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.

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. Graphical abstract 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.