The novel DNA biosensor detecting human AP-endonuclease (hAPE1) for early diagnosis of cancer

Abstract

Human (Apurinic / Apyrimidic) AP-endonuclease (hAPE1) which is involved in Base Excision Repair (BER) system to repair the endo/exogenous reagents-driven AP site is over-expressed in various cancer. In this reasons, hAPE1 have been proposed as a prognostic and diagnostic biomarker for early diagnosis of cancer since a few decades. Therefore, to quantify the hAPE1 level in cells is very important for early diagnosis and cure of cancer. We have developed the hAPE1-targerted novel DNA biosensor which is composed of biotinylated double strand (ds) DNA containing AP site, labeled with Cy3 and NeutrAvidin to improve the specificity. This novel DNA biosensor has some advantages such as a non-invasiveness, high sensitivity, high specificity and low cost comparing other general biosensors through the Fluorescence-Enhancement (FE) effect. The FE effect in DNA biosensor is the increase of Cy3 intensity on the DNA due to enzymatic-driven conformational DNA structure change from dsDNA to ssDNA labeled with Cy3, promoting isomerization in trans state of Cy3. We tested the detectability of DNA biosensor to check whether the FE effect-based DNA biosensor can detect the biomarker, hAPE1 and EXOIII (E.coli type AP-endonuclease) or not in-vitro. As a results, we could check the FE-based DNA biosensor can detect the level of hAPE1 from 10pM and EXOIII from 1nM on average. Additionally, we checked the specificity of DNA biosensor in other non-AP site sensitive nucleases (Phi29, Lambda exonuclease, T7 exonuclease, T4 DNA polymerase - No FE effect at all). According to this results, we expect that the novel DNA biosensor can be a good biosensor for early diagnosis of cancer.