Construction of dual exponential amplification accompanied by multi-terminal signal output method for convenient detection of tumor biomarker FEN1 activity

Abstract

As an important 5′-nuclease in DNA replication and damage repair, Flap endonuclease 1 (FEN1) has been considered as a potential tumor biomarker due to its overexpression in different human cancer cells. Here, we developed a convenient fluorescent method based on dual enzymatic repairing exponential amplification accompanied by multi-terminal signal output to realize the rapid and sensitive detection of FEN1. In the presence of FEN1, the double-branched substrate could be cleaved to produce 5′ flap single strand DNA (ssDNA) which subsequently was used as a primer to initiate the dual exponential amplification (EXPAR) to generate abundant ssDNAs (X′ and Y′), then the ssDNAs can respectively hybridize with the 3′ and 5′ ends of the signal probe to form partially complementary double strands (dsDNAs). Subsequently, the signal probe on the dsDNAs could be digested under the assistance of Bst. polymerase and T7 exonuclease, as well as releasing the fluorescence signals. The method displayed high sensitivity with the detection limit of 9.7 × 10−3 U mL−1 (1.94 × 10−4 U) and also exhibited good selectivity towards FEN1 under the challenge from complicated samples including extracts of normal and cancer cells. Furthermore, it was successfully applied to screen FEN1 inhibitors, holding great promise in the screening of potential drugs targeting FEN1. This sensitive, selective and convenient method could be used for FEN1 assay without the complicated nanomaterial synthesis/modification, showing great potential in FEN1- related prediction and diagnosis.