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Updated: Aug 19, 2025
Fluorescence-Based Detection of FEN1 Nuclease Activity and Screening of Small-Molecule Inhibitors
Published on: June 27, 2025
Double-wing switch nanodevice-mediated primer exchange reaction for the activity analysis of cancer biomarker FEN1
Siyi Chen1, Zuowei Xie1, Wenxiu Zhang1
1Key Laboratory of Medical Diagnostics, Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, 400016, PR China.
Abstract:
DNA damage repair is one of the foremost factors leading to changes in tumor drug resistance. The analysis of Flap endonuclease 1 (FEN1), a kind of pivotal enzyme in various DNA metabolic pathways, has been of great support to tumor research and the development of chemotherapeutics. Nevertheless, few analytical techniques can achieve quantitative and simplified FEN1 measurement. Here, we constructed a double-wing switch nanodevice (DWSN)-mediated primer exchange technique for rapid and label-free quantification of FEN1 activity. Target FEN1 triggered the generation of numerous telomeric repeat fragments in different lengths through recognizing the three-base mismatched sites on the DWSN to release the 5'-Flaps. Further binding to the fluorescent dye ThT resulted in significantly enhanced fluorescence. This study broke the limitation of traditional single-site identification and demonstrated good sensitivity and specificity with detection limits up to 0.55 mU. Besides, the extraordinary analytical performance allowed the method to be utilized to monitor FEN1 extracted from cells and clinical serum samples and to compare the effect of targeted FEN1 inhibitors.
Insights
This study introduces a novel nanodevice for rapid, label-free quantification of Flap endonuclease 1 (FEN1) activity. This method offers a sensitive and specific tool for cancer research and drug development.
Area of Science:
- Biochemistry
- Molecular Biology
- Nanotechnology
Background:
- DNA damage repair significantly influences tumor drug resistance.
- Flap endonuclease 1 (FEN1) is a key enzyme in DNA metabolism, crucial for cancer research and chemotherapy development.
- Existing methods for FEN1 measurement lack simplicity and quantitative accuracy.
Purpose of the Study:
- To develop a rapid, label-free, and quantitative method for measuring FEN1 activity.
- To utilize a novel double-wing switch nanodevice (DWSN) for FEN1 detection.
- To establish a sensitive and specific analytical technique for FEN1 quantification.
Main Methods:
- Construction of a double-wing switch nanodevice (DWSN).
- FEN1-triggered primer exchange reaction releasing 5'-Flaps.
- Label-free fluorescence enhancement upon binding of telomeric repeat fragments to Thioflavin T (ThT).
Main Results:
- The DWSN-mediated primer exchange technique achieved rapid and label-free quantification of FEN1 activity.
- The method demonstrated high sensitivity and specificity, with a detection limit as low as 0.55 mU.
- The technique was successfully applied to monitor FEN1 in cell extracts and clinical serum samples, and to evaluate FEN1 inhibitors.
Conclusions:
- The developed nanodevice-based assay provides a simplified and quantitative approach for FEN1 activity measurement.
- This method overcomes limitations of traditional single-site identification techniques.
- The assay shows potential for applications in cancer diagnostics, drug discovery, and monitoring therapeutic responses.
