Fluorescence determination of the activity of O 6 -methylguanine-DNA methyltransferase based on the activation of restri
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ORIGINAL PAPER
Fluorescence determination of the activity of O6-methylguanine-DNA methyltransferase based on the activation of restriction endonuclease and the use of graphene oxide Dinh-Vu Le 1 & Jian-Hui Jiang 2 Received: 17 November 2019 / Accepted: 13 April 2020 # Springer-Verlag GmbH Austria, part of Springer Nature 2020
Abstract A fluorescence method is described for the determination of the activity of O6-methylguanine-DNA methyltransferase (MGMT). It is based on the activation of restriction endonuclease PvuII and the adsorbing a fluorophore-labelled DNA onto the surface of graphene oxide (GO). MGMT activity removes the methyl group from O6-methylguanine (O6MeG) in the fluorophore-labelled DNA to unblock the specific recognition site for further hydrolysis reaction of restriction endonuclease PvuII. The endonuclease catalytic reaction releases fluorophores (5-carboxyfluorescein) from fluorophore-labelled DNA, which can avoid fluorescence quenching by GO, creating an abundance of the fluorescence signal. The fluorescence increase in the assay is thus directly dependent on the MGMT activity. Under the optimal conditions with the emission wavelength of 519 nm (exitation at 494 nm), the activity of the MGMT can be determined in the range 0.5 to 35 ng mL−1 with a detection limit of 0.15 ng mL−1. This is extremely sensitive for the determination of MGMT. The short time of analysis (2 h) is superior to many reported strategies. The method can also be extended for the rapid and sensitive activity assay of other DNA repair enzymes by designing a proper substrate DNA. Conceivably, the technique represents a powerful tool for diagnosis and drug exploitation. Keywords MGMT . DNA repair enzyme . PvuII . Nanomaterial . Fluorescence quenching
Introduction O6-Methylguanine-DNA methyltransferase is a central DNA repair enzyme that repairs the pre-mutagenic, precarcinogenic and pre-toxic DNA damage O6-methylguanine by one-step transfer alkyl adduct from O6-guanine in DNA to itself [1–3]. The determination of the activity of O 6 methylguanine-DNA methyltransferase (MGMT) is an Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00604-020-04280-0) contains supplementary material, which is available to authorized users. * Dinh-Vu Le [email protected] 1
Faculty of Chemical Engineering, Industrial University of Ho Chi Minh City, 12 Nguyen Van Bao St. Go Vap, Ho Chi Minh 70000, Viet Nam
2
State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, Hunan, China
essential step to investigate DNA damage O6-methylguanine (O6MeG)–induced chromosomal aberrations, mutations and cancer [4–6]. Radiolabelled methylation is the standard method in MGMT activity assay by using 3H- or 32P-labelled DNA as the MGMT substrate [7, 8]. Still, the widespread use of these methods is limited by the presence of radioactive reagents and is not suitable for high-throughput assay. The other commonly used assays include methy
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