Terminal Deoxynucleotidyl Transferase and T7 Exonuclease-Aided Amplification Strategy for Ultrasensitive Detection of Uracil-DNA Glycosylase
Terminal Deoxynucleotidyl Transferase and T7 Exonuclease-Aided Amplification Strategy for Ultrasensitive Detection of Uracil-DNA Glycosylase
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DOI：
10.1021/acs.analchem.8b01928
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发表时间：
2018-07-17
影响因子：
7.4
通讯作者：
Kong, De-Ming
中科院分区：
文献类型：
--
作者：
Du, Yi-Chen;Cui, Yun-Xi;Kong, De-Ming
尿嘧啶-DNA糖基酶(UDG)作为碱基切除修复过程的关键启动子之一，在维持基因组完整性方面起着重要作用。研究发现，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具有重要意义。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外切酶(T7 Exo)辅助循环扩增策略的UDG活性检测的新型荧光传感平台。在这个策略中，只使用了两个DNA寡核苷酸(DNA底物含有一个尿嘧啶碱基和用荧光团/猝灭剂对标记的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，形成脱嘧啶位点，底物寡核苷酸在该位点被核酸内切酶N切割。释放的3‘端可被TDT拉长，形成富含脱氧腺嘌呤的长尾巴(Poly Da)，该尾巴可作为可回收模板，启动Poly DT探针的T7外显式杂交-消化循环，从而显著增强荧光输出。该UDG传感策略具有良好的选择性和高灵敏度，检测下限为1.5×10~(-4)U/mL。该传感平台还可用于UDG抑制剂的筛选和抑制活性评价，在UDG相关疾病的诊断和药物发现方面具有巨大的潜力。该方法只需改变DNA底物上的识别位点，即可用于其他DNA修复相关酶的检测，也可推广到一些DNA/RNA加工酶的分析，包括限制性内切酶、DNA甲基转移酶、多核苷酸激酶等。
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 N. 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 Exomediated 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 x 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.