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Creating a Single Sensing Zone Within an Alpha-Hemolysin Pore via Site-Directed Mutagenesis

BioNanoScience volume 4, pages 78–84 (2014)Cite this article

Abstract

Although significant progress has recently been made towards realizing the goal of direct nanopore-based DNA sequencing (Manrao et al. in Nature Biotechnology 30:349–353, 2012), there are still numerous hurdles that need to be overcome. One such hurdle associated with the use of the biological nanopore α-hemolysin (αHL) is the fact that the wild-type channel contains three very distinct recognition or sensing regions within the β-barrel (Stoddart et al. in Proceedings of the National Academy of Sciences of the United States of America 106:7702–7707, 2009; Stoddart et al. in Nano Letters 10(9):3633–3637, 2010), making identification of the bases residing within or moving through the pore very difficult. Through site-directed mutagenesis, we have been able to selectively remove one of two sensing regions while simultaneously enhancing the third. Our approach has led to the creation of αHL pores containing single sensing zones and provides the basis for engineering αHL pores suitable for direct DNA sequencing.

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Acknowledgments

This work was supported by NIH grant 1R01HG005095 and DHS grant HSHQDC-09-C-0009.

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  1. Electronic BioSciences, Inc, 5754 Pacific Center Blvd. Suite 204, San Diego, CA, 92121, USA

    Eric N. Ervin, Geoffrey A. Barrall, Prithwish Pal, Megan K. Bean, Anna E. P. Schibel & Andrew D. Hibbs

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  1. Eric N. Ervin
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  2. Geoffrey A. Barrall
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Correspondence to Eric N. Ervin.

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Ervin, E.N., Barrall, G.A., Pal, P. et al. Creating a Single Sensing Zone Within an Alpha-Hemolysin Pore via Site-Directed Mutagenesis. BioNanoSci. 4, 78–84 (2014). https://doi.org/10.1007/s12668-013-0119-0

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  • DOI: https://doi.org/10.1007/s12668-013-0119-0

Keywords

  • Nanopore
  • DNA sequencing
  • Alpha-hemolysin
  • Site-directed mutagenesis
  • Sensing zone