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Terminal Deoxynucleotidyl Transferase and T7 Exonuclease-Aided Amplification Strategy for Ultrasensitive Detection of Uracil-DNA Glycosylase

Du, Yi-Chen, Cui, Yun-Xi, Li, Xiao-Yu, Sun, Guo-Ying, Zhang, Yu-Peng, Tang, An-Na, Kim, Kwangil, Kong, De-Ming
Analytical chemistry 2018 v.90 no.14 pp. 8629-8634
DNA, DNA repair, catalytic activity, disease diagnosis, drugs, fluorescence, fluorescent dyes, genomics, methyltransferases, oligonucleotides, restriction endonucleases, screening, uracil, uracil-DNA glycosylase
As one of the key initiators of the base excision repair process, uracil-DNA glycosylase (UDG) plays an important role in maintaining genomic integrity. It has been found that aberrant expression of UDG is associated with a variety of diseases. Thus, accurate and sensitive detection of UDG activity is of critical significance for biomedical research and early clinical diagnosis. Here, we developed a novel fluorescent sensing platform for UDG activity detection based on a terminal deoxynucleotidyl transferase (TdT) and T7 exonuclease (T7 Exo)-aided recycling amplification strategy. In this strategy, only two DNA oligonucleotides (DNA substrate containing one uracil base and Poly dT probe labeled with a fluorophore/quencher pair) are used. UDG catalyzes the removal of uracil base from the enclosed dumbbell-shape DNA substrate to give an apyrimidinic site, at which the substrate oligonucleotide is cleaved by endonuclease IV. The released 3′-end can be elongated by TdT to form a long deoxyadenine-rich (Poly dA) tail, which may be used as a recyclable template to initiate T7 Exo-mediated hybridization–digestion cycles of the Poly dT probe, giving a significantly enhanced fluorescence output. The proposed UDG-sensing strategy showed excellent selectivity and high sensitivity with a detection limit of 1.5 × 10–⁴ U/mL. The sensing platform was also demonstrated to work well for UDG inhibitor screening and inhibitory activity evaluation, thus holding great potential in UDG-related disease diagnosis and drug discovery. The proposed strategy can be easily used for the detection of other DNA repair-related enzymes by simply changing the recognition site in DNA substrate and might also be extended to the analysis of some DNA/RNA-processing enzymes, including restriction endonuclease, DNA methyltransferase, polynucleotide kinase, and so on.