RESUMEN
The base-excision repair enzyme uracil-DNA glycosylase (UDG) plays a crucial role in the maintenance of genome integrity. The authors describe a fluorometric method for the detection of the activity of UDG. It is making use of (a) a 3'-FAM-labeled hairpin DNA probe with two uracil deoxyribonucleotides in the self-complementary duplex region of its hairpin structure, (b) exonuclease I (Exo I) that catalyzes the release of FAM from the UDG-induced stretched ssDNA probe, and (c) graphene oxide that quenches the green FAM fluorescence of the intact hairpin DNA probe in the absence of UDG. If Exo I causes the release of FAM from the hairpin DNA probe, the fluorescence peaking at 517 nm is turned off in the absence of UDG but turned on in its presence. The resulting assay has a wide linear range (0.008 to 1 U·mL-1) and a detection limit as low as 0.005 U·mL-1. It has good specificity for UDG over potentially interfering enzymes and gave satisfactory results when applied to biological samples. Conceivably, the method may be used in a wide range of applications such as in diagnosis, drug screening, and in studying the repair of DNA lesions. Graphical abstract Schematic presentation of a fluorometric strategy for detection of the activity of uracil-DNA glycosylase by using on graphene oxide and exonuclease I assisted signal amplification.
Asunto(s)
Pruebas de Enzimas/métodos , Exodesoxirribonucleasas/metabolismo , Fluorometría/métodos , Grafito/química , Óxidos/química , Uracil-ADN Glicosidasa/metabolismo , Sondas de ADN/química , Sondas de ADN/genética , Sondas de ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Secuencias Invertidas Repetidas , Técnicas de Amplificación de Ácido Nucleico , Uracil-ADN Glicosidasa/antagonistas & inhibidores , Uracil-ADN Glicosidasa/sangreRESUMEN
Uracil-deoxyribonucleic acid glycosylase (UDG) is known to function as an important base-excision repair enzyme and eliminate uracil from DNA molecules to maintain genomic integrity. A new small organic molecule (DID-VP) with interesting structural properties was synthesized as a G-quadruplex selective ligand and was demonstrated to be a sensitive luminescent switch-on probe in a convenient luminescent assay specifically for UDG detection in fetal bovine serum samples under rapid and simple conditions. This newly developed analytical method is based on the UDG enzymatic activity to unwind a duplex DNA substrate, and comprises a G-quadruplex-forming sequence (ON1) and uracil-containing DNA strand (ON2) to generate a remarkable fluorescence signal through the specific interaction of DID-VP with ON1. This luminescent switch-on assay is able to achieve high sensitivity and specificity for UDG over other enzymes. The application range of the present analytical system is found to be 0.05 to 1.00 U mL(-1) UDG with a very low detection limit of 0.005 U mL(-1). The recovery study of UDG in real samples gave a very good performance with 75.05%-102.7% recovery. In addition, an extended application of the assay in screening of UDG inhibitors is demonstrated. A good dose-dependence of the luminescence response with respect to the concentration of UDG inhibitors in samples was observed.
