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Fluorometric determination of the activity of uracil-DNA glycosylase by using graphene oxide and exonuclease I assisted signal amplification

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Abstract

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.

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.

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Acknowledgments

This work was supported by National Natural Science Foundation of China (No. 21205142, 31370104), State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University (2017006), The Research Innovation Program for Graduates of Central South University (2018zzts384, 2018zzts399).

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  1. Mingjian Chen and Wenkai Li contributed equally to this work.

Authors and Affiliations

  1. School of Life Sciences, Central South University, Changsha, 410013, China

    Mingjian Chen, Wenkai Li, Changbei Ma, Kefeng Wu & Hailun He

  2. State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha, 410081, China

    Changbei Ma & Kemin Wang

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  1. Mingjian Chen
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Correspondence to Changbei Ma.

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Chen, M., Li, W., Ma, C. et al. Fluorometric determination of the activity of uracil-DNA glycosylase by using graphene oxide and exonuclease I assisted signal amplification. Microchim Acta 186, 110 (2019). https://doi.org/10.1007/s00604-019-3247-6

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