Synthesis and Investigation of Multi-Phase DNA Microspheres

Abstract

This thesis presents a new method for the formation of DNA nanostar (NS) synthetic cells, with chemically and structurally distinct core and shell regions. First, modifications to the physical structure of nanostars were investigated. The inter-related effects of NS size, shape, inter-star binding strength, and environmental conditions on coacervate properties were investigated. A method for the experimental determination of NS phase-separation temperature was presented.

Orthogonal nanostars were combined in solution and shown to form distinct populations of coacervates. Selective modification of one NS population to induce limited interaction with the other resulted in increasing interfacial contact. The degree of contact was dependent on the energy profiles of each nanostar and the strength of inter-phase complementarity. Techniques for the prediction and formation of patchy, two-layer, and three-layer coacervates were presented, with each phase consisting of distinct nanostars. The majority of experimental micro-architectures aligned with predictions. Effects of key NS variables on the resulting coacervate micro-architecture were investigated.

Nanostar design allowed for control over the physical properties of the micro-scale coacervates. The encapsulation of a nanostar droplet in a two-layer core-shell system was shown to prevent core fusion over time at elevated temperatures. The targeted capture of cargo in multi-phase coacervates was demonstrated, using ssDNA-functionalised biomolecules. The location and degree of capture was controlled through the selective functionalisation of the cargo, along with selective release of cargo through toehold-mediated strand displacement reactions. The shell of a core-shell coacervate was shown to selectively prevent the infiltration of core-targeted cargo, acting as a switchable filter. Finally, proof-of concept designs were presented for the dynamic switching of cargo compartmentalisation and coacervate micro-architecture.