Dr Stefan Howorka
My group uses chemistry to facilitate the structural elucidation of proteins and to enhance bioanalysis with nanopores or fluorescence assays.
S-layer proteins: Structural investigations and protein engineering
S-layers are the outermost cell wall component of bacteria and are formed by self-assembly of identical proteins. These aesthetically pleasing planar crystalline arrays can perform various biological roles and are of interest in nanobiotechnology. My group examines the molecular structure of an S-layer protein with chemical tools including targeted chemical modification of cysteines and cross-linking. These investigations enable the advanced structural elucidation and the engineering of lattices with tuneable assembly for biophysics and biomedicine.
Membrane protein pores: Engineering and single-molecule detection
Nanoscale protein pores are a powerful tool to detect individual molecules for basic science and diagnostic applications. In nanopore recordings, single molecules pass and temporarily block a pore leading to modulations in the ionic pore current. My contribution to the field of nanopores has been to create analyte-specific biosensors. In collaboration with Prof. Bayley, sensors were generated by covalently attaching receptors for DNA and proteins to a membrane protein pore and then applying these rationally designed structures to detect biomedically relevant DNA sequences or to study kinetic and thermodynamics of biomolecular interactions at the single-molecule level. My group has recently equipped a pore with a molecular filter composed of a hyperbranched dendrimer with applications in separation and sensing.
DNA: Chemical tagging and single-molecule biophysics
Targeted chemical modification enhances the properties of DNA. In the field of analytical chemistry, chemical tags have been attached to individual bases and shown to facilitate the sensing of DNA with nanopore recordings. The use of chemically modified analytes represents a new paradigm which is different to the previous approach of sensing via pore engineering. In addition, chemically modified DNA can be exploited for nanoscale engineering via rational design. A DNA-based nanostructure was shown to act as a supramolecular scaffold to combine different chemical groups at defined nanoscale distance and at tuneable stoichiometry yielding rationally designed functional properties for applications in biosensing.
Solid substrates: Surface modification and applications for ultrasensitive sensing
Modifying solid substrates with organic or polymer films is an attractive strategy to tailor the surfaces for applications in ultrasensitive DNA sensing and materials science.My group has found that dense poly(ethylene glycol) films on microarray glass supports enhance the sensitivity of DNA arrays while reducing the non-specific binding of analyte DNA to the chip surface. Modified substrates are applied to the microarray analysis of RNA expression levels at the single-molecule level (collaboration with Prof. Schütz). Polymer coatings are also an attractive strategy to tune the electronic properties of ITO substrates.
- Kinns,H., Badelt-Lichtblau,H., Egelseer,E.M., Sleytr,U.B., Howorka,S. (2010). Identifying Assembly-Inhibiting and Assembly-Tolerant Sites in the SbsB S-Layer Protein from Geobacillus stearothermophilus. J. Mol. Biol. 395, 742-753
- Borsenberger,V., Mitchell,N., Howorka,S. (2009). Chemically Labeled Nucleotides and Oligonucleotides Encode DNA for Sensing with Nanopores. J. Am. Chem. Soc. 131(22), 7530-7531
- Howorka,S., Ziwy,S. (2009). Nanopore Analytics: Sensing of Single Molecules. Chemical Society Reviews 38(8), 2360-2384.
- Mitchell,N., Schlapak,R., Kastner,M., Armitage,D., Chrzanowski,W., Riener,J., Hinterdorfer,P., Ebner,A., Howorka,S. (2009). A DNA nanostructure for the functional assembly of chemical groups with tunable stoichiometry and defined nanoscale geometry.Angew. Chem Int. Ed. 48(3), 525-527.
- Schlapak,R., Armitage,D., Saucedo-Zeni,N., Chrzanowski,W., Hohage,M., Caruana,D., Howorka,S. (2009). Selective protein and DNA adsorption on PLL-PEG films modulated by ionic strength. Soft Matter 5(3), 613-621
- Kinns, H., and Howorka, S. (2008) The surface location of individual residues in a bacterial S-layer protein. J. Mol. Biol.377, 589-604.
- Mitchell, N., and Howorka, S. (2008) Chemical tags facilitate the sensing of individual DNA strands with nanopores. Angew. Chem. Int. Ed. .47, 5476-5479.
- Howorka, S. (2007) Creating regular arrays of nanoparticles with self-assembling protein building blocks. J. Mater. Chem.17, 2049-2053.
- Martin, H., Kinns, H., Mitchell, N., Astier, Y., Madathil, R., and Howorka, S. (2007) Nanoscale protein pores modified with PAMAM dendrimers. J. Am. Chem. Soc.129, 9640-9649.
- Schlapak, R., Armitage, D., Saucedo-Zeni, N., Latini, G., Gruber, H. J., Samotskaya, Y., Mesquida, P., Cacialli, F., Hohage, M., and Howorka, S. (2007) Preparation and characterization of dense films of poly(amidoamine) dendrimers on indium tin oxide.Langmuir23, 8916-8924.
- Schlapak, R., Pammer, P., Armitage, D., Zhu, R., Hinterdorfer, P., Vaupel, M., Fruhwirth, T., and Howorka, S. (2006) Glass surfaces grafted with high-density poly(ethylene glycol) as substrates for DNA oligonucleotide microarrays. Langmuir22, 277-285.
- Howorka, S., Cheley, S., and Bayley, H. (2001) Sequence-specific detection of individual DNA-strands using engineered nanopores.Nat. Biotechnol.19, 636-639.
- Movileanu, L., Howorka, S., Braha, O., and Bayley, H. (2000) Detecting protein analytes that modulate transmembrane movement of a polymer chain within a single protein pore. Nat. Biotechnol.18, 1091-1095.