Department of Laboratory Medicine, State Key Laboratory of Biotherapy, Med-X Center for Manufacturing, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu, 610041, China.
Department of Laboratory Medicine, State Key Laboratory of Biotherapy, Med-X Center for Manufacturing, West China Hospital, Sichuan University and Collaborative Innovation Center, Chengdu, 610041, China; School of Pharmacy, North Sichuan Medical College, Nanchong, 637000, China.
Biosens Bioelectron. 2022 Mar 15;200:113894. doi: 10.1016/j.bios.2021.113894. Epub 2021 Dec 17.
Current tools for dNTP analysis mainly rely on expensive fluorescent labeling, mass spectrometry or electrochemistry. Single-molecule assay by protein nanopores with an internal diameter of ca. 1-3.6 nm provides a useful tool for dNTP sensing. However, the most commonly used protein nanopores require additional modifications to enable dNTP detection. In this study, the PaMscS channel (mechanosensitive channel of small conductance from Pseudomonas aeruginosa) embedded in the bilayer lipid membrane (BLM) of E. coli polar lipid extract was applied as a nanopore for single molecular sensing. Two mutants of PaMscS nanopores on the side portal region (PaMscS W130A and PaMscS K180R) were selected for direct dNTP or pyrophosphoric acid (PPi) detection without aptamer or protein modification. Notably, the PaMscS mutant pore can be adjusted by regulation of osmolarity differences, which is crucial for the optimal detection of specific molecules. In addition, we established a PaMscS-based diagnosis method for the rapid sensing of disease-associated nucleic acids by monitoring the consumption of dNTPs, with 86% specificity and 100% sensitivity among 22 clinical samples. This protein nanopore, without aptamer or modification, paves a new way for dNTPs, PPi direct sensing and nucleic acid detection with low cost but high versatility.
目前的 dNTP 分析工具主要依赖于昂贵的荧光标记、质谱或电化学。具有约 1-3.6nm 内径的蛋白质纳米孔的单分子分析为 dNTP 传感提供了一种有用的工具。然而，最常用的蛋白质纳米孔需要额外的修饰才能实现 dNTP 检测。在本研究中，嵌入大肠杆菌极性脂质提取物双层脂质膜（BLM）中的 PaMscS 通道（来自铜绿假单胞菌的机械敏感小电导通道）被用作纳米孔进行单分子传感。选择 PaMscS 纳米孔侧门区域的两个突变体（PaMscS W130A 和 PaMscS K180R）用于直接检测 dNTP 或焦磷酸（PPi），而无需适体或蛋白质修饰。值得注意的是，PaMscS 突变体孔可以通过调节渗透压差异进行调节，这对于最佳检测特定分子至关重要。此外，我们建立了一种基于 PaMscS 的诊断方法，通过监测 dNTP 的消耗来快速感测与疾病相关的核酸，在 22 个临床样本中具有 86%的特异性和 100%的敏感性。这种无需适体或修饰的蛋白质纳米孔为 dNTP、PPi 直接传感和核酸检测开辟了一条新途径，具有低成本但高通用性的特点。
