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

We developed a fluorescence-based method to detect FEN1 nuclease activities and screen the small-molecule inhibitors, advancing the diagnostics and targeted therapeutic research.

Current expression challenges include the complexity and the safety concerns of conventional methods along with the high cost and specialized equipment required for existing fluorescence-based techniques.

This protocol addressed the shortcoming of traditional radiological method and high-cost fluorescence techniques, provide a safer, efficient, and easy-to-use detection solution.

Our laboratory will focus on optimization of a high-throughput screening platform to explore in vivo applications and mechanisms of FEN1 inhibitors in cancer therapy.

[Instructor] To begin, design DNA oligonucleotide strands based on the specific cleavage activity of FEN1. Mix the single-stranded DNA oligonucleotides in an annealing buffer. Add 10 microliters of annealing buffer and deionized water to make a total volume of 50 microliters. Then, heat the reaction mixture at 100 degrees Celsius for one minute in a metal bath. Immediately transfer the tube to 72 degrees Celsius and incubate for 10 minutes. Then, turn off the heat source and allow the solution to gradually cool to room temperature to form the complete double-stranded DNA substrate. For the FEN1 nuclease activity assay, first combine two microliters of the DNA substrate with FEN1 nuclease in the reaction buffer. Incubate the reaction mixture in a metal bath at 37 degrees Celsius for 15 minutes. Add 20 microliters of 2X termination buffer to stop the reaction. Then, load 20 microliters of the terminated sample onto a pre-prepared 12% denaturing polyacrylamide gel. Electrophorese the gel at 100 volts for 60 minutes. Once complete, visualize the electrophoresis results under a fluorescence imaging system. With a pipette, transfer two microliters of dimethyl sulfoxide into a 1.5 milliliter microcentrifuge tube. Add two microliters of FEN1 nuclease and 10 microliters of 2X reaction buffer to the tube and six microliters of deionized water. Mix the solution thoroughly, then incubate it on ice for 10 minutes. After incubation, pipette two microliters of DNA substrate to reach a final reaction volume of 20 microliters. Next, prepare a working dilution of the FEN1 nuclease inhibitor FEN1-IN-4 by performing a twofold serial dilution from a one molar stock solution in dimethyl sulfoxide. Pipette two microliters each of FEN1 nuclease and the diluted FEN1-IN-4 inhibitor into a new 1.5 milliliter microcentrifuge tube. Then, pipette 10 microliters of 2X reaction buffer and four microliters of deionized water. Mix gently and incubate the mixture on ice for 10 minutes. Now, add two microliters of DNA substrate to the tube to make the final volume to 20 microliters. Incubate the reaction mixture at 37 degrees Celsius in a metal bath for 15 minutes. Then, terminate the reaction by adding 20 microliters of termination buffer. A clear protein band at approximately 45 kilodaltons confirmed successful purification of the FEN1 nuclease from induced expression. The fluorescein-labeled double-stranded DNA substrate appeared at 80 kilodaltons, confirming successful synthesis compared to the single-stranded substrate at 59 kilodaltons. FEN1 nuclease cleaved the 80-nucleotide, double-stranded DNA substrate in a dose-dependent manner, with complete cleavage observed at 0.5 micrograms per microliter. The FEN1-IN-4 compound significantly inhibited the cleavage of double-stranded DNA substrate by FEN1, as shown by the retention of the 80-nucleotide band. FEN1-IN-4 reduced FEN1 nuclease activity in a concentration-dependent manner, with a half maximal inhibitory concentration of 14.03 micromolar.