College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University, Jinan 250014, China.
Nanoscale. 2020 Feb 20;12(7):4519-4526. doi: 10.1039/c9nr10376g.
CpG island methylation plays an important role in diverse biological processes including the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer. Due to the dependence of DNA methylation on DNA methyltransferase (MTase) activity, DNA MTases have become the potential targets in anticancer therapy. Herein we demonstrate for the first time the construction of a single quantum dot (QD) nanosensor with the capability of sensing methylcytosine sites for sensitive quantification of M.SssI CpG methyltransferase (M.SssI MTase). We design a biotin-/phosphate-modified double-stranded DNA (dsDNA) substrate with a 5'-G-C-G-mC-3'/3'-mC-G-mC-G-5' site for sensing M.SssI MTase. In the presence of M.SssI MTase, the methylation-responsive sequence of the dsDNA substrate is methylated and cleaved by GlaI endonuclease, producing two dsDNA fragments with a free 3'-OH terminus. In the presence of terminal deoxynucleotidyl transferase (TdT), multiple Cy5-dATPs can be sequentially added to the free 3'-OH terminus of dsDNA fragments to obtain biotin-/multiple Cy5-labeled dsDNAs. The resultant biotin-/multiple Cy5-labeled dsDNAs can assemble on the surface of the streptavidin-coated QD to obtain a QD-dsDNA-Cy5 nanostructure in which the fluorescence resonance energy transfer (FRET) from the QD to Cy5 can occur. The emission of Cy5 can be simply quantified by single-molecule detection. By the integration of sensing methylcytosine sites and enzymatic polymerization, the sensitivity of this nanosensor has been significantly enhanced. This nanosensor can detect as low as 2.1 × 10-7 U μL-1 M.SssI MTase with good selectivity against other cytosine MTases, and it can be further applied for the screening of MTase inhibitors and complex biological sample analysis, holding great potential in clinical diagnosis and drug discovery.
CpG 岛甲基化在多种生物学过程中发挥着重要作用，包括印迹基因的调控、X 染色体失活和人类癌症中肿瘤抑制基因的沉默。由于 DNA 甲基化依赖于 DNA 甲基转移酶（MTase）的活性，因此 DNA MTase 已成为抗癌治疗的潜在靶点。本文首次构建了一种单量子点（QD）纳米传感器，该传感器具有检测甲基胞嘧啶位点的能力，可用于灵敏定量 M.SssI CpG 甲基转移酶（M.SssI MTase）。我们设计了一种带有 5'-G-C-G-mC-3'/3'-mC-G-mC-G-5' 位点的生物素/磷酸化修饰双链 DNA（dsDNA）底物，用于检测 M.SssI MTase。在 M.SssI MTase 的存在下，dsDNA 底物的甲基化反应序列被甲基化，并被 GlaI 内切酶切割，产生两个带有游离 3'-OH 末端的 dsDNA 片段。在末端脱氧核苷酸转移酶（TdT）的存在下，多个 Cy5-dATP 可以依次添加到 dsDNA 片段的游离 3'-OH 末端，得到生物素/多个 Cy5 标记的 dsDNA。所得的生物素/多个 Cy5 标记的 dsDNA 可以组装在链霉亲和素涂覆的 QD 表面上，从而获得 QD-dsDNA-Cy5 纳米结构，其中 QD 到 Cy5 的荧光共振能量转移（FRET）可以发生。Cy5 的发射可以通过单分子检测简单地定量。通过检测甲基胞嘧啶位点和酶聚合的集成，该纳米传感器的灵敏度得到了显著提高。该纳米传感器可以检测低至 2.1×10-7 U μL-1 的 M.SssI MTase，对其他胞嘧啶 MTase 具有良好的选择性，并且可以进一步用于 MTase 抑制剂的筛选和复杂生物样品分析，在临床诊断和药物发现方面具有巨大潜力。
