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

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Terminal Deoxynucleotidyl Transferase and T7 Exonuclease-Aided Amplification Strategy for Ultrasensitive Detection of Uracil-DNA Glycosylase
Analytical Chemistry ( IF 6.7 ) Pub Date : 2018-06-18 00:00:00 , DOI: 10.1021/acs.analchem.8b01928
Yi-Chen Du _{1,

2} , Yun-Xi Cui ₁ , Xiao-Yu Li ₁ , Guo-Ying Sun ₁ , Yu-Peng Zhang ₁ , An-Na Tang ₁ , Kwangil Kim _{2,

3} , De-Ming Kong _{1,

2}

Affiliation

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.

中文翻译：

末端脱氧核苷酸转移酶和T7核酸外切酶辅助的扩增策略，用于尿嘧啶-DNA糖基化酶的超灵敏检测

作为碱基切除修复过程的关键发起者之一，尿嘧啶DNA糖基化酶（UDG）在维持基因组完整性方面起着重要作用。已经发现UDG的异常表达与多种疾病有关。因此，准确，灵敏地检测UDG活性对于生物医学研究和早期临床诊断至关重要。在这里，我们开发了一种基于末端脱氧核苷酸转移酶（TdT）和T7核酸外切酶（T7 Exo）辅助回收扩增策略的UDG活性检测的新型荧光传感平台。在此策略中，仅使用两个DNA寡核苷酸（包含一个尿嘧啶碱基的DNA底物和标记有荧光团/猝灭剂对的Poly dT探针）。UDG催化从封闭的哑铃形DNA底物上去除尿嘧啶碱基，从而得到一个嘧啶环糊精位点，在该位点，底物寡核苷酸被核酸内切酶IV裂解。释放的3'末端可被TdT延长，形成一条长的富含脱氧腺嘌呤（Poly dA）的尾巴，可将其用作可回收利用的模板，以启动Poly dT探针的T7 Exo介导的杂交-消化循环，从而获得显着增强了荧光输出。拟议的UDG传感策略具有出色的选择性和高灵敏度，检测限为1.5×10 它可以用作可回收模板，以启动Poly dT探针的T7 Exo介导的杂交-消化循环，从而显着增强荧光输出。拟议的UDG传感策略具有出色的选择性和高灵敏度，检测限为1.5×10 它可以用作可回收模板，以启动Poly dT探针的T7 Exo介导的杂交-消化循环，从而显着增强荧光输出。拟议的UDG传感策略具有出色的选择性和高灵敏度，检测限为1.5×10^–4 U / mL。该传感平台还被证明可以很好地用于UDG抑制剂的筛选和抑制活性评估，因此在与UDG相关的疾病诊断和药物发现方面具有巨大的潜力。只需更改DNA底物中的识别位点，即可轻松将拟议的策略用于检测其他与DNA修复相关的酶，并且还可以扩展到分析某些DNA / RNA处理酶，包括限制性内切核酸酶，DNA甲基转移酶，多核苷酸激酶等。

更新日期：2018-06-18

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