DNA origami assembly

This thesis describes my investigations into the principles underlying self-assembly of DNA origami nanostructures and discusses how these principles may be applied. To study the origami folding process I designed, synthesized and characterized a polymorphic tile, which could adopt various shapes. The distribution of tile shapes provided new insights into assembly. The origami tiles I studied were based on scaffolds derived from customized plasmids, which I prepared using recombinant DNA technology. I developed a technique to monitor incorporation of individual staples in real time using fluorescence, measuring small differences in staple binding temperatures (~0.5-5 degrees C). I examined the tiles using Atomic Force Microscopy and I found that a remarkably high proportion of polymorphic tiles folded well, which suggests that there are assembly pathways, arising from strong cooperation between staples. In order to analyse the tile shapes quantitatively, I developed a specialized image processing technique. For validation of the method, I generated and analysed simulated data, and the results confirmed that I could measure individual tile parameters with sub-pixel resolution. I studied eleven variants of the polymorphic tile, and I proved that minor staple modifications can be used to change the folding pathway dramatically. The strength of cooperation between staples affects their behaviour, which is also influenced by their length and base sequences. Paired staples are particularly significant in assembly, and there are clear parallels with protein folding. I describe in an Appendix how I applied origami assembly principles in the development of my concept for an autonomous rotary nanomotor utilizing the sequential opening of DNA hairpins (already used for linear motors). This device represents an advance over non-autonomous rotary motors and I have simulated its performance. In this thesis I have answered important questions about DNA origami assembly, and my findings could enable the development of more sophisticated DNA nanostructures for specific purposes.This thesis is not currently available on ORA