Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, Shaanxi Province, PR China.
Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi'an 710062, Shaanxi Province, PR China.
Talanta. 2019 Apr 1;195:320-326. doi: 10.1016/j.talanta.2018.11.083. Epub 2018 Nov 24.
As an important base excision repair (BER) enzyme, uracil-DNA glycosylase (UDG) can repair the uracil-induced DNA lesion and maintain the genomic integrity. Herein, we have proposed a sensitive UDG assay based on the terminal deoxynucleotidyl transferase (TdT)-assisted formation of fluorescent copper nanoclusters (CuNCs). In this study, a uracil-containing stem-loop DNA substrate is rationally designed with its 3'-end blocked with 2', 3'-dideoxycytosine (ddC). UDG can remove the uracil in the DNA substrate to generate an apurinic/apyrimidinic (AP) site, which can then be specifically cleaved by endonuclease IV (Endo IV) to expose a 3'-OH terminus. TdT will initiate the template-free DNA extension along the exposed 3'-OH terminus to produce a quite long poly(T) tail, which will perfectly template the production of fluorescent CuNCs. By recording the fluorescence of the CuNCs, the UDG activity can be faithfully detected. In contrast, if UDG is absent, the 3'-ddC terminus of the DNA substrate cannot be recognized by TdT and thus no TdT-based extension and formation of CuNCs will occur. The use of ddC as a 3'-end blocker can greatly decrease the nonspecific DNA extension and improve the signal-to-noise ratio. Furthermore, TdT is a template-free and sequence-independent DNA polymerase, which can effectively catalyze the tailing process up to a maximum of thousands of thymines, and each tail can form many fluorescent CuNCs. Therefore, an ultrahigh sensitivity is achieved and as low as 0.00005 U/mL of UDG can be clearly detected. Moreover, with an additional AP site-contained poly(A) oligonucleotide during TdT-mediated extension, a branched amplification mechanism is also preliminarily devised, which can further push the detection limit of UDG to an extremely low level of 0.000002 U/mL.
作为一种重要的碱基切除修复（BER）酶，尿嘧啶-DNA 糖基化酶（UDG）可以修复尿嘧啶诱导的 DNA 损伤，维持基因组的完整性。在此，我们提出了一种基于末端脱氧核苷酸转移酶（TdT）辅助形成荧光铜纳米簇（CuNCs）的灵敏 UDG 测定法。在本研究中，我们合理设计了一个含有尿嘧啶的茎环 DNA 底物，其 3'端被 2'，3'-二脱氧胞苷（ddC）封闭。UDG 可以去除 DNA 底物中的尿嘧啶，产生无嘌呤/无嘧啶（AP）位点，然后内切核酸酶 IV（Endo IV）特异性切割该位点，暴露出 3'-OH 末端。TdT 将沿着暴露的 3'-OH 末端起始无模板的 DNA 延伸，产生相当长的聚（T）尾巴，该尾巴将完美地模板生成荧光 CuNCs。通过记录 CuNCs 的荧光，可以准确地检测 UDG 活性。相反，如果没有 UDG，DNA 底物的 3'-ddC 末端不能被 TdT 识别，因此不会发生 TdT 依赖的延伸和 CuNCs 的形成。使用 ddC 作为 3'端封阻物可以大大减少非特异性 DNA 延伸，提高信号噪声比。此外，TdT 是一种无模板和序列非依赖性的 DNA 聚合酶，可以有效地催化延伸过程，最多可延伸数千个胸腺嘧啶，并且每个尾巴都可以形成多个荧光 CuNCs。因此，实现了超高的灵敏度，能够清晰地检测到低至 0.00005 U/mL 的 UDG。此外，通过在 TdT 介导的延伸过程中添加一个含有 AP 位点的聚（A）寡核苷酸，可以初步设计出一种分支扩增机制，将 UDG 的检测限进一步推至极低的 0.000002 U/mL。
