Lighting-up aptamer transcriptional amplification for highly sensitive and label-free FEN1 detection

Abstract

Specific and sensitive detection of flap endonuclease 1 (FEN1), an enzyme biomarker involved in DNA replications and several metabolic pathways, is of high values for the diagnosis of various cancers. In this work, a fluorescence strategy based on transcriptional amplification of lighting-up aptamers for label-free, low background and sensitive monitoring of FEN1 is developed. FEN1 cleaves the 5' flap of the DNA complex probe with double flaps to form a notched dsDNA, which is ligated by T4 DNA ligase to yield fully complementary dsDNA. Subsequently, T7 RNA polymerase binds the promoter region to initiate cyclic transcriptional generation of many RNA aptamers that associate with the malachite green dye to yield highly amplified fluorescence for detecting FEN1 with detection limit as low as 0.22 pM in a selective way. In addition, the method can achieve diluted serum monitoring of low concentrations of FEN1, exhibiting its potential for the diagnosis of early-stage cancers.

Keywords: FEN1; Fluorescence; Lighting-up aptamer; RNA transcription.