Construction of a Multiple Cyclic Ligation-Promoted Exponential Recombinase Polymerase Amplification Platform for Sensitive and Simultaneous Monitoring of Cancer Biomarkers Fpg and FEN1

Formamidopyrimidine DNA glycosylase (Fpg) and flap endonuclease 1 (FEN1) are essential to sustaining genomic stability and integrity, while the abnormal activities of Fpg and FEN1 may lead to various diseases and cancers. The development of simple methods for simultaneously monitoring Fpg and FEN1 is highly desirable. Herein, we construct a multiple cyclic ligation-promoted exponential recombinase polymerase amplification (RPA) platform for sensitive and simultaneous monitoring of Fpg and FEN1 in cells and clinical tissues. We designed two programmable substrate probes with 8-oxo-7,8-dihydroguanine (8-oxoG) damage sites and 5' flaps that can be identified/cleaved by Fpg and FEN1 to produce nicking sites. The juxtaposition of the cleavage sites is ligated by DNA ligase to form intact double-stranded DNA (dsDNA) templates that can be amplified via RPA to produce abundant dsDNA products labeled with Cy5 and Cy3 fluorophores and biotin, respectively. The resultant dsDNA can be captured by magnetic beads and subsequently disassembled into dispersed Cy3 and Cy5 molecules upon heat treatment, generating significant fluorescence signals. This assay exhibits a limit of detection of 1.12 × 10^-10 U μL^-1 for Fpg and 1.66 × 10^-9 U μL^-1 for FEN1, and it can be used for the analysis of enzymatic kinetic parameters, screening of inhibitors, and simultaneous monitoring of Fpg and FEN1 in a single cell and in clinic tissue samples. Moreover, the proposed strategy can be applied to monitor other DNA repair proteins by merely changing the recognition sites of dsDNA substrate probes, providing a promising platform for clinical diagnosis, biomedical research, and drug discovery.