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Discrimination of oligonucleotides of different lengths with a wild-type aerolysin nanopore

Nature Nanotechnology volume 11, pages 713–718 (2016)Cite this article

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Abstract

Protein nanopores offer an inexpensive, label-free method of analysing single oligonucleotides. The sensitivity of the approach is largely determined by the characteristics of the pore-forming protein employed, and typically relies on nanopores that have been chemically modified or incorporate molecular motors. Effective, high-resolution discrimination of oligonucleotides using wild-type biological nanopores remains difficult to achieve. Here, we show that a wild-type aerolysin nanopore can resolve individual short oligonucleotides that are 2 to 10 bases long. The sensing capabilities are attributed to the geometry of aerolysin and the electrostatic interactions between the nanopore and the oligonucleotides. We also show that the wild-type aerolysin nanopores can distinguish individual oligonucleotides from mixtures and can monitor the stepwise cleavage of oligonucleotides by exonuclease I.

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Figure 1: Electrical recording of dAn (n = 2, 3, 4, 5 and 10) with aerolysin nanopores.
Figure 2: Voltage and pH dependence of dAn (n = 3, 4, 5 or 10).
Figure 3: Discrimination among mixtures of dAn with aerolysin nanopore.
Figure 4: Real-time monitoring of the stepwise cleavage of dA5 induced by ExoI with WT aerolysin.

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References

  1. Kasianowicz, J., Brandin, E., Branton, D. & Deamer, D. Characterization of individual polynucleotide molecules using a membrane channel. Proc. Natl Acad. Sci. USA 93, 13770–13773 (1996).

    Article  CAS  Google Scholar 

  2. Reiner, J. E. et al. Disease detection and management via single nanopore-based sensors. Chem. Rev. 112, 6431–6451 (2012).

    Article  CAS  Google Scholar 

  3. Cherf, G. M. et al. Automated forward and reverse ratcheting of DNA in a nanopore at 5-A precision. Nature Biotechnol. 30, 344–348 (2012).

    Article  CAS  Google Scholar 

  4. Laszlo, A. H. et al. Decoding long nanopore sequencing reads of natural DNA. Nature Biotechnol. 32, 829–833 (2014).

    Article  CAS  Google Scholar 

  5. Wendell, D. et al. Translocation of double-stranded DNA through membrane-adapted phi29 motor protein nanopores. Nature Nanotech. 4, 765–772 (2009).

    Article  CAS  Google Scholar 

  6. Haque, F. et al. Single pore translocation of folded, double-stranded, and tetra-stranded DNA through channel of bacteriophage phi29 DNA packaging motor. Biomaterials 53, 744–752 (2015).

    Article  CAS  Google Scholar 

  7. Franceschini, L., Soskine, M., Biesemans, A. & Maglia, G. A nanopore machine promotes the vectorial transport of DNA across membranes. Nature Commun. 4, 2415 (2013).

    Article  Google Scholar 

  8. Ying, Y. L., Zhang, J. J, Gao, R. & Long, Y. T. Nanopore-based sequencing and detection of nucleic acids. Angew. Chem. Int. Ed. 52, 13154–13161 (2013).

    Google Scholar 

  9. Parker, M. W. et al. Structure of the aeromonas toxin proaerolysin in its water-soluble and membrane-channel states. Nature 367, 292–295 (1994).

    Article  CAS  Google Scholar 

  10. Song, L. et al. Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 274, 1859–1865 (1996).

    Article  CAS  Google Scholar 

  11. Stefureac, R., Long, Y. T., Kraatz, H. B., Howard, P. & Lee, J. S. Transport of α-helical peptides through α-hemolysin and aerolysin pores. Biochemistry 45, 9172–9179 (2006).

    Article  CAS  Google Scholar 

  12. Pastoriza-Gallego, M. et al. Evidence of unfolded protein translocation through a protein nanopore. ACS Nano 8, 11350–11360 (2014).

    Article  CAS  Google Scholar 

  13. Cressiot, B. et al. Dynamics and energy contributions for transport of unfolded pertactin through a protein nanopore. ACS Nano 9, 9050–9061 (2015).

    Article  CAS  Google Scholar 

  14. Pastoriza-Gallego, M. et al. Dynamics of unfolded protein transport through an aerolysin pore. J. Am. Chem. Soc. 133, 2923–2931 (2011).

    Article  CAS  Google Scholar 

  15. Payet, L. et al. Thermal unfolding of proteins probed at the single molecule level using nanopores. Anal. Chem. 84, 4071–4076 (2012).

    Article  CAS  Google Scholar 

  16. Merstorf, C. et al. Wild type, mutant protein unfolding and phase transition detected by single-nanopore recording. ACS Chem. Biol. 7, 652–658 (2012).

    Article  CAS  Google Scholar 

  17. Fennouri, A. et al. Kinetics of enzymatic degradation of high molecular weight polysaccharides through a nanopore: experiments and data-modeling. Anal. Chem. 85, 8488–8492 (2013).

    Article  CAS  Google Scholar 

  18. Robertson, J. W. et al. Single-molecule mass spectrometry in solution using a solitary nanopore. Proc. Natl Acad. Sci. USA 104, 8207–8211 (2007).

    Article  CAS  Google Scholar 

  19. Reiner, J. E., Kasianowicz, J. J., Nablo, B. J. & Robertson, J. W. Theory for polymer analysis using nanopore-based single-molecule mass spectrometry. Proc. Natl Acad. Sci. USA 107, 12080–12085 (2010).

    Article  CAS  Google Scholar 

  20. Balijepalli, A., Robertson, J. W., Reiner, J. E., Kasianowicz, J. J. & Pastor, R. W. Theory of polymer-nanopore interactions refined using molecular dynamics simulations. J. Am. Chem. Soc. 135, 7064–7072 (2013).

    Article  CAS  Google Scholar 

  21. Baaken, G. et al. High-resolution size-discrimination of single nonionic synthetic polymers with a highly charged biological nanopore. ACS Nano 9, 6443–6449 (2015).

    Article  CAS  Google Scholar 

  22. Meller, A., Nivon, L. & Branton, D. Voltage-driven DNA translocations through a nanopore. Phys. Rev. Lett. 86, 3435–3438 (2001).

    Article  CAS  Google Scholar 

  23. Nakane, J., Wiggin, M. & Marziali, A. A nanosensor for transmembrane capture and identification of single nucleic acid molecules. Biophys. J. 87, 615–621 (2004).

    Article  CAS  Google Scholar 

  24. Meller, A., Nivon, L., Brandin, E., Golovchenko, J. & Branton, D. Rapid nanopore discrimination between single polynucleotide molecules. Proc. Natl Acad. Sci. USA 97, 1079–1084 (2000).

    Article  CAS  Google Scholar 

  25. Rincon-Restrepo, M., Mikhailova, E., Bayley, H. & Maglia, G. Controlled translocation of individual DNA molecules through protein nanopores with engineered molecular brakes. Nano Lett. 11, 746–750 (2011).

    Article  CAS  Google Scholar 

  26. Degiacomi, M. T. et al. Molecular assembly of the aerolysin pore reveals a swirling membrane-insertion mechanism. Nat. Chem. Biol. 9, 623–629 (2013).

    Article  CAS  Google Scholar 

  27. Payet, L. et al. Temperature effect on ionic current and ssDNA transport through nanopores. Biophys. J. 109, 1600–1607 (2015).

    Article  CAS  Google Scholar 

  28. Benner, S. et al. Sequence-specific detection of individual DNA polymerase complexes in real time using a nanopore. Nature Nanotech. 2, 718–724 (2007).

    Article  CAS  Google Scholar 

  29. Jin, Q., Fleming, A. M., Burrows, C. J. & White, H. S. Unzipping kinetics of duplex DNA containing oxidized lesions in an alpha-hemolysin nanopore. J. Am. Chem. Soc. 134, 11006–11011 (2012).

    Article  CAS  Google Scholar 

  30. Cao, C., Ying, Y. L., Gu, Z. & Long, Y. T. Enhanced resolution of low molecular weight poly(ethylene glycol) in nanopore analysis. Anal. Chem. 86, 11946–11950 (2014).

    Article  CAS  Google Scholar 

  31. Balijepalli, A. et al. Quantifying short-lived events in multistate ionic current measurements. ACS Nano 8, 1547–1553 (2014).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank M. Dal Peraro and F. G. van der Goot for the helpful discussions on the structure of aerolysin. This work was supported by the National Natural Science Foundation of China (grant nos 21327807, 21421004), the 111 Project (grant no. B16017) and National Basic Research Program of China (973 Program) (Grant no. 2013CB733700). Y.-T. L. is supported by the Chang Jiang Scholars Program.

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  1. Key Laboratory for Advanced Materials & Department of Chemistry, East China University of Science and Technology, Shanghai, 200237, China

    Chan Cao, Yi-Lun Ying, Zheng-Li Hu, Dong-Fang Liao, He Tian & Yi-Tao Long

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  1. Chan Cao
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Contributions

C.C. and Y.-T.L. conceived and designed the study; C.C., Z.-L.H. and D.-F.L. conducted the experiments; C.C., Z.-L.H. and D.-F.L. analysed the data; C.C., Y.-L.Y., H.T. and Y.-T.L. wrote the paper.

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Correspondence to Yi-Tao Long.

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Cao, C., Ying, YL., Hu, ZL. et al. Discrimination of oligonucleotides of different lengths with a wild-type aerolysin nanopore. Nature Nanotech 11, 713–718 (2016). https://doi.org/10.1038/nnano.2016.66

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