clinical research on disease mechanisms and the detection of genotoxic substances.