title: Tungsten disulfide nanosheet and exonuclease III co-assisted amplification strategy for highly sensitive fluorescence polarization detection of DNA glycosylase activity.
abstract: Herein, we introduced a tungsten disulfide (WS2) nanosheet and exonuclease III (Exo III) co-assisted signal amplification strategy for highly sensitive fluorescent polarization (FP) assay of DNA glycosylase activity. Two DNA glycosylases, uracil-DNA glycosylase (UDG) and human 8-oxoG DNA glycosylase 1 (hOGG1), were tested. A hairpin-structured probe (HP) which contained damaged bases in the stem was used as the substrate. The removal of damaged bases from substrate by DNA glycosylase would lower the melting temperature of HP. The HP was then opened and hybridized with a FAM dye-labeled single strand DNA (DP), generating a duplex with a recessed 3'-terminal of DP. This design facilitated the Exo III-assisted amplification by repeating the hybridization and digestion of DP, liberating numerous FAM fluorophores which could not be adsorbed on WS2 nanosheet. Thus, the final system exhibited a small FP signal. However, in the absence of DNA glycosylases, no hybridization between DP and HP was occurred, hampering the hydrolysis of DP by Exo III. The intact DP was then adsorbed on the surface of WS2 nanosheet that greatly amplified the mass of the labeled-FAM fluorophore, resulting in a large FP value. With the co-assisted amplification strategy, the sensitivity was substantially improved. In addition, this method was applied to detect UDG activity in cell extracts. The study of the inhibition of UDG was also performed. Furthermore, this method is simple in design, easy in implementation, and selective, which holds potential applications in the DNA glycosylase related mechanism research and molecular diagnostics.
authors: Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources of Education Ministry, Guangxi Normal University, Guilin, 541004, China.
authors: Jingjin
authors: J
authors: Zhao
authors: Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources of Education Ministry, Guangxi Normal University, Guilin, 541004, China.
authors: Yefei
authors: Y
authors: Ma
authors: The Key Laboratory of Life-Organic Analysis, College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu, Shandong 273165, China.
authors: Rongmei
authors: R
authors: Kong
authors: Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources of Education Ministry, Guangxi Normal University, Guilin, 541004, China. Electronic address: liangzhang319@163.com.
authors: Liangliang
authors: L
authors: Zhang
authors: Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources of Education Ministry, Guangxi Normal University, Guilin, 541004, China.
authors: Wen
authors: W
authors: Yang
authors: Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources of Education Ministry, Guangxi Normal University, Guilin, 541004, China.
authors: Shulin
authors: S
authors: Zhao
mesh_terms: D015374:Biosensing Techniques / Q000379:methods; D045647:DNA Glycosylases / Q000032:analysis / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry*; D057075:Enzyme Assays / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology*; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D049329:Nanostructures / Q000737:chemistry; D014414:Tungsten / Q000737:chemistry*; D051981:Uracil-DNA Glycosidase / Q000032:analysis / Q000378:metabolism*
chemical_list: D004220:Disulfides; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D045647:DNA Glycosylases; D051981:Uracil-DNA Glycosidase; C479058:oxoguanine glycosylase 1, human; D014414:Tungsten
keywords: DNA glycosylase; Exonuclease III; Fluorescence polarization; Tungsten disulfide nanosheet