Switchable DNA-origami nanostructures that respond to their environment and their applications

Structural DNA nanotechnology, in which Watson-Crick base pairing drives the formation of self-assembling nanostructures, has rapidly expanded in complexity and functionality since its inception in 1981. DNA nanostructures can now be made in arbitrary three-dimensional shapes and used to scaffold many other functional molecules such as proteins, metallic nanoparticles, polymers, fluorescent dyes and small molecules. In parallel, the field of dynamic DNA nanotechnology has built DNA circuits, motors and switches. More recently, these two areas have begun to merge—to produce switchable DNA nanostructures, which change state in response to their environment. In this review, we summarise switchable DNA nanostructures into two major classes based on response type: molecular actuation triggered by local chemical changes such as pH or concentration and external actuation driven by light, electric or magnetic fields. While molecular actuation has been well explored, external actuation of DNA nanostructures is a relatively new area that allows for the remote control of nanoscale devices. We discuss recent applications for DNA nanostructures where switching is used to perform specific functions—such as opening a capsule to deliver a molecular payload to a target cell. We then discuss challenges and future directions towards achieving synthetic nanomachines with complexity on the level of the protein machinery in living cells.This work was supported by Australian Research Council Discovery Early Career Research Fellowship DE180101635 (SW), University of Sydney Nano Institute Scholarship (JKDS, MTL)

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

Life Cycle Assessment of Disposed and Recycled End-of-Life Photovoltaic Panels in Australia

This study presents a life cycle assessment (LCA) of end-of-life (EoL) photovoltaic (PV) systems in Australia. Three different EoL scenarios are considered for 1 kWh of electricity generation across a 30-year PV system lifespan: (i) disposal to landfill, (ii) recycling by laminated glass recycling facility (LGRF), and (iii) recycling by full recovery of EoL photovoltaics (FRELP). It is found that recycling technologies reduce the overall impact score of the cradle-to-grave PV systems from 0.00706 to 0.00657 (for LGRF) and 0.00523 (for FRELP), as measured using the LCA ReCiPe endpoint single score. The CO2 emissions to air decrease slightly from 0.059 kg CO2 per kWh (landfill) to 0.054 kg CO2 per kWh (for LGRF) and 0.046 kg CO2 per kWh (for FRELP). Increasing the PV system lifespan from 30 years to 50 and 100 years (a hypothetical scenario) improves the ReCiPe endpoint single-score impact from 0.00706 to 0.00424 and 0.00212, respectively, with corresponding CO2 emissions reductions from 0.059 kg CO2 per kWh to 0.035 and 0.018 kg CO2 per kWh, respectively. These results show that employing recycling slightly reduces the environmental impact of the EoL PV systems. It is, however, noted that recycling scenarios do not consider the recycling plant construction step due to a lack of data on these emerging PV panel recycling plants. Accounting for the latter will increase the environmental impact of the recycling scenarios, possibly defeating the purpose of recycling. Increasing the lifespan of the PV systems increases the longevity of the use of panel materials and is therefore favorable towards reducing environmental impacts. Our findings strongly suggest that PV recycling steps and technologies be carefully considered before implementation. More significantly, it is imperative to consider the circular design step up front, where PV systems are designed via circular economy principles such as utility and longevity and are rolled out through circular business models

Minimizing Cholesterol-Induced Aggregation of Membrane-Interacting DNA Origami Nanostructures

DNA nanotechnology provides methods for building custom membrane-interacting nanostructures with diverse functions, such as shaping membranes, tethering defined numbers of membrane proteins, and transmembrane nanopores. The modification of DNA nanostructures with hydrophobic groups, such as cholesterol, is required to facilitate membrane interactions. However, cholesterol-induced aggregation of DNA origami nanostructures remains a challenge. Aggregation can result in reduced assembly yield, defective structures, and the inhibition of membrane interaction. Here, we quantify the assembly yield of two cholesterol-modified DNA origami nanostructures: a 2D DNA origami tile (DOT) and a 3D DNA origami barrel (DOB), by gel electrophoresis. We found that the DOT assembly yield (relative to the no cholesterol control) could be maximised by reducing the number of cholesterols from 6 to 1 (2 ± 0.2% to 100 ± 2%), optimising the separation between adjacent cholesterols (64 ± 26% to 78 ± 30%), decreasing spacer length (38 ± 20% to 95 ± 5%), and using protective ssDNA 10T overhangs (38 ± 20% to 87 ± 6%). Two-step folding protocols for the DOB, where cholesterol strands are added in a second step, did not improve the yield. Detergent improved the yield of distal cholesterol configurations (26 ± 22% to 92 ± 12%), but samples re-aggregated after detergent removal (74 ± 3%). Finally, we confirmed functional membrane binding of the cholesterol-modified nanostructures. These findings provide fundamental guidelines to reducing the cholesterol-induced aggregation of membrane-interacting 2D and 3D DNA origami nanostructures, improving the yield of well-formed structures to facilitate future applications in nanomedicine and biophysics