Aerolysin纳米孔道对寡聚核苷酸的高灵敏单分子检测

Detection of Single Oligonucleotide by an Aerolysin Nanopore

自纳米孔道单分子电化学技术提出以来,为了构建性能良好的纳米孔道,研究人员一直在寻找不同的孔道材料.本研究探索了Aerolysin生物纳米孔道在寡聚核苷酸检测方面的可能性.实验结果表明,与常用的α-溶血素纳米孔道相比,Aerolysin纳米孔道在寡聚核苷酸检测方面表现出更强的空间和时间分辨能力.三个碱基长度的寡聚核苷酸可对Aerolysin纳米孔道造成约为40%的电流阻断.阻断时间表现出电压相关性,随电压的升高而减小.与其他生物纳米孔道相比,Aerolysin纳米孔道无需任何基因突变、化学修饰即可实现对单个寡聚核苷酸的超灵敏分析.未来,Aerolysin纳米孔道将有可能应用于DNA损伤检测、micro RNA分析以及其他基于纳米孔道的单分子分析检测.

Since the nanopore single-molecule technology has been proposed, it remains a big challenge to generate a sensi- tive and stable nano-scale pore. In order to achieve this goal, membrane proteins, solid-state nanopore and other materials such as DNA origami have been involved to fabricate a suitable nanopore. Compared to the solid-state nanopores, biological nanopores perform a higher resolution for single molecule analysis. Therefore, the investigation of finding new biological nanopores is very important to realize the discrimination of single oligonucleotide. Aerolysin biological nanopore has been applied for the study of oligosaccharides, peptides, protein unfolding and small organic molecules so far. Here, we report that Aerolysin could be utilized for oligonucleotide analysis. The data demonstrated that Aerolysin nanopore has a high resolution both for current and time compared with other most widely used wild-type biological nanopores, such as a-hemolysin and Mycobacterium smegmatis porin A （MspA）. It may be because of its narrow diameter and positive charged amino acids in the lumen. One Aerolysin pore generates a 50 pA constant ion current in 1 mol/L KCl solution, as a three nucleotides length oli- gonucleotides （5＇-AGG-3＇） traversing through nanopore could induce nearly 40% current blockage. In comparison, no current blockage signals were observed when 5＇-AGG-3＇ driven from either cis or trans side of the a-hemolysin nanopore. Further- more, the statistical analysis of duration time of single oligonucleotide through Aerolysin indicates a relationship scale with applied voltage, as the voltage increased from 80 to 160 mV, the duration gradually decreased. Although Aerolysin nanopore has been investigated for nearly 10 years, its ability to detect oligonucleotide was not highlighted. Our findings explored high sensing capabilities of Aerolysin nanopore in the analysis of single oligonucleotide, and extended its application to sin- gle-molecule nucleic acid analysis. Aerolysin is a promising candidate for the DNA sensing, DNA damage detection, mi- croRNA analysis and other single molecule investigations.