A novel DNAzyme-based colorimetric assay for the detection of hOGG1 activity with lambda exonuclease cleavage

摘要

The generation of 8-oxo-7,8-dihydroguanine (8-oxoG) in DNA is a common type of DNA damage after exposure to reactive oxygen species or drugs. Human 8-oxoG DNA glycosylase/AP lyase (hOGG1) is a kind of base excision repair enzyme specifically used to repair the base excision of 8-oxoG. In this paper, we develop a novel, simple and sensitive strategy for the detection of hOGG1 activity based on the self-assembly of the active HRP-mimicking DNAzyme coupled with lambda exonuclease (X exo) cleavage. We designed two DNA oligonucleotides that are fully complementary to each other. One is modified with 8-oxoG, the other contains the G-quadruplex DNAzyme sequence. The two single-stranded DNA (ssDNA) firstly hybridize to form a DNA duplex containing an 8-oxoG. In the presence of hOGG1, the formed DNA duplex is selectively cleaved at the 8-oxoG site, yielding a new DNA duplex with a recessed 5'-phosphate terminus. Upon treatment with X exo, the 5'-phosphoryl ssDNA of the new DNA duplex is digested by X exo, releasing the G-quadruplex DNAzyme sequence. After addition of hemin, the G-quadruplex-hemin complex is used as a peroxidase-mimicking DNAzyme, catalyzing H2O2-mediated oxidation of 2,2'-azinobis(3-ethylbenzothiozoline)-6-sulfonic acid (ABTS~(2-)) to generate a colorimetric signal. The activity of hOGG1 is directly related to UV/Vis absorption intensity. The results revealed that the method allowed a sensitive quantitative assay of the hOGG1 concentration with a wide range from 0.05-32 U mL~(-1) and a low detection limit of 0.01 U mL~(-1).