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Atomic Force Microscopy Studies of DNA Origami Nanostructures: from Structural Stability to Molecular Patterning

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Atomic Force Microscopy Studies of DNA Origami Nanostructures: from Structural Stability to Molecular Patterning Atomic force microscopy studies of DNA origami nanostructures: from structural stability to molecular patterning Dissertation zur Erlangung des Grades „Doktor rerum naturalium“ an der Universität Paderborn Fakultät für Naturwissenschaften Department Chemie von Saminathan Ramakrishnan Geb. 22.04.1989 in Aranthangi, India Gutachter: PD Dr. Adrian Keller Adjunct Professor, PhD Veikko Linko Eingereicht am: Tag der Verteidigung: ii Contents Abstract ............................................................................................................................................ 1 1. Introduction .................................................................................................................................. 3 1.1 Genetic material ...................................................................................................................... 3 1.2 DNA as a genetic material ...................................................................................................... 3 1.3 Molecular structure of nucleic acids ....................................................................................... 4 1.4 Nanotechnology and Atomic Force Microscopy (AFM)........................................................ 7 1.5 DNA nanotechnology ........................................................................................................... 11 1.6 DNA origami ........................................................................................................................ 14 1.7 Structural stability of DNA origami ..................................................................................... 17 1.7.1 Biological factors ........................................................................................................... 17 1.7.2 Stability of DNA origami in biological conditions ........................................................ 18 1.7.3 Physico-chemical factors ............................................................................................... 23 1.7.4 Mechanical stability ....................................................................................................... 25 1.8 DNA origami for molecular patterning ................................................................................ 27 1.8.1 Surface-assisted hierarchical assembly .......................................................................... 27 1.8.2 Directed adsorption of DNA origami on surfaces ......................................................... 29 2. Structural stability of DNA origami nanostructures in the presence of chaotropic agents ........ 31 2.1 Introduction .......................................................................................................................... 31 2.2 Methods ................................................................................................................................ 33 iii 2.3 Results and Discussion ......................................................................................................... 34 2.4 Denaturation of biotin-streptavidin complex on DNA origami ............................................ 44 2.5 Conclusions .......................................................................................................................... 47 3. Cation-induced stabilization and denaturation of DNA origami nanostructures in urea and guanidinium chloride ...................................................................................................................... 49 3.1 Introduction .......................................................................................................................... 49 3.2 Methods ................................................................................................................................ 50 3.3 Results and Discussion ......................................................................................................... 51 3.4 Conclusion ............................................................................................................................ 58 4. Regular Nanoscale Protein Patterns via Directed Adsorption through Self-Assembled DNA Origami Masks ............................................................................................................................... 59 4.1 Introduction .......................................................................................................................... 59 4.2 Methods ................................................................................................................................ 61 4.3 Results and Discussion ......................................................................................................... 63 4.3.1 Assembly of DNA origami hole masks ......................................................................... 63 4.3.2 Redβ patterning .............................................................................................................. 64 4.3.3 Time-dependent adsorption of Redβ inside DNA origami mask ................................... 69 4.3.4 Irregular adsorption of Redβ inside DNA origami mask ............................................... 70 4.3.5 GFP-Redβ patterning ..................................................................................................... 71 4.3.6 Sak patterning ................................................................................................................ 73 4.3.7 Ferritin patterning .......................................................................................................... 75 iv 4.3.8 BSA patterning ............................................................................................................... 77 4.3.9 Desorption experiments with Redβ protein ................................................................... 78 4.3.10 Factors limiting protein adsorption specificity ............................................................ 79 4.4 Conclusion ............................................................................................................................ 80 5. On the Adsorption of DNA Origami Nanostructures in Nanohole Arrays ................................ 83 5.1 Introduction .......................................................................................................................... 83 5.2 Methods ................................................................................................................................ 84 5.3 Results and discussion .......................................................................................................... 85 5.4 Conclusion ............................................................................................................................ 97 6. Summary .................................................................................................................................. 100 7. References ................................................................................................................................ 104 8. Appendix .................................................................................................................................. 128 8.1 Abbreviations ...................................................................................................................... 128 8.2 List of Publications ............................................................................................................. 130 8.2.1 Part of this work have been / will be published in ....................................................... 130 8.2.2 Other publications ........................................................................................................ 130 8.3 Affidavit .............................................................................................................................. 132 8.4 Acknowledgements ............................................................................................................ 133 v Abstract After their introduction in 2006 by Rothemund, DNA origami nanostructures have emerged as an important molecular tool for various applications in biophysics, medicine, and engineering. The efficient addressability and simple synthesis protocol for a variety of two- and three-dimensional DNA origami structures allow the single-molecule investigation of various biochemical and biophysical processes. However, under a variety of experimental conditions, the question on the structural stability of DNA origami nanostructures is of importance and pose significant limitations regarding their applicability, for instance in protein folding studies that require strongly denaturing conditions. In the first half of this thesis, the structural stability of DNA origami triangles under chaotropic agents induced denaturing conditions is studied. Chaotropic agents such as urea and guanidinium chloride have been used to induce the denaturation of biomolecules. To this end, the effect of denaturing agents on the structural organization of DNA origami triangles is studied with dependent on the temperature and cation concentration. At a 6M concentration of urea and guanidinium chloride, DNA origami structures are found to be stable at 23°C but completely denatured at 42°C. The structural damage of DNA origami triangles varies with a chaotropic agent. Importantly the DNA origami were found to be stable overnight under 6M urea and guanidium chloride at room temperature. Since the stability of DNA
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