DNA Three-way Junctions as Scaffolds for Hybrid Catalysis

Abstract

The industrial production of chemicals is one of the largest contributors to global greenhouse gas emissions. The principles of green chemistry outline a route to transform chemistry into a sustainable, environmentally benign sector. As part of this, the development of novel catalysts and new approaches to catalysis is essential. Bio-functionalised DNA is known to catalyse a number of industrially important chemical reactions, and DNA supported catalysts have the advantage of being biodegradable and non-toxic, and are therefore of interest as green alternatives to traditional chemical reagents. This work explores the use of DNA nanotechnology as a platform for enzyme inspired catalysis. The historical background and theoretical underpinning of hybrid catalysis shows that there is a good case for exploring DNA nanostructures for catalysis; DNA is significantly more temperature stable than enzymes, can be modified with extrinsic functional groups, and obeys simple hybridisation rules that determine the tertiary conformation of nanostructures. This means DNA nanostructures can act as scaffolds for the precise placement of chemical moieties, which in linear DNA has been shown as an effective method of catalyst design. To evaluate the catalytic properties of non-canonical DNA, DNA three-way junctions were designed, synthesised and characterised in detail. A number of chemically modified oligonucleotides were synthesised to introduce non-canonical functionality into DNA nanostructures. These include histidine, arginine, serine and flavin analogues. Experiments with flavinated DNA suggest a unique chemical environment at the centre of the three-way junction branch point, causing a change in both the fluorescence emission and fluorescence lifetime of the flavin moiety. Imidazole modified oligonucleotides were assembled into three-way junctions using a rational design approach incorporating computer simulation. Combinatorial experiments demonstrate a new method for producing variation in modified functional DNA nanostructures, and eight modified DNA junctions were studied for esterase-like activity. Kinetic experiments found seven variants of imidazole modified junctions that enhance the rate of ester hydrolysis. This is the first time such structures have been shown to perform hydrolytic catalysis. All variants accelerate ester hydrolysis compared to unhybridised yet chemically modified oligonucleotides, showing that the formation of the three-way junction is essential for catalysis. Experiments demonstrate how junctions can be efficiently recovered and recycled after catalytic reactions, emphasising their potential use in industrial processes. A combinatorial workflow for a psuedo-evolutionary high throughput experiments called Machine Aided Combinatorial Screening (MACS) is outlined as a solution to multi-strand DNA catalyst selection. The work presented for this thesis shows the significant potential of DNA nanostructures in catalysis, and provides a road map for future experiments.