Signal-Amplified Detection of the Tumor Biomarker FEN1 Based on Cleavage-Induced Ligation of a Dumbbell DNA Probe and Rolling Circle Amplification

Abstract

Flap endonuclease 1 (FEN1), an endogenous nuclease with the ability to cleave the 5′ overhang of branched dsDNA, is of significance in DNA replication and repair. The overexpression of FEN1 is common in cancer because of the ubiquitous upregulation of DNA replication; thus, FEN1 has been recognized as a potential biomarker in oncological investigations. However, few analytical methods targeting FEN1 with high sensitivity and simplicity have been developed. This work developed a signal-amplified detection of FEN1 based on the cleavage-induced ligation of a dumbbell DNA probe and rolling circle amplification (RCA). A flapped dumbbell DNA probe (FDP) was rationally designed with a FEN1 cleavable flap at the 5′ end. The cleavage generated a nick site with juxtaposed 5′ phosphate and 3′ hydroxyl ends, which were linkable by T4 DNA ligase to form a closed dumbbell DNA probe (CDP) with a circular conformation. The CDP functioned as a template for RCA, which produced abundant DNA that could be probed using SYBR Green I. The highly sensitive detection of FEN1 with a limit of detection of 15 fM was achieved, and this method showed high specificity, which enabled the quantification of FEN1 in real samples. The inhibitory effects of chemicals on FEN1 were also evaluated. This study represents the first attempt to develop an FEN1 assay that involves signal amplification, and the novel biosensor method enriches the tools for FEN1-based diagnostics.

Flap endonuclease 1 Facilitated Hepatocellular Carcinoma Progression by Enhancing USP7/MDM2-mediated P53 Inactivation

“…Previous studies have indicated that FEN1 was overexpressed in multiple solid tumors like lung cancer and breast cancer with aggressive clinical implications 11 , 12 . In addition to its significance, in situ sensing of oncogenic FEN1 with DNA nanosphere or dumbbell DNA probe has been developed for early diagnosis and precision medicine 23 , 24 . Actually, a recent study noted that FEN1 expression was elevated in HCC tissues by analyzing bioinformatic data 25 .…”

A Pax-5a gene analysis approach enabled by selective digestion with lambda exonuclease

“…In recent years, nucleic acid detection technology has been a research hotspot for investigators, because identifying cancer markers is very signicant for cancer diagnosis and prognosis. [1][2][3] Gene mutations and abnormal gene expression are early molecular manifestations of some cancers. 4 At the same time, studies have shown that gene mutations play an important role in 5-10% of malignant tumors.…”

Label-Free and Homogeneous Electrochemical Biosensor for Flap Endonuclease 1 Based on the Target-Triggered Difference in Electrostatic Interaction between Molecular Indicators and Electrode Surface

“…Traditional assays, including western blot, reverse transcription-polymerase chain reaction (RT-PCR), and enzyme-linked immunosorbent assay (ELISA) [ 1 , 3 , 4 ] had already been utilized to detect FEN1. Several novel strategies were also designed for FEN1 detection [ 9 , 10 , 11 , 12 , 13 , 14 , 15 ]. For instance, Zhang et al [ 9 ] proposed a DNA-based fluorescent biosensor to evaluate FEN1 activity in living cells.…”