A one-pot transcriptional assay method that detects the tumor biomarker FEN1 based on its flap cleavage activity

Abstract

Background: Detection of tumor biomarkers in body fluids is a significant advancement in cancer treatment because it allows diagnosis without invasive tissue biopsies. Nucleases have long been regarded as a potential class of biomarkers that can indicate the occurrence and progression of cancers. Among these, flap endonuclease 1 (FEN1) plays an important role in DNA replication and repair, and also overexpressed in abnormally proliferating cells such as cancer cells. FEN1 is thus considered to be a potential biomarker as well as a target for cancer therapy.

Results: We developed a novel method for detecting FEN1 based on its specific endonuclease activity which incises bifurcated nucleic acids (flaps), in combination with in vitro transcription. Developed method uses a simple DNA structure (substrate DNA) carrying a short 5'-flap sequence, and a single-stranded sensor DNA encoding the Broccoli light-up aptamer. When the assay mixture was supplied with a FEN1-containing sample, the flap sequence encoding the sense sequence of T7 promoter was cleaved and released from the substrate DNA. Because the sensor DNA was designed to carry the Broccoli RNA aptamer under the antisense sequence of T7 promoter, hybridization of the excised flap onto the sensor DNA initiated the transcription of the Broccoli RNA aptamer, enabling determination of the FEN1 titer based on the fluorescence of transcribed Broccoli aptamer. By using a combination of FEN1-mediated generation of a short oligonucleotide and subsequent oligonucleotide-dependent in vitro transcription, this method could detect FEN1 in biological samples within 1 h.

Significance and novelty: Developed method enables the detection of FEN1 by a simple one-pot reaction. It can detect sub-nanomolar concentrations of FEN1 within an hour, and has the potential to be used for cancer diagnosis, prognosis, and drug screening. It also enables easy identification of compounds that inhibit FEN1 activity and is thus a versatile platform for screening anti-cancer drugs. We anticipate that the basic principles of this assay can be applied to detect other biomolecules, such as nucleic acids.

Keywords: Broccoli light-Up RNA aptamer; Cancer biomarker; Flap endonuclease 1; In vitro diagnostics; In vitro transcription.