Simultaneous Analysis Of Multiple Glycosylation Enzymes In Lung Cancer Cells

DNA glycosylases involve in the base excision repair pathway,and all mammals express multiple DNA glycosylases to maintain the genome stability.However,simultaneous detection of multiple DNA glycosylase still remains a great challenge.Here,we develop a single-molecule detection method for simultaneous detection of human 8-oxoguanine DNA glycosylase 1?hOGG1?and human alkyladenine DNA glycosylase?hAAG?on the basis of DNA glycosylases-mediated cleavage of molecular beacons.We design a Cy3-labeled molecular beacon modified with a 8-oxoguanine?8-oxoG?for hOGG1 assay and a Cy5-labeled molecular beacon modified with a deoxyinosine for hAAG assay.The hOGG1 may catalyze the removal of 8-oxoG from 8-oxoG/C base pairs to generate an apurinic/apyrimidinic?AP?site,and the hAAG may catalyze the removal of deoxyinosine from deoxyinosine/T base pairs to generate an AP site.With the assistance of apurinic/apyrimidinic endonuclease?APE1?,the cleavage of AP sites results in the cleavage of molecular beacons,with Cy3 indicating the presence of hOGG1 and Cy5indicating the presence of hAAG.Both Cy3 and Cy5 signals can be simply quantified by total internal reflection fluorescence-based single-molecule detection.This method can simultaneously detect multiple DNA glycosylases with a detection limit of 2.23×10^-6 U/?L for hOGG1 and 8.69×10^-7 U/?L for hAAG without the involvement of any target amplification.Moreover,this method can be used for the screening of enzyme inhibitors and simultaneous detection of hOGG1 and hAAG in lung cancer cells,holding great potential for further application in early clinical diagnosis.