title: Target-mediated consecutive endonuclease reactions for specific and sensitive homogeneous fluorescence assay of O6-methylguanine-DNA methyltransferase.
abstract: O(6)-Methylguanine-DNA methyltransferase (MGMT) is one of the most important DNA-repair enzymes. Herein, a simple, sensitive and selective homogeneous fluorescence assay strategy is developed for the detection of MGMT on the basis of target-mediated two consecutive endonuclease reactions. The activity assay of MGMT is firstly accomplished using a hairpin-structured DNA substrate to offer a specific recognition site on the substrate DNA for restriction endonuclease PvuII, and thus to initiate the first endonuclease reaction. The product which activates the second endonuclease reaction allows an efficient amplification approach to create an abundance of fluorescence signal reporters. The first endonuclease reaction offers the method high specificity and the second one furnishes the assay improved sensitivity. The results reveal that the MGMT assay strategy shows dynamic responses in the concentration range from 1 to 120 ng mL(-1) with a detection limit of 0.5 ng mL(-1). By simply altering the alkylated bases, this strategy can also be extended for the detection of other alkyltransferases. Therefore, the developed strategy might provide an intrinsically convenient, sensitive and specific platform for alkyltransferase activate assay and related biochemical studies due to its label-free, homogeneous, and fluorescence-based detection format.
authors.affiliation: State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China; Faculty of Chemical Engineering, Industrial University of Hochiminh City, Viet Nam.
authors.forename: Dinh-Vu
authors.initials: DV
authors.lastname: Le
authors.forename: Dian-Ming
authors.initials: DM
authors.lastname: Zhou
authors.forename: Li-Juan
authors.initials: LJ
authors.lastname: Tang
authors.forename: Jian-Hui
authors.initials: JH
authors.lastname: Jiang
authors.forename: Ru-Qin
authors.initials: RQ
authors.lastname: Yu
authors.forename: Yu-Zhi
authors.initials: YZ
authors.lastname: Wang
mesh_terms: D005453:Fluorescence; D057230:Limit of Detection; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry*
chemical_list: D019853:O(6)-Methylguanine-DNA Methyltransferase
keywords: Alkyltransferase; DNA-repair enzyme; Fluorescence; O(6)-Methylguanine DNA methyltransferase; Restriction endonuclease