Label-free and homogeneous detection of flap endonuclease 1 by ligation-promoted hyperbranched rolling circle...

The structure-specific endonuclease FEN1 participates in various genome maintenance pathways in eukaryotes and is associated with different human diseases. Herein, we demonstrate label-free and homogeneous detection of FEN1 based on ligation-promoted hyperbranched rolling circle amplification (HRCA). This assay can be performed isothermally with the involvement of primers 1 and 2 and a circular DNA substrate with a 5'-flap. When FEN1 is present, it cleaves 5'-flap of circular DNA substrate to obtain a circular padlock probe with the assistance of Taq DNA ligase. The circular padlock probe can serve as a template to initiate HRCA in the presence of primers 1 and 2 and Vent (exo^-) DNA polymerase. The obtained dsDNA fragments can produce an enhanced fluorescence signal with SYBR Green I as indicator. This method displays good specificity and high sensitivity, and it can be employed to screen FEN1 inhibitors and quantitatively detect FEN1 activity in human cancer cells, with potential applications in early diagnosis and drug discovery.