Key Laboratory of Luminescence Analysis and Molecular Sensing, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing, 400715, PR China.
School of Chemistry and Chemical Engineering, Chongqing University of Technology, Chongqing, 400054, PR China.
Anal Chim Acta. 2023 Apr 22;1251:341025. doi: 10.1016/j.aca.2023.341025. Epub 2023 Feb 27.
The sensitive detection of ten-eleven translocation (TET) dioxygenase is of significance for understanding the demethylation mechanism of 5-methylocytosine (5mC), which is responsible for a wide range of biological functions that can affect gene expression in eukaryotic species. Here, a non-label and sensitive fluorescence biosensing method for TET assay using TET1 as the model target molecule is established on the basis of target-triggered Mg-dependent DNAzyme and catalytic hairpin assembly (CHA)-mediated multiple signal amplification cascades. 5mC sites in the hairpin DNA probe are first oxidized by TET1 into 5-carboxycytosine, which are further reduced by pyridine borane into dihydrouracil, followed by its recognition and cleavage by the USER enzyme to liberate active DNAzyme and G-quadruplex sequences from the probe. The DNAzyme further cyclically cleaves the substrate hairpins to trigger subsequent CHA reaction and DNAzyme cleavage cycles for yielding many G-quadruplex strands. Thioflavin T dye then intercalates into G-quadruplexes to cause a magnificent increase of fluorescence for high sensitivity assay of TET1 with 47 fM detection limit. And, application of this method for TET1 monitoring in diluted serum has also been confirmed.
TET 双加氧酶的灵敏检测对于理解 5-甲基胞嘧啶（5mC）的去甲基化机制具有重要意义，该机制负责广泛的生物学功能，可影响真核生物的基因表达。在此，基于目标触发的 Mg 依赖性 DNA 酶和催化发夹组装（CHA）介导的多重信号放大级联，建立了一种用于 TET 测定的非标记和灵敏荧光生物传感方法，以 TET1 作为模型靶分子。发夹 DNA 探针中的 5mC 位点首先被 TET1 氧化为 5-羧基胞嘧啶，然后被硼氢化吡啶进一步还原为二氢尿嘧啶，随后由 USER 酶识别并切割，从探针中释放活性 DNA 酶和 G-四链体序列。DNA 酶进一步循环切割底物发夹，引发随后的 CHA 反应和 DNA 酶切割循环，从而产生许多 G-四链体链。硫黄素 T 染料随后嵌入 G-四链体中，导致 TET1 的高灵敏度测定，检测限为 47 fM。并且，已经证实该方法可用于稀释血清中的 TET1 监测。
