An electrochemiluminescence (ECL) sensing for sensitive assaying of active DNA glycosylase was developed using the enhanced host-guest recognition technique. The α-cyclodextrin was selected as the host molecule which captured the guest-labeled ECL probe to the surface of electrode by the host-guest recognition. The ECL probe can be protected from the digestion of exonuclease I (Exo I) and exonuclease III (Exo III) with the presence of the target enzyme human 8-oxoguanine DNA glycosylase 1 (hOGG 1), resulting in the ECL emission intensity was correlated with the quantity of hOGG 1. The α-cyclodextrin functionalized gold/silica (Au/SiO 2 ) cell-shell nanoparticles was prepared to enhance the host-guest recognition sensitivity. Because of the increased recognition sites provided by α-cyclodextrin functionalized Au/SiO 2 cell-shell nanoparticles, the nanoparticle-modified electrode displayed a high capacity for the guest ECL probe, and four fold enhancement of the ECL signals was achieved. The as-prepared ECL sensing exhibits excellent analytical property for the detection of hOGG 1 in the linear range of 2–100 U mL −1 with a detection limit of 0.225 U mL −1 (S/N = 3). Interference tests show that the ECL intensity of the interferents human apurinic/apyrimidinic endonuclease (APE 1), T4 endonuclease V (T4 PDG) and endonuclease III (Endo III) is within 36.5 to 44.7% of that of hOGG 1. The ECL sensing exhibits long-term stability with a relative standard deviation (RSD) of 1.6% for 16 cycles of continuous potential scans, and the life time of the ECL sensing is up to 20 days. The obtained results in this study indicate that the proposed ECL sensing possesses high sensitivity, specificity and stability and provides a powerful tool for assaying hOGG 1 activity.
Junjie Wu,Mengmeng Zhang,Lulu Song,Yahong Tan,Yosuke Taniguchi,Christopher John Hipolito,Youming Zhang,Yizhen Yin
Oxidative lesions, such as 8-oxo-dG and 8-oxo-dA, are continuously generated from exposure to reactive oxygen species. While 8-oxo-dG has been extensively studied, 8-oxo-dA has not received as much attention until recently. Herein, we report the synthesis of duplex DNAs incorporating dA, 8-oxo-dA, 7-deaza-dA, 8-Br-dA, and 8-Br-7-deaza-dA, which have different substitutions at 7- and 8-position, for the investigation into the implications of N7-hydrogen and C8-keto on the base pairing preference, mutagenic potential and repair of 8-oxo-dA. Base pairing study suggested that the polar N7-hydrogen and C8-keto of 8-oxo-dA, rather than the syn -preference, might be essential for 8-oxo-dA to form a stable base pair with dG. Insertion and extension studies using KF-exo − and human DNA polymerase β indicated that the efficient dGTP insertion opposite 8-oxo-dA and extension past 8-oxo-dA:dG are contingent upon not only the stable base pair with dG, but also the flexibility of the active site in polymerase. The N7-hydrogen in 8-oxo-dA or C7-hydrogen in 7-deaza-dA and 8-Br-7-deaza-dA was suggested to be important for the recognition by hOGG1, although the excision efficiencies of 7-deaza-dA and 8-Br-7-deaza-dA were much lower than 8-oxo-dA. This study provides an insight into the structure-function relationship of 8-oxo-dA by nucleotide analogues.
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