ArticleMikrochimica acta2019
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.
Article in Mikrochimica acta, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 1 paper.
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1 citing paper in PubMed.
- A Single-Enzyme Activated CRISPR-Cas12a Nano System via Subtly Balanced dsDNA for Kinetic-Gated UDG Detection and Spatiotemporal Cellular Imaging.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2026Article
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6 authors.
Funding
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
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