Detection of Locus-Specific N6-Methyladenosine Modification Based on Ag+-Assisted Ligation and Supersandwich Signal Amplification.
Detection of Locus-Specific N6-Methyladenosine Modification Based on Ag+-Assisted Ligation and Supersandwich Signal Amplification.
复制标题
基于 Ag 辅助连接和超三明治信号放大的位点特异性 N6-甲基腺苷修饰检测。
作者：
Yanxia Wang;Ji Zheng;Chengjie Duan;Jin Jiao;Youjing gong;Hai Shi;Yang Xiang
新的证据表明，表位转录组标记N6-甲基腺苷（m6 A）在生物体中起着重要作用，包括基因调控和疾病进展。然而，开发敏感的方法来检测m6 A修饰，特别是在单位点水平上鉴定m6 A标记，仍然是一个挑战。因此，基于靶特异性触发信号放大，我们开发了一种高灵敏度的电化学方法来检测DNA中的位点特异性m6 A修饰。在这项工作中，DNA中的m6 A位点可以限制Ag+辅助的连接，这种限制作用可以激活随后的链置换反应和杂交链反应（HCR），从而实现来自m6 A位点的信号放大，最终实现m6 A甲基化的高灵敏度分析。利用碱基对的高度特异性和Ag+与m6 A之间极弱的结合亲和力，该方法不仅可用于检测具有推定m6 A位点的靶DNA，而且可用于在单位点水平上识别DNA中的m6 A标记。此外，这项研究不依赖于抗体和放射性标记，因此具有成本效益的优势。因此，我们认为，所提出的策略可能为甲基化研究提供一个新的视角，可以在进一步的研究中用于测试更多的临床样本。
Emerging evidence reveals that the epitranscriptomic mark N6-methyladenosine (m6A) plays vital roles in organisms, including gene regulation and disease progression. However, developing sensitive methods to detect m6A modification, especially the identification of m6A marks at the single-site level, remains a challenge. Therefore, based on target-specific triggered signal amplification, we developed a highly sensitive electrochemical method to detect site-specific m6A modifications in DNA. In this work, the m6A site in DNA can restrict the ligation assisted by Ag+, and this restriction effect can activate the subsequent strand displacement reaction and hybridization chain reaction (HCR), thus achieving signal amplification from the m6A site, and finally realizing high sensitivity analysis of m6A methylation. Benefiting from the high specificity of base pairs and the extremely weak binding affinity between Ag+ and m6A, the proposed method was used for not only detecting the target DNA with a putative m6A site, but also identifying m6A marks at the single-site level in DNA. In addition, this study does not rely on antibodies and radiolabeling, so it has the advantage of cost-effectiveness. Therefore, we believe that the proposed strategy may provide a new perspective for methylation research, which can be used to test more clinical samples in further research.