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

Summary

This protocol describes a gel shift biochemical assay for measuring FEN1 (Flap Endonuclease 1) activity and inhibitor development.

Abstract

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.

Introduction

DNA replication is not always flawless and can be disrupted by both internal and external factors. Consequently, the mechanisms of DNA replication and damage repair are vital for cell survival and genome stability. In this process, the human branching structure-specific endonuclease FEN1 (Flap Endonuclease 1), a core DNA nuclease, plays a crucial role^1,2,3.

FEN1 is primarily involved in DNA replication and is indispensable for the maturation of Okazaki fragments on the lagging strand^4,5. This ensures the efficient progression of DNA replication and the accurate repair of damaged DNA. However, abnormal increases in FEN1 levels have been closely linked to the development of various cancers^6,7, such as lung and breast cancer⁷. Thus, FEN1 is not only essential for DNA replication and repair but has also garnered significant attention as a biomarker for cancer diagnosis and a potential therapeutic target^8,9. The detection of FEN1 activity is essential for the early diagnosis of cancers and for advancing research on targeted therapies.

Given the importance of FEN1, and the need to study its activity for early cancer diagnosis and targeted therapy development, the primary goal of the method developed in this experiment was to accurately and efficiently measure FEN1 nuclease activity. This method aims to provide a reliable platform for screening small-molecule inhibitors targeting FEN1. By achieving these goals, the study seeks to contribute to a better understanding of FEN1's role in cancer and to facilitate the discovery of novel therapeutic strategies.

Numerous techniques exist for detecting FEN1 nuclease activity, each with distinct characteristics, principles, advantages, limitations, and applicable scenarios. For instance, gel electrophoresis and HPLC-based substrate separation methods provide intuitive results but involve cumbersome procedures and low throughput^10,11. Fluorescence polarization (FP) assays face challenges in evaluating FEN1 activity due to interference from compound autofluorescence or light scattering, which affects FP signals. Although counter-screening strategies can partially exclude false positives, approximately 15% of primary active compounds are still misidentified due to technical interference¹². The combined use of fluorescence donor/quencher pairs (e.g., TAMRA/BHQ-2) and AlphaScreen chemiluminescence presents drawbacks, such as the high synthesis cost of complex triple-flap fluorescent substrates, particularly limiting their utility in large-scale screening. Furthermore, the dual-flap structure of substrates may impede optimal binding with FEN1, reducing enzymatic cleavage efficiency and compromising detection sensitivity. Additionally, this approach heavily relies on specialized instruments (e.g., ViewLux, EnVision), restricting its adoption in resource-limited laboratories^13,14,15. Fluorescent detection methods based on dual-flap dumbbell-shaped DNA nanoprobes functionalized with silver nanoclusters (DNA-AgNCs) suffer from batch-to-batch variability due to their dependence on precise annealing and in situ nucleation steps, hindering reproducibility and scalability. High costs and lengthy procedures further limit their practical application¹⁶.

In this article, a fluorescence labeling-based assay is developed to address current technical limitations and meet the growing demand for safer, user-accessible methods for studying FEN1 activity. This technique takes advantage of the specificity and high sensitivity of fluorescence detection, as well as the ability to engineer DNA substrates with defined structures that can be recognized and cleaved by FEN1 (Figure 1). This method streamlines experimental procedures while maintaining high accuracy in assessing FEN1 nuclease activity. Compared with traditional gel electrophoresis and HPLC-based techniques, it offers simplified operation and faster turnaround. Unlike fluorescence polarization assays, this approach enables rapid qualitative screening of compounds, establishing an efficient strategy for preliminary enzymatic evaluation.

This method is suitable for studying FEN1 nuclease activity in laboratory settings where access to radioactive materials is limited. It is also well-suited for screening a large number of small-molecule inhibitors in a relatively short time, especially when high sensitivity and specificity are required. However, it is not ideal for studies requiring absolute quantification with very high precision or for laboratories lacking basic fluorescence detection equipment and expertise in fluorescence signal analysis. For studies focusing on in vivo applications-where FEN1 behavior may be influenced by complex biological factors not replicated in current in vitro assay designs-further refinement or alternative methods may be necessary.

This fluorescence assay is highly sensitive and specific, providing a safe, convenient, and effective tool for studying FEN1 activity and developing its inhibitors.

Protocol

The reagents and the equipment used in this study are listed in the Table of Materials.

1. FEN1 protein expression purification

1. Protein purification
   1. Remove the bacterial stock containing the target plasmid (see Table of Materials) from −80 °C. Thaw at room temperature and inoculate 200 µL from the frozen 1.5 mL microcentrifuge tubes into 20 mL LB medium containing 50 µg/mL kanamycin using a 200 µL pipette tip.
      NOTE: The strain is E. coli BL21(DE3)-FEN1.
   2. Incubate at 37 °C with shaking at 160 rpm for 12 h.
   3. Transfer the bacterial suspension into a 200 mL Erlenmeyer flask containing LB medium supplemented with 50 µg/mL kanamycin. Continue incubation at 37 °C with shaking at 160 rpm for 2-3 h.
   4. When the OD₆₀₀ of the culture reaches approximately 0.6, add 2 mL of IPTG (final concentration: 1 mM). Continue incubation at 25 °C, 160 rpm for 10 h.
   5. Transfer the induced culture to a pre-weighed 50 mL centrifuge tube. Centrifuge at 4 °C and 2500 × g for 15 min. Discard the supernatant. Weigh the pellet-containing tube and subtract the weight of the empty tube to calculate the pellet mass.
   6. Add 7 mL of lysis buffer per gram of wet cell pellet. Use a pipette to resuspend the pellet thoroughly by pipetting up and down.
      NOTE: Lysis buffer (10 mL total) contains 20 mM of Tris-HCl, 250 mM of NaCl, 2% Triton X-100, and ddH₂O. Perform this step in a fume hood to ensure safe handling of chemical reagents.
   7. Transfer the bacterial suspension in lysis buffer to a −80 °C freezer and freeze overnight. On the following day, thaw the suspension on a 4 °C shaker for 1 h to enhance lysis efficiency.
   8. Lyse cells using ultrasound at 75% power for 45 min. Centrifuge the lysate at 12,000 × g for 20 min at 4 °C to separate the supernatant from the pellet.
      NOTE: If the lysate remains turbid, increase the volume of the lysis buffer or repeat freeze-thaw cycles. A nuclease inhibitor may also be added to preserve protein integrity and prevent degradation.
   9. Apply the clarified supernatant along the wall of a tube containing prepared protein purification resin. Wash with 10 mL of elution buffer to remove non-specifically bound proteins.
      NOTE: Elution buffer contains 20 mM of Tris-HCl (pH 7.5), 250 mM of NaCl, 2% Triton X-100, 200 mM of imidazole, and distilled water.
   10. Elute the FEN1 protein by adding 5 mL of elution buffer along the inner wall of the tube.
2. Identification of target proteins by rapid coomassie brilliant blue staining
   1. Prepare SDS-PAGE gels using 12% separating gel and 5% stacking gel according to the expected size of FEN1.
   2. Mix the target protein sample and BSA standard with 5× sample buffer at a 1:4 ratio. Heat the mixture at 95 °C for 5 min to fully denature the proteins.
   3. Load 20 µL each of the BSA standard and denatured protein samples into separate wells of the SDS-PAGE gel.
   4. Connect the electrophoresis apparatus. Set the voltage to 90 V and run for 1 h and 40 min.
   5. After electrophoresis, transfer the gel to a staining container. Rinse with deionized water, then add 30 mL of protein staining solution and stain for 25 min. Recover the staining solution, rinse the gel with 20 mL of deionized water, and decolorize overnight on a horizontal shaker.
   6. After decolorization, place the gel on a transparent plastic plate and observe the protein bands under white light.
      NOTE: If the bands are faint or protein concentration is low, concentrate the sample using ultrafiltration tubes. This step removes excess solvent and small molecules, improving protein concentration and ensuring accurate analysis in subsequent experiments.

2. FEN1 TAMRA-labeled DNA substrate preparation

1. TAMRA-labeled DNA substrate preparation
   1. Design DNA oligonucleotide strands based on the specific cleavage activity of FEN1, referring to published methods¹⁷(see Table 1). The 5′ end of the D1 sequence is modified with a fluorescent TAMRA label.
   2. Add mixed single-stranded DNA oligonucleotides to 10 μL of annealing buffer in the following volumes: 5 μL of D1 (10 pmol/μL), 10 μL of T1 (10 pmol/μL), and 7.5 μL of U2 (10 pmol/μL). Add deionized water to bring the total volume to 50 μL.
      NOTE: The annealing buffer contains 100 mM of Tris-HCl (pH 7.5) and 10 mM of MgCl₂.
   3. Heat the reaction mixture in a metal bath at 100 °C for 1 min, then immediately transfer it to 72 °C for 10 min¹⁸.
      NOTE: The final DNA substrate is 80 nucleotides in length. The high initial temperature (100 °C) ensures denaturation, while the annealing temperature (72 °C) promotes accurate strand pairing.
   4. Turn off the heat source and allow the solution to cool gradually to room temperature, forming the complete double-stranded DNA substrate.
2. TAMRA-labeled DNA substrate assay
   1. Prepare a 12% denaturing polyacrylamide gel by mixing 4.8 mL of 30% acrylamide (29:1), 4.32 mL of 6 M urea, 2.4 mL of 5× TBE, 50 µL of 10% APS, 5 µL of TEMED, and 0.48 mL of deionized water. Mix thoroughly to prepare the gel.
   2. Place the gel in a 37 °C oven for 30 min to solidify.
   3. For detection, pipette 2 µL of the synthesized DNA substrate into 18 µL of distilled water and add 7.7 µL Stop Buffer.
   4. For reference, pipette 2 µL of TAMRA-labeled D1 into 18 µL of distilled water and add 7.7 µL of Stop Buffer.
   5. Load 20 µL of each prepared sample onto the gel and perform SDS-PAGE electrophoresis to analyze DNA substrate formation.

3. FEN1 nuclease activity assay

1. FEN1 enzyme activity assay
   1. Prepare 20 µL of reaction mixture by combining 2 µL of DNA substrate with FEN1 nuclease in the reaction buffer.
      NOTE: The reaction buffer consists of 100 mM of Tris-HCl (pH 7.5), 200 mM of MgCl₂, 2 mM of DTT, 0.2 mg/mL BSA, and deionized water.
   2. Incubate the reaction mixture at 37 °C in a metal bath for 15 min.
   3. Terminate the reaction by adding 20 µL of 2× termination buffer.
   4. Load 20 µL of the reacted sample onto a pre-prepared 12% denaturing polyacrylamide gel and perform electrophoresis at 100 V for 60 min.
   5. After electrophoresis, visualize the results using a fluorescence imaging system (600 pathway).

4. Screening of small molecule inhibitors of FEN1 nuclease

1. Small molecule inhibitor screening
   1. Use a pipette to transfer 2 µL of dimethyl sulfoxide (DMSO) into a 1.5 mL microcentrifuge tube.
   2. Add 2 µL of FEN1 nuclease and 10 µL of 2× reaction buffer to the tube.
   3. Mix thoroughly and incubate the mixture on ice for 10 min.
   4. Add 2 μL of DNA substrate and 4 μL of deionized water, mix thoroughly to bring the total reaction volume to 20 μL.
   5. Prepare a working dilution of the FEN1 nuclease inhibitor, FEN1-IN-4, by performing a two-fold serial dilution of a 1 M stock solution in DMSO.
   6. Pipette 2 µL of FEN1 nuclease (0.03 µg/µL) and 2 µL of the diluted FEN1-IN-4 inhibitor into a new 1.5 mL microcentrifuge tube. Add 10 µL of 2× reaction buffer and 4 µL of deionized water. Mix gently and incubate on ice for 10 min to ensure sufficient interaction between the inhibitor and enzyme.
   7. Add 2 µL of DNA substrate to the tube to form a final 20 µL reaction mixture.
   8. Incubate the reaction at 37 °C in a metal bath for 15 min.
   9. Terminate the reaction by adding 20 µL of 2× termination buffer.

5. Evaluation of inhibitory potency of FEN1--IN-4 small molecule inhibitor

1. Use a micropipette to take up 10 µL of 2× reaction buffer and add it to a 1.5 mL microcentrifuge tube.
2. Add 2 µL of FEN1 nuclease at a concentration of 0.03 µg/µL, as well as 2 µL of FEN1 inhibitor FEN1-IN-4 at different concentration.
   NOTE: Multiple concentration gradients should be established according to experimental requirements.
3. Add 4 μL of deionized water and mix the solution thoroughly. Place the 1.5 mL microcentrifuge tube on ice and incubate for 10 min.
4. Add 2 µL of double-stranded DNA substrate to the tube to form a 20 µL reaction system.
5. Place the EP tube in a 37 °C metal bath for 15 min.
6. After the reaction is complete, add 20 µL of 2× stop buffer to the reaction mixture to terminate the reaction.
7. Observe the results using a fluorescence imaging device and plot the IC₅₀ curve¹⁹.
   NOTE: Fluorescence intensity quantification was performed using Fiji/ImageJ²⁰, followed by statistical analysis and visualization with graphing and analysis software²¹.

Results

As shown in Figure 2A, the staining reagent binds to protein, causing the protein to appear as a blue band on the gel. The first lane contains the labeled protein used as a molecular weight reference, in which the BSA standard protein band corresponds to a molecular weight of 70 kDa, and the FEN1 nucleic acid protein band corresponds to a molecular weight of 45 kDa. The experimental results showed that the FEN1 nuclease appeared as a clear band at approximately 45 kDa, indicating successful ...

Discussion

The FEN1 nuclease activity assay and small molecule inhibitor screening are central to research on DNA damage repair and cancer development. Compared to traditional radiolabeling techniques, the present method offers a cost-effective, efficient, easy-to-operate, and safer alternative. Additionally, unlike fluorescence burst-based FEN1 assays, this approach provides a more intuitive visualization of substrate cleavage, thereby allowing a more accurate assessment of enzyme activity and offering a more reliable platform for...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 M Tris-HCL	Beyotime	ST774
30% Acr-Bis (29:1)	Beyotime	ST003-500ml
5M Nacl	Beyotime	ST347
BeyoBlue Plus	Beyotime	P0003S
BL21(DE3)-FEN1	Vazyme	C504-F1
BSA	Beyotime	P0007
Decoloring shaker	Kylin-Bell	TS-100
DL-Dithiothreitol	Beyotime	ST041
Dry Bath Incubator	RUICHENG	DH200
Electrophoresis apparatus	Bei jing Liuyi Biotechnology	DDY-6D
FEN1-IN-4	TargetMOL	T8545
IPTG	Takaba	9030
Kanamycin Slufate	Biosharp	BS152
Laboratory Centrifuge	Thermo	SL 16R
LB Broth	Solarbio	L8291
Ni-NTA His Bind Resin	7se biotech	PANF001-001C
Odyssey FC	ODYSSEY	LI-COR
SDS-PAGE	Beyotime	P0015L
Trition-x-100	Shyuanye	S15022