Lipid-interacting switchable DNA origami nanostructures

DNA nanotechnology allows for the programmable self-assembly of nanostructures of arbitrary shapes and sizes. DNA nanostructures can be hydrophobically modified for integration with lipid bilayers. Lipid-integrated DNA nanotechnology can allow for the study of membrane proteins and other fundamental biological processes. In this thesis, switchable lipid-interacting DNA origami nanostructures are introduced. First, dimeric DNA origami nanostructures are successfully programmed to monomerise upon switching. The design space of switchable DNA origami nanostructures is explored using molecular switching mechanisms such as strand displacement, ionic switching and pH switching, as well as photoswitching, an external switching mechanism. Following that, lipid-interacting DNA origami nanostructures are introduced. These include previously studied DNA origami tile and a novel DNA origami barrel nanopore. The DNA origami tile is decorated with cholesterols and its membrane binding is characterised. The optimal number of cholesterols for membrane binding is shown to be between four and eight cholesterols, and the optimal position of the cholesterols is at the edge of the tiles. The spacing between cholesterols and the tiles also affects membrane binding, with a larger spacing increasing membrane binding. Furthermore, these parameters are also shown to affect the aggregation of the cholesterol-modified tiles during folding, and hence the yield of correctly formed tiles. Reversible membrane binding of the tiles is demonstrated using a strand displacement mechanism. A toehold positioned proximal to the cholesterol group is found to decrease the efficiency of strand displacement for tiles not bound to a membrane. However, for membrane bound tiles, the toehold position does not affect strand displacement. Next, a novel switchable lipid-interacting DNA origami barrel nanopore (DOBN) is developed. The design of the DOBN is optimised to maximise the yield of the correctly folded structure. Following that, the switchability of the DOBN in response to strand displacement, pH switching and photoswitching is explored. Logic gates combining the different switching mechanisms are also developed and validated. Finally, selective membrane binding of the DOBN upon switching is successfully demonstrated. Ultimately, the findings in this thesis establish design guidelines for integrating complex switching mechanisms with membrane-binding DNA nanostructures. This paves the way for achieving dynamic control of complex membrane-interacting DNA nanostructures with potential applications in nanomedicine, biophysics, and nucleic acids research