Beijing Key Laboratory for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing 100083, China.
Anal Chem. 2024 Aug 13;96(32):13335-13343. doi: 10.1021/acs.analchem.4c03638. Epub 2024 Aug 2.
Function as a potential cancer biomarker, DNA methylation shows great significance in cancer diagnosis, prognosis, and treatment monitoring. While the lack of an ultrasensitive, specific, and accurate method at the single-molecule level hinders the analysis of the exceedingly low levels of DNA methylation. Herein, based on the outstanding recognition and digestion ability of methylation-sensitive restriction endonuclease (MSRE), we established a single MSRE-based cascade exponential amplification method, which requires only two ingeniously designed primers and only one recognition site of MSRE for the detection of DNA methylation. Differentiated by MSRE digestion, the cleaved unmethylated DNA is too short to induce any amplification reactions, while methylated DNA remains intact to trigger cascade exponential amplification and the subsequent CRISPR/Cas12a system. By integrating the two exponential amplification reactions, as low as 1 aM methylated DNA can be accurately detected, which corresponds to 6 molecules in a 10 μL system, indicating that our method is more sensitive than single amplification-based methods with the ability to detect DNA methylation at the single-molecule level. In addition, 0.1% methylated DNA can be effectively distinguished from large amounts of unmethylated DNA. Our method is further introduced to exploit the expression difference of DNA methylation among normal cells and cancer cells. Moreover, the visual detection of DNA methylation is also realized by the full hybridization between amplification products and the crRNA of CRISPR/Cas12a. Therefore, the proposed method has great potential to be a promising and robust bisulfite-free method for the detection of DNA methylation at the single-molecule level, which is of great importance for early diagnosis of cancer.
作为一种潜在的癌症生物标志物，DNA 甲基化在癌症的诊断、预后和治疗监测中具有重要意义。然而，由于缺乏在单分子水平上具有超灵敏、特异和准确的方法，限制了对极低水平的 DNA 甲基化的分析。在此，基于甲基化敏感限制性内切酶（MSRE）的出色识别和消化能力，我们建立了一种基于单个 MSRE 的级联指数扩增方法，该方法仅需要两个巧妙设计的引物和 MSRE 的一个识别位点即可检测 DNA 甲基化。通过 MSRE 消化区分，被切割的未甲基化 DNA 太短而不能引发任何扩增反应，而甲基化 DNA 保持完整以触发级联指数扩增和随后的 CRISPR/Cas12a 系统。通过整合两种指数扩增反应，我们可以准确检测低至 1 aM 的甲基化 DNA，相当于在 10 μL 体系中检测到 6 个分子，表明我们的方法比基于单一扩增的方法更灵敏，能够在单分子水平上检测 DNA 甲基化。此外，我们的方法还可以有效区分 0.1%甲基化 DNA 和大量未甲基化 DNA。我们的方法还进一步用于利用正常细胞和癌细胞中 DNA 甲基化的表达差异。此外，通过扩增产物与 CRISPR/Cas12a 的 crRNA 完全杂交，实现了 DNA 甲基化的可视化检测。因此，该方法具有成为一种有前途的、稳健的、无需亚硫酸氢盐的单分子水平 DNA 甲基化检测方法的潜力，对癌症的早期诊断具有重要意义。
