Flap endonuclease I (FEN1) is a structure-specific nuclease that participates in DNA repair, and its overexpression is related to an increased risk of cancer. Currently, few FEN1 assays can meet the requirements for diagnostic purposes due to their high complexity, low specificity, and limited sensitivity. Herein, we developed a biosensor adopting synthetic biology strategy, called CLAIM (cleavage-ligation-activation-involved monitoring) for FEN1 quantification. CLAIMintegrates ligation-based T7 promoter repair, RNA generation, and CRISPR/Cas13a. FEN1 cleavages the DNA probe with a 5′-flap to generate a nick site that can be ligated to form a T7 promoter. Subsequent transcription produces abundant RNA that can activate Cas13a, generating a fluorescence signal due to the trans -cleavage of Cas13a to a fluorogenic reporter. CLAIM was able to detect FEN1 with low femtomolar sensitivity, and its accuracy was validated using samples of cancer and non-tumorigenic cell lines. Interestingly, we found that the utilization of thermostable T4 DNA ligase is critical to make the cascade of FEN1 cleavage, ligation, and transcription take place in one-pot. CLAIM was also repurposed for validating FEN1 inhibitors, indicating its potential in drug screening. This work provides a diagnostic tool for FEN1 analysis and opens up opportunities for FEN1 biosensing and related drug discovery.
Flap endonuclease 1 (FEN1) is a nuclease overexpressed in cells with a high replicate rate, which has been recognized as a potential biomarker in the molecular diagnosis of cancers. In this work, we developed a fluorescent biosensor based on exponential amplification reaction (EXPAR) to realize the rapid and sensitive detection of FEN1. A DNA probe specific for FEN1 is used for the generation of reporter DNA (rDNA), and then the rDNA hybridizes with a template, triggering EXPAR to produce substantial rDNA exponentially. However, when no FEN1 is in the system, the EXPAR will not be initiated because of the lack of rDNA. Derived from the high amplification efficiency of EXPAR, the rapid quantification of FEN1 with a limit of detection (LOD) of 0.5 fM was realized. It is also revealed that this biosensor exhibits good selectivity towards FEN1 under the challenge from real samples including extracts of cells and solid tumors. Furthermore, this sensor was reengineered for the analysis of FEN1 inhibitors, which shows promise in the screening and evaluation of potential drugs targeting FEN1. This work integrates EXPAR into the biosensing of FEN1 for the first time, which provides a promising tool for FEN1-related biomedical investigations.
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