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Determination of the activity of uracil-DNA glycosylase by using two-tailed reverse transcription PCR and gold nanoparticle-mediated silver nanocluster fluorescence: a new method for gene therapy-related enzyme detection

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Abstract

The authors present a fluorometric method for ultrasensitive determination of the activity of uracil-DNA glycosylase (UDG). It is based on the use of two-tailed reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and an entropy-driven reaction. The assay involves the following steps: (1) UDG-driven uracil excision repair, (2) two-tailed RT-qPCR-mediated amplification, (3) RNA polymerase-aided amplification, and (4) DNA-modified silver nanoclusters (AgNCs) as a transducer to produce a fluorescent signal. UDG enables uracil to be removed from U·A pairs in DNA1 and produces a depurinated/depyrimidinated site that is readily cleaved by endonuclease IV (Endo IV). The cleaved DNA contains the T7 RNA polymerase primer for the T7 RNA polymerase amplification which produces a large number of microRNA sequences. Subsequent two-tailed RT-qPCR leads to the formation of a prolonged DNA termed DNA3. The prolonged part of DNA3 is then hybridized with an added DNA4/DNA5 duplex, where DNA5 is labeled with gold nanoparticles (AuNPs), and DNA 4 is labeled with AgNCs. The AuNPs quench the fluorescence of the AgNCs. The duplex has a toehold to hybridize the prolong part of DNA3. This results in the formation of a DNA5/DNA3 duplex due to strand displacement (by replacing the DNA4 in the DNA4/DNA5 duplex). DNA4 is released and moves away from the AuNPs. This results in restored AgNC fluorescence, best measured at excitation/emission wavelengths of 575/635 nm. The method has a detection limit as low as 0.1 mU mL−1 of UDG activity (3σ criterion) with a range of 0.001–0.01 U mL−1. It was used to measure UDG activity in cell lysates. Conceivably, it may be used to screen for UDG inhibitors such as Ugi.

Schematic presentation of the two-tailed RT-qPCR assay platform for ultrasensitive detection of uracil-DNA glycosylase (UDG). Two-tailed RT-qPCR-mediated amplification and RNA polymerase-aided amplification are utilized for signal amplification. DNA-modified silver nanoclusters (AgNCs) are used as a transducer to produce a fluorescent signal.

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Acknowledgments

This work was supported by the National Natural Science Foundation of China (21705061), the Jiangsu Provincial Key Medical Discipline (Laboratory) (ZDXKA2016017) and the Innovation Capacity Development Plan of Jiangsu Province (BM2018023).

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Authors and Affiliations

  1. Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, 214063, Jiangsu, China

    Kai Zhang, Wanting Huang, Ke Wang, Xue Zhu & Minhao Xie

  2. College of Light Industry and Food Engineering, Nanjing Forestry University, Nanjing, 210037, China

    Yue Huang

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  1. Kai Zhang
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  2. Wanting Huang
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  4. Ke Wang
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Correspondence to Kai Zhang or Minhao Xie.

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Zhang, K., Huang, W., Huang, Y. et al. Determination of the activity of uracil-DNA glycosylase by using two-tailed reverse transcription PCR and gold nanoparticle-mediated silver nanocluster fluorescence: a new method for gene therapy-related enzyme detection. Microchim Acta 186, 181 (2019). https://doi.org/10.1007/s00604-019-3307-y

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