title: Base-Excision-Repair-Induced Construction of a Single Quantum-Dot-Based Sensor for Sensitive Detection of DNA Glycosylase Activity.
abstract: DNA glycosylase is an initiating enzyme of cellular base excision repair pathway which is responsible for the repair of various DNA lesions and the maintenance of genomic stability, and the dysregulation of DNA glycosylase activity is associated with a variety of human pathology. Accurate detection of DNA glycosylase activity is critical to both clinical diagnosis and therapeutics, but conventional methods for the DNA glycosylase assay are usually time-consuming with poor sensitivity. Here, we demonstrate the base-excision-repair-induced construction of a single quantum dot (QD)-based sensor for highly sensitive measurement of DNA glycosylase activity. We use human 8-oxoguanine-DNA glycosylase 1 (hOGG1), which is responsible for specifically repairing the damaged 8-hydroxyguanine (8-oxoG, one of the most abundant and widely studied DNA damage products), as a model DNA glycosylase. In the presence of biotin-labeled DNA substrate, the hOGG1 may catalyze the removal of 8-oxo G from 8-oxoG·C base pairs to generate an apurinic/apyrimidinic (AP) site. With the assistance of apurinic/apyrimidinic endonuclease (APE1), the cleavage of the AP site results in the generation of a single-nucleotide gap. Subsequently, DNA polymerase β incorporates a Cy5-labeled dGTP into the DNA substrate to fill the gap. With the addition of streptavidin-coated QDs, a QD-DNA-Cy5 nanostructure is formed via specific biotin-streptavidin binding, inducing the occurrence of fluorescence resonance energy transfer (FRET) from the QD to Cy5. The resulting Cy5 signal can be simply monitored by total internal reflection fluorescence (TIRF) imaging. The proposed method enables highly sensitive measurement of hOGG1 activity with a detection limit of 1.8 × 10(-6) U/μL. Moreover, it can be used to measure the enzyme kinetic parameters and detect the hOGG1 activity in crude cell extracts, offering a powerful tool for biomedical research and clinical diagnosis.
authors.affiliation: College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University , Jinan 250014, China.
authors.forename: Li-Juan
authors.initials: LJ
authors.lastname: Wang
authors.affiliation: College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University , Jinan 250014, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Ma
authors.affiliation: College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University , Jinan 250014, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Tang
authors.affiliation: College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University , Jinan 250014, China.
authors.forename: Chun-Yang
authors.initials: CY
authors.lastname: Zhang
mesh_terms: D000072283:A549 Cells; D002232:Carbocyanines / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D045647:DNA Glycosylases / Q000032:analysis* / Q000378:metabolism; D019951:DNA Polymerase beta / Q000378:metabolism; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D057230:Limit of Detection; D045663:Quantum Dots / Q000737:chemistry*
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; C085321:cyanine dye 5; D004247:DNA; D019951:DNA Polymerase beta; C000625660:POLB protein, human; D045647:DNA Glycosylases; C479058:oxoguanine glycosylase 1, human; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase