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Construction of an aerolysin nanopore in a lipid bilayer for single-oligonucleotide analysis

Nature Protocols volume 12, pages 1901–1911 (2017)Cite this article

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Abstract

Nanopore techniques offer the possibility to study biomolecules at the single-molecule level in a low-cost, label-free and high-throughput manner. By analyzing the level, duration and frequency of ionic current blockades, information regarding the structural conformation, mass, length and concentration of single molecules can be obtained in physiological conditions. Aerolysin monomers assemble into small pores that provide a confined space for effective electrochemical control of a single molecule interacting with the pore, which significantly improves the temporal resolution of this technique. In comparison with other reported protein nanopores, aerolysin maintains its functional stability in a wide range of pH conditions, which allows for the direct discrimination of oligonucleotides between 2 and 10 nt in length and the monitoring of the stepwise cleavage of oligonucleotides by exonuclease I (Exo I) in real time. This protocol describes the process of activating proaerolysin using immobilized trypsin to obtain the aerolysin monomer, the construction of a lipid membrane and the insertion of an individual aerolysin nanopore into this membrane. A step-by-step description is provided of how to perform single-oligonucleotide analyses and how to process the acquired data. The total time required for this protocol is ∼3 d.

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Figure 1: Structure of the wild-type aerolysin.
Figure 2: Schematic of the experimental setup.
Figure 3: I–V curves and conductance of aerolysin in various pH solutions.
Figure 4: I–V curve and current traces of a single aerolysin pore (Step 20).
Figure 5: Translocation of dAn (n = 2, 3, 4, 5 and 10) through an aerolysin nanopore (Step 22A).
Figure 6: Real-time monitoring of stepwise cleavage of dA5 by Exo I (Step 22B).

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Acknowledgements

This research was supported by the National Natural Science Foundation of China (21421004 and 21327807), the Program of Introducing Talents of Discipline to Universities (B16017), the Program of Shanghai Subject Chief Scientist (15XD1501200) and the Fundamental Research Funds for the Central Universities (222201718001 and 222201717003).

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  1. Chan Cao and Dong-Fang Liao: These authors contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory for Advanced Materials, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai, China

    Chan Cao, Dong-Fang Liao, Jie Yu, He Tian & Yi-Tao Long

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Contributions

C.C. and Y.-T.L. conceived and designed the research; D.-F.L. and J.Y. performed nanopore experiments and analyzed the data; C.C., D.-F.L. and Y.-T.L. drew and summarized the figures; C.C., D.-F.L., H.T. and Y.-T.L. wrote the manuscript. All authors discussed the results and commented on the manuscript at all stages.

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Integrated supplementary information

Supplementary Figure 1 pH effect on duration and frequency of dA4 translocation

(a) The duration of dA4 exponentially decreased with the applied voltage at pH levels of 4.0-10.0. The blockades revealed the longest duration time at pH 8.0, and the duration significantly decreased in acidic conditions, while it was unchanged in basic conditions. (b) The frequency of dA4 linearly increased with the applied voltage at pH levels between 4.0-10.0. The error-bars in panel a and b indicate standard deviation from data derived from five independent experiments. Figures reproduced with permission from ref. 9.

Supplementary Figure 2 Total internal reflection fluorescence (TIRF) measurements of dA2-FAM translocation

(a) The translocation event counts (red) and the TIRF intensity (blue) exhibit the consistent normalized slope values (9.87%/h and 9.89%/h, respectively). The error-bars in panel a indicates standard deviation from data derived from three independent experiments. (b-e) TIRF images of the collected trans-solution at the recording time of 4 h (b), 7 h (c), 9 h (d) and 11 h (e). Reproduced with permission from ref. 9.

Supplementary information

Supplementary Figures and Tables

Supplementary Figures 1 and 2, and Supplementary Tables 1 and 2. (PDF 365 kb)

Translocation of dAn (n = 2, 3, 4 and 5) through an aerolysin nanopore

The successive addition of dA2, dA3, dA4 and dA5 to the cis side of the aerolysin pore produced distinguishable blockades. The data were acquired in 1.0 M KCl, 10 mM Tris and 1.0 mM EDTA, pH = 8.0. (MOV 13804 kb)

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Cao, C., Liao, DF., Yu, J. et al. Construction of an aerolysin nanopore in a lipid bilayer for single-oligonucleotide analysis. Nat Protoc 12, 1901–1911 (2017). https://doi.org/10.1038/nprot.2017.077

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