Synthetic membrane nanopores from self-assembled and chemically modified DNA

Nanopores are widespread in nature and facilitate the essential transport of water-soluble molecules across bilayers. Replicating this key property with engineered or de-novo pores is scientifically intriguing and additionally leads to powerful biomedical research tools and biosensor components.

The Howorka group creates synthetic nanopores from self-assembled DNA carrying hydrophobic lipid anchors to insert the otherwise hydrophilic structures into lipid bilayer membranes [1-4]. The new DNA-nanopores open up the design of entirely new molecular devices for a broad range of applications including sensing, killing of cancer cells, catalysis, and controlled release. The group’s publications on DNA nanopores have been featured on two Angewandte Chemie and a ACS Nano cover and have been highlighted in a Nature Chemistry article [5].


Biological protein pores of biomedical and biotechnological relevance

The Howorka group is engaged in biophysically analysing biological channels to understand their function. A recent break-through has been achieved in collaboration with structural biologist Prof. Han Remaut on the CsgG channel which shuttles proteins out of the pathogenic bacteria to form curli-fibres and antibiotic-resistant biofilms, as published in Nature [6]. The insight may help design strategies to interfere with biofilm formation. The analysis of the CsgG pores with single-channel electrical recordings complements current  [7-9] and continues previous research interests [10, 11] the engineering of and sensing with the protein nanopore α-hemolysin.

In related research, the structure of a multi-porous protein assembly was investigated to achieve a world-first. The S-layer protein lattices are major two-dimensional cell-wall component in archaea and many pathogenic and biotechnologically relevant bacteria. Initiated by our group’s examination of their molecular architecture with chemical and genetics tools [12, 13], structure of an S-layer protein has been elucidated to atomic resolution (see illustration) in collaboration with Prof. Han Remaut, as published in Nature [14]. This unprecedented structural insight will help guide the rational engineering of the protein lattices into vaccine carriers [15].


Nucleic acids probes and linker chemistries for research

Nucleic acids and linker chemistries are important to expand the functional range of DNA strands. For example, fluorophores are chemically attached to facilitate the detection of DNA. The Howorka group has created new nucleotide derivatives which enable the facile and highly efficient labelling of DNA strands with fluorophores and several other biotags [16-18]. The linker chemistry under mild conditions involves e.g. Diels Alder reaction.

In addition, we have developed linker and surface chemistries which enable the immobilisation of DNA and other molecular receptors onto sensor surface and biophysical research platforms. The key benefit is the retention of the receptors’ specific recognition activity for analytes. At the same time, the sensor surface is rendered resistant to the undesired adsorption of non-target molecules. Different linker chemistries have been successfully used including poly(ethylene glycol) but also molecularly defined DNA nanostructures resembling a tetrahedron (see illustration) [19, 20]. In microarrays, this dual advantage has achieved the ultrasensitive sensing of diagnostically important RNA species which remain undetected in conventional analytical schemes [21]. As further highlight, epigenetically relevant methylated DNA strands could be analysed at the single-molecule level in collaboration with Prof. Hinterdorfer as published in Nature Nanotechnology [22].

DNA tetrahedron
Selected Publications

[1] J. Burns, E. Stulz, S. Howorka, Nano Lett. 2013, 13, 2351-2356.

[2] J. R. Burns, K. Göpfrich, J. W. Wood, V. V. Thacker, E. Stulz, U. F. Keyser, S. Howorka, Angew. Chem. Int. Ed. 2013, 52, 12069–12072.

[3] J. R. Burns, N. Al-Juffali, S. M. Janes, S. Howorka, Angew. Chem. Int. Ed. 2014, 53, 12466-12470.

[4] A. Seifert, K. Gopfrich, J. R. Burns, N. Fertig, U. F. Keyser, S. Howorka, ACS Nano 2015, 9, 1117-1126.

[5] S. Krishnan, F. C. Simmel, Nat. Chem. 2014, 7, 17-18.

[6] P. Goyal, P. V. Krasteva, N. Van Gerven, F. Gubellini, I. Van den Broeck, A. Troupiotis-Tsaïlaki, W. Jonckheere, G. Péhau-Arnaudet, J. S. Pinkner, M. R. Chapman, S. J. Hultgren, S. Howorka, R. Fronzes, H. Remaut, Nature 2014, 516, 250-253.

[7] S. Buchsbaum, N. Mitchell, M. Hugh, M. Wiggin, A. Marziali, P. V. Coveney, S. Ziwy, S. Howorka, Nano Lett. 2013, 13, 3890-3896.

[8] S. Howorka, Z. S. Siwy, Nat. Biotechnol. 2012, 30, 506-507.

[9] S. Howorka, Z. Siwy, Chem. Soc. Rev. 2009, 38, 2360-2384.

[10] S. Howorka, S. Cheley, H. Bayley, Nat. Biotechnol. 2001, 19, 636-639.

[11] L. Movileanu, S. Howorka, O. Braha, H. Bayley, Nat. Biotechnol. 2000, 18, 1091-1095.

[12] H. Kinns, S. Howorka, J. Mol. Biol. 2008, 377, 589-604.

[13] H. Kinns, H. Badelt-Lichtblau, E. M. Egelseer, U. B. Sleytr, S. Howorka, J. Mol. Biol. 2010, 395, 742-753

[14] E. Baranova, R. Fronzes, A. Garcia-Pino, N. Van Gerven, D. Papapostolou, G. Péhau-Arnaudet, E. Pardon, J. Steyaert, S. Howorka, H. Remaut, Nature 2012, 487, 119-122.

[15] S. Howorka, Curr. Opin. Biotechnol. 2011, 22, 485-491.

[16] V. Borsenberger, S. Howorka, Nucleic Acids Res. 2009, 37, 1477-1485.

[17] V. Borsenberger, M. Kukwikila, S. Howorka, Org. Biomol. Chem. 2009, 7, 3826 - 3835.

[18] V. Borsenberger, N. Mitchell, S. Howorka, J. Am. Chem. Soc. 2009, 131, 7530-7531.

[19] R. Schlapak, J. Danzberger, D. Armitage, D. Morgan, A. Ebner, P. Hinterdorfer, P. Pollheimer, H. J. Gruber, F. Schaffler, S. Howorka, Small 2012, 8, 89-97.

[20] N. Mitchell, R. Schlapak, M. Kastner, D. Armitage, W. Chrzanowski, J. Riener, P. Hinterdorfer, A. Ebner, S. Howorka, Angew. Chem. Int. Ed. 2009, 48, 525-527.

[21] S. Howorka, J. Hesse, Soft Matt. 2014, 10, 931-941.

[22] R. Zhu, S. Howorka, J. Proll, F. Kienberger, J. Preiner, J. Hesse, A. Ebner, V. P. Pastushenko, H. J. Gruber, P. Hinterdorfer, Nat. Nanotechnol. 2010, 5, 788-791.