Fluorometric detection of cancer marker FEN1 based on double-flapped dumbbell DNA nanoprobe functionalized with silver nanoclusters

Abstract

Flap endonuclease 1 (FEN1), a ubiquitous enzyme involved in DNA repair and replication, is overexpressed in highly proliferative cancer cells. FEN1 has been recognized as a promising diagnostic marker of cancers; however, very few analytical techniques have been developed for the convenient detection of FEN1. To realize the simplified quantification of FEN1, we developed a FEN1-responsive fluorescent nanoprobe based on DNA-silver nanoclusters (DNA-AgNCs). The nanoprobe was rationally designed with a double-flapped dumbbell conformation, where its 5' flap was produced with DNA-AgNCs, and the 3' flap was elongated by a guanine-rich enhancer sequence (GRS). Rigidified by the DNA scaffold, DNA-AgNCs and the GRS are in close proximity, resulting in high fluorescence because of the GRS-induced activation of DNA-AgNCs. Upon the addition of FEN1, the 5' flap of the nanoprobe is cleaved due to the structure-specific endonuclease activity of FEN1. This cleavage released the DNA-AgNCs from the nanoprobe, broke the proximity between DNA-AgNCs and the GRS, and caused decreased fluorescence. This nanoprobe can be applied in the sensitive detection of FEN1 with a detection limit of 40 fM, and it showed high specificity for the monitoring of FEN1 in clinical samples. As the first attempt to develop biosensors targeting FEN1 based on DNA-AgNCs, this work provided a potent platform for monitoring FEN1 and screening FEN1 inhibitors.

Keywords: Aptamer; Biosensor; Cancer biomarker; Flap endonuclease 1; Functional nucleic acid; Silver nanocluster.