Aerolysin纳米孔核酸检测灵敏区域的协同相互作用探索研究

doi:10.6023/A19060202

摘要/Abstract

纳米孔单分子检测技术以其简便、快速、高通量及无需标记等特点, 应用于DNA及蛋白质测序, 更有望实现单分子动态构象变化的研究. Aerolysin(气单胞菌溶素)纳米孔道由于其特有的较长的β-桶限域区(β-barrel)及孔内壁丰富的带电荷氨基酸残基, 在单个寡聚核苷酸分子分析中展现出极高的灵敏性. 本设计利用dA₁₄-4-X, dA₁₄-11-X, dA₁₄-4-X-11-X (X=C, T, G)等单个寡聚核苷酸探针分子, 研究了Aerolysin的两个灵敏区域R1和R2, 探索了R1灵敏区域对单个碱基弱相互作用的差异, 实现区分单个碱基差异. 进一步实验证明, R1灵敏区域对单个碱基类型差异的灵敏区分不受R2灵敏区域被碱基A、C、T占位所影响. 然而, 当R2区域被碱基G占位时, 会使R1区域丧失对整个孔道电流的主导性. 本研究有助于理解Aerolysin对单个寡聚核苷酸分子的超灵敏测量机制.

关键词: 纳米孔, 单分子界面, 电化学限域, 气单胞菌溶素, 寡聚核苷酸

Nanopore technology are being developed for large areas in life science, not only in DNA sequencing and protein sequencing, but also in biomolecule detection, bio-interaction measurement and drug screening. Aerolysin is regarded as new powerful tool for oligonucleotide sensing and peptide sensing due to its high charged pore lumen. Applied a transmembrane potential with a pair of Ag/AgCl electrodes, the negatively charged oligonucleotides are driven into the aerolysin nanopore, inducing a series of ionic current blockages, which could distinguish the oligonucleotides with different length or single base variation. However, due to the lack of high-resolution structure of aerolysin nanopore, the mechanism of its high sensing capability is not clear, limiting the further applications of aerolysin. Recently, we presented two sensing regions inside aerolysin, R1 (near R220) and R2 (near K238), having huge influences on oligonucleotide sensing. Especially, the R1 is responsible for distinguished all 4 bases and 2 modified based in the mixture. However, the detailed mechanism of synergistic effect for these two regions in detection of single nucleotides is still unclear. Here, we use dA₁₄-4-X, dA₁₄-11-X, dA₁₄-4-X-11-X (X=C, T, G) as probes to investigate the effects of base types on the sensing ability of R1 and R2. The results show that the A, C or T in R2 region did not change the sensing ability of R1 region, while G in R2 would hinder the base discrimination in R1 region. This may be caused by the large volume of G that would nearly fully occupy the R2 region and the stronger non-covalent interaction between G and R2 region, resulting in determining the residual current of the whole nanopore. Moreover, we evaluated the interaction between different bases with the sensing region. The results show that the interaction is independent with the volume of the bases, which is ordered by A>G>C>T, suggesting the interaction inside the aerolysin lumen is a considerable factor for its sensing capability. These results would guide us to directly design the mutant Aerolysin nanopore that aims for DNA sequencing and peptide sequencing.

Key words: nanopore, single-molecule interface, electro confinement, aerolysin, oligonucleotide

引用此文

李孟寅, 应佚伦, 龙亿涛. Aerolysin纳米孔核酸检测灵敏区域的协同相互作用探索研究[J]. 化学学报, 2019, 77(10): 984-988.

Li, Mengyin, Ying, Yilun, Long, Yi-Tao. Unveiling the Synergistic Effect from Key Sensing Regions in Aerolysin-Based Single Oligonucleotide Detection[J]. Acta Chimica Sinica, 2019, 77(10): 984-988.

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图/表 5

图1. (a) Aerolysin纳米孔检测体系示意图、(b)不同寡聚核苷酸穿过Aerolysin纳米孔的电流轨迹示意图

Figure 1. (a) Illustration of Aerolysin nanopore sensing system. (b) Diagram of current traces with different oligonucleotides translocating through aerolysin nanopore The β-barrel of aerolysin was colored yellow, while the two sensing regions were marked green (R1) and red (R2), respectively

图2. (a) dA14-4-T-11-T, (b) dA14-4-T-11-T和dA14-4-T的混合物、(c) dA14-4-T-11-T、dA14-4-T和dA14-11-T的混合物的残余电流直方图、(d)在加入3.0 μmol/L dA14-11-T前后P1、P2两个峰的占比

Figure 2. Histogram of residual current of (a) dA14-4-T-11-T, (b) the mixture of dA14-4-T-11-T and dA14-4-T, (c) the mixture of dA14-4-T-11-T, dA14-4-T and dA14-11-T. (d) Percentage of P1 and P2 before and after the addition of 3.0 μmol/L dA14-11-T All data were acquired in 3.0 mol/L KCl, 10 mmol/L TRIS, 1.0 mmol/L EDTA at pH 8.0 at the applied voltage of 100 mV. The final concentration of each oligonucleotide is 3.0 μmol/L. The current was sampled at 100 kHz and filtered at 5 kHz

图3. (a) dA14-4-C、(b) dA14-4-C和dA14-4-C-11-C混合物的残余电流直方图

Figure 3. Histogram of residual current of (a) dA14-4-C and (b) the mixture of dA14-4-C and dA14-4-C-11-C All data were acquired in 3.0 mol/L KCl, 10 mmol/L TRIS, 1.0 mmol/L EDTA at pH 8.0 at the applied voltage of 100 mV. The final concentration of each oligonucleotide is 3.0 μmol/L. The current was sampled at 100 kHz and filtered at 5 kHz

图4. (a) dA14-4-G-11-G, (b) dA14-4-G-11-G 和dA14-4-G混合物的残余电流直方图

Figure 4. Histogram of residual current of (a) dA14-4-G-11-G and (b) the mixture of dA14-4-G-11-G and dA14-4-G The data were acquired in 3.0 mol/L KCl, 10 mmol/L TRIS, 1.0 mmol/L EDTA at pH 8.0 at the applied voltage of 100 mV. The final concentration of each oligonucleotide is 3.0 μmol/L. The current was sampled at 100 kHz and filtered at 5 kHz

图5. 待测物dA14-4-X-11-X (X=G, A, T, C)的穿孔时间对比图

Figure 5. Translocation duration of dA14-4-X-11-X (X=G, A, T, C) The data were acquired at the applied voltage of 100 mV in 3.0 mol/L KCl, 10 mmol/L TRIS, 1.0 mmol/L EDTA at pH 8.0. The error bars were based on at least three separated experiments

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