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Fluorescence determination of the activity of O6-methylguanine-DNA methyltransferase based on the activation of restriction endonuclease and the use of graphene oxide

Authors
  • Le, Dinh-Vu1
  • Jiang, Jian-Hui2
  • 1 Industrial University of Ho Chi Minh City, 12 Nguyen Van Bao St. Go Vap, Ho Chi Minh, 70000, Viet Nam , Ho Chi Minh (Vietnam)
  • 2 Hunan University, Changsha, Hunan, 410082, China , Changsha (China)
Type
Published Article
Journal
Microchimica Acta
Publisher
Springer-Verlag
Publication Date
Apr 29, 2020
Volume
187
Issue
5
Identifiers
DOI: 10.1007/s00604-020-04280-0
Source
Springer Nature
Keywords
License
Yellow

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. Graphical abstractSchematic representation of the fluorescence method for MGMT activity assay

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