Unveiling the Synergistic Effect from Key Sensing Regions in Aerolysin-Based Single Oligonucleotide Detection

doi:10.6023/A19060202

Abstract

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

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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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Fig. & Tab. 5

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

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

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

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

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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Aerolysin纳米孔核酸检测灵敏区域的协同相互作用探索研究