Oligofunktionale DNA-Protein-Nanostrukturen für die Untersuchung von Multienzymkaskaden und die Selbstassemblierung oberflächengebundener DNA-Monolagen
Zusammenfassung der Projektergebnisse
This project concerned the implementation of structural DNA nanotechnology with surface-based techniques to realize applications in the life sciences. As the first goal, novel DNA nanostructures based on either double-crossover (DX) DNA tiles or scaffolded DNA origami constructs were developed. Both types of DNA nanostructures contain single-stranded protruding arms (PAs) to facilitate their site-selective immobilization on solid substrates through Watson-Crick hybridization. Specific results of this part of the project include the design, synthesis and characterization of the DNA nanostructures as well as the establishment of methods for their large-scale preparation. Furthermore, novel reconfigurable DNA origami nanochambers were developed, which can be switched under physiological conditions by aid of strand-displacement techniques. As the second goal of this project, recombinant proteins, in particular, enzymes, along with methodologies for their selective attachment on DNA nanostructures were developed. Specific results include the cloning and expression of DNA-conjugatable ketoreductases and oxidoreductases, their exploration in biocatalytic settings, and their exploitation for studying reaction cascades conducted by synthetic multienzyme complexes on DNA nanostructures. We also established a novel approach to significantly increase the efficacy of protein immobilization on DNA nanostructures by engineering electrostatic interactions and we developed a series of photocleavable hapten-modifiers as affinity tags for DNA nanostructures. As the third goal, the bottom-up assembly of DNA nanostructures should be implemented with surface-based technologies, using top-down fabricated DNA microarrays. We successfully demonstrated the site-selective immobilization of both DX- and origami-based nanostructures on solid surfaces bearing complementary DNA micropatterns. Furthermore, to extend our knowledge on DNA nanostructure-surface interactions, we established a novel concept to modify the backbone properties of DNA nanostructures through non-covalent binding of designed intercalators and we studied the interactions of DNA nanostructures with lipid membranes. The fourth and most important goal of the project concerned the exploration and exploitation of the aforementioned surface-bound DNA architectures in cell culture and molecular cell biology. Indeed, we were able to establish that (i) such DNA surfaces are stable under cell culture conditions, (ii) the ligands presented on these surfaces are able to bind and activate their cognate transmembrane receptor, and (iii) the ligand architecture at the nanometer length-scale influences the response of the adhered cells. The latter findings were obtained in proof-of-concept studies for epidermal growth factor signalling. They suggest that the newly developed “multiscale origami structures as interface for cells” (MOSAIC) have the potential to present ligands to living cells on surfaces with a full control over their stoichiometry and nanoscale orientation. This opens the door to addressing fundamental questions in cell signalling, which cannot be tackled by conventional technologies. In conclusion, various issues have been solved in this project to harnessing the advantages of the self-assembly of protein–DNA nanostructures and micropatterning of solid surfaces. As the most important result, the combination of bottom-up and top-down fabrication has led to the innovative MOSAIC tool, which promises novel applications in the life sciences.
Projektbezogene Publikationen (Auswahl)
- (2011) DNA-mediated assembly of Cytochrome P450 BM3 subdomains. J. Am. Chem. Soc. 133, 16111-16118
Erkelenz, M., Kuo, C. H., Niemeyer, C. M.
- (2013) Biochips for Cell Biology by Combined Dip-Pen Nanolithography and DNA-Directed Protein Immobilization. Small 9, 4243-4249
Arrabito, G., Reisewitz, S., Dehmelt, L., Bastiaens, P. I., Pignataro, B., Schroeder, H., Niemeyer, C. M.
(Siehe online unter https://doi.org/10.1002/smll.201300941) - (2014) A Facile Method for Preparation of Tailored Scaffolds for DNA-Origami. Small 10, 73-77
Erkelenz, M., Bauer, D. M., Meyer, R., Gatsogiannis, C., Raunser, S., Sacca, B., Niemeyer, C. M.
(Siehe online unter https://doi.org/10.1002/smll.201300701) - (2015) Designed intercalators for modification of DNA origami surface properties. Chem. Eur. J. 21, 9440-9446
Brglez, J., Nikolov, P. M., Angelin, A., Niemeyer, C. M.
(Siehe online unter https://doi.org/10.1002/chem.201500086) - (2015) Multiscale Origami Structures as Interface for Cells. Angew Chem Int Ed Engl 54, 15813-15817
Angelin, A., Weigel, S., Garrecht, R., Meyer, R., Bauer, J., Kumar, R. K., Hirtz, M., Niemeyer, C. M.
(Siehe online unter https://doi.org/10.1002/anie.201509772) - (2015) Reversible Reconfiguration of DNA Origami Nanochambers Monitored by Single-Molecule FRET. Angew Chem Int Ed Engl 54, 3592-3597
Sacca, B., Ishitsuka, Y., Meyer, R., Sprengel, A., Schoneweiss, E. C., Nienhaus, G. U., Niemeyer, C. M.
(Siehe online unter https://doi.org/10.1002/anie.201408941) - (2015) Site-Directed, On-Surface Assembly of DNA Nanostructures. Angew Chem Int Ed Engl 54, 12039-12043
Meyer, R., Sacca, B., Niemeyer, C. M.
(Siehe online unter https://doi.org/10.1002/ange.201505553) - (2016) A rationally designed connector for assembly of protein-functionalized DNA nanostructures. Chembiochem 17
Koßmann, K. J., Ziegler, C., Angelin, A., Meyer, R., Skoupi, M., Rabe, K. S., Niemeyer, C. M.
(Siehe online unter https://doi.org/10.1002/cbic.201600039)
