Li Yunxuan, Meng Siong Chen, Wang Yesheng, Platnich Casey M, Earle Max K, Mylona Elli, Naydenova Plamena, Baker Stephen, Zhu Jinbo, Keyser Ulrich F
Cavendish Laboratory, University of Cambridge, Cambridge, UK.
School of Biomedical Engineering, Faculty of Medicine, Dalian University of Technology, Dalian, China.
Nat Nanotechnol. 2025 Jun 27. doi: 10.1038/s41565-025-01965-6.
RNA mutations and modifications have been implicated in a wide range of pathophysiologies. However, current RNA detection methods are hindered by data complexity and error-prone protocols, restricting their widespread use. Here we present a solid-state nanopore-based approach, RNA single-nucleotide characterization and analysis nanolatch (RNA-SCAN) system, which simplifies the detection of nucleotide mutations and modifications in RNA with high resolution. Using phage RNA as a template, we tested multiple sequences and chemical modifications on nanolatches, allowing the detection of mismatches caused by nucleotide mutations through significant changes in positive event ratios using single-molecule nanopore measurements. This approach is also sensitive to modifications that either strengthen or weaken the interaction between the target RNA sequence and the nanolatch. As a proof-of-concept, we demonstrate successful discrimination of Escherichia coli and Salmonella spp. from total RNA based on nucleotide variations in their 16S rRNA, as well as quantification of different Salmonella spp. and detection of mC1407 modification on E. coli 16S rRNA. The RNA-SCAN approach demonstrates the feasibility of combining RNA/DNA hybrid nanotechnology with nanopore sensing and diagnosing RNA-related health conditions.
RNA突变和修饰与多种病理生理过程有关。然而，当前的RNA检测方法受到数据复杂性和易出错协议的阻碍，限制了它们的广泛应用。在此，我们提出了一种基于固态纳米孔的方法，即RNA单核苷酸表征与分析纳米锁（RNA-SCAN）系统，该系统以高分辨率简化了RNA中核苷酸突变和修饰的检测。以噬菌体RNA为模板，我们在纳米锁上测试了多个序列和化学修饰，通过单分子纳米孔测量中正向事件比率的显著变化，能够检测由核苷酸突变引起的错配。这种方法对增强或减弱目标RNA序列与纳米锁之间相互作用的修饰也很敏感。作为概念验证，我们展示了基于大肠杆菌和沙门氏菌16S rRNA中的核苷酸变异，成功地从总RNA中区分出这两种菌，以及对不同沙门氏菌的定量分析和对大肠杆菌16S rRNA上mC1407修饰的检测。RNA-SCAN方法证明了将RNA/DNA杂交纳米技术与纳米孔传感相结合以及诊断RNA相关健康状况的可行性。
