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DNA glycosylases are involved in the base excision repair pathway, and all mammals express multiple DNA glycosylases to maintain genome stability. However, the simultaneous detection of multiple DNA glycosylase still remains a great challenge. Here, we develop a single-molecule detection method for the simultaneous detection of human 8-oxoguanine DNA glycosylase 1 (hOGG1) and human alkyladenine DNA glycosylase (hAAG) on the basis of DNA glycosylase-mediated cleavage of molecular beacons. We designed a Cy3-labeled molecular beacon modified with 8-oxoguanine (8-oxoG) for a hOGG1 assay and a Cy5-labeled molecular beacon modified with deoxyinosine for a hAAG assay. hOGG1 may catalyze the removal of 8-oxoG from 8-oxoG/C base pairs to generate an apurinic/apyrimidinic (AP) site, and 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 Cy5 indicating the presence of hAAG. Both of the 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 μL -1 for hOGG1 and 8.69 × 10 -7 U μL -1 for hAAG without the involvement of any target amplification. Moreover, this method can be used for the screening of enzyme inhibitors and the simultaneous detection of hOGG1 and hAAG from lung cancer cells, having great potential for further application in early clinical diagnosis.

Simultaneous sensitive detection of multiple DNA glycosylases from lung cancer cells at the single-molecule level