Custom-tailored DNA origami mechanics for cellular applications

DNA molecules have been used as the building block for the self-assembly of artificial nanostructures. In particular, the DNA origami method has made the design of DNA nanostructures more robust and approachable. Different design approaches have been created and DNA origami has been used in a variety of fields, from plasmonic, to drug delivery, to biology and biophysics. In recent years, DNA nanotechnology has shown very promising uses in studying forces in biological contexts, both by measuring them and applying them. Mechanosensitive systems in biology are widespread and the study of their complex regulation is increasing in importance, and DNA origami has recently been used as a tool to study them. In paper I, we implement an unsupervised software to simulate wireframe DNA origami structures and evaluate their rigidity. After this evaluation, the software produces mutant structures and then the process is started again, iteratively. In this way the software creates an in-silico evolution towards more rigid wireframe DNA origami. The structures are modified following one of two schemes. In the first one, the individual edges are evaluated and then modified by adding or removing individual bases; in the second scheme, the structures have internal supports, and the software can modify the position of these internal supports to create mutants. We show that these two schemes have different results on the rigidity of the structures, with the internal supports-based scheme increasing the rigidity of structures to up to 50%, after several iterations. In paper II, we compare the mechanical characteristics of a lattice-based DNA origami nanostructure and a wireframe DNA origami nanostructure, exploring how the differences between the two affect their interaction with cancer cells. The wireframe structure showed a higher local flexibility when compared to the lattice-based structure. These physical differences play an important role in the interaction between DNA nanostructures and human cancer cells, in particular thanks to the differences in interaction with scavenger receptors. We show that wireframe origamis are more likely to stay on the cell membrane, while the lattice-based origami are more likely to be internalized. This is also reflected in a deeper penetration of the wireframe structures into cell spheroid tissue models. With these observations, we show that the design method should be considered when applying DNA origami for biological applications. In paper III, we aim to expand the design space of wireframe DNA origami, by designing structures with four-helix bundles (4HB) as edges. This is possible thanks to the addition of additional helices to the edge of the wireframe structures, to create 4HB on a square lattice: this results in increased rigidity of the edges. We developed the software for the design of the new type of structures and then we successfully folded a library of five structures, investigating the rigidity of the new type of structures. In addition, we designed a new type of hybrid structures, presenting more rigid 4HB edges and less rigid single helix edges. We think that the development of new ways of designing DNA origami structures can pave the way for the design of nanostructures more suited for specific applications. In paper IV, we design a DNA origami nanoactuator with the aim of pulling on molecular targets. DNA origami is a promising technology in this field because of its high throughput and the relative simplicity when compared with other force spectroscopy techniques. We designed a barrel-like structure with an internal block connected to ssDNA or dsDNA strands, depending on the activation mode of the mechanism. We estimated that the structure can create forces of up to 40 pN, and coarse-grained molecular dynamics simulations in oxDNA and Förster resonance energy transfer experiments confirm the successful activation of the structure. We also demonstrated that the structure, modified with Cy5, cholesterol, and anti-CD3 aptamer, can interact with T cells. We think that DNA origami can become an important tool in the study of mechanosensitive cellular receptors