Engineered organic templates for controlling crystallisation

Nature has developed unique strategies for controlling the morphology, size, mechanical properties and polymorph of biominerals. Being able to emulate these approaches would enable mineralised structures to be synthesised with properties optimised for applications in different areas of nanotechnology. One of the most important strategies – which can be translated to synthetic systems – is the use of molecular and supramolecular templates to control mineralisation. This thesis explores the use of engineered proteins and DNA nanostructures to control ice and calcium carbonate crystallisation. The thesis consists of six different chapters. Chapter 1 introduces the general concepts of crystallisation, providing an overview of the main theories currently used to describe and understand the crystallisation processes. Chapter 2 provides a description of the main techniques used in this thesis. This is followed by three chapters describing in detail the projects carried out in this work. Chapter 3 investigates the use of phage display to identify Affimer proteins that strongly bind to ice. The proteins identified after several biopanning rounds showed recurring amino acids that are considered important for the activity of natural antifreeze and ice nucleating proteins. Tests of the activity of the selected proteins showed that some promote ice formation at higher temperatures than the controls. This is one of the first examples of using biopanning to identify ice-binding proteins from a phage library. Chapter 4 explores the use of phage display to identify proteins that can direct calcium carbonate formation and, in particular, polymorph selection. A library of M13 pIIIconjugated Affimer proteins was screened against two of the polymorphs of calcium carbonate – calcite and aragonite – identifying proteins with binding affinity for the two phases. The proteins identified showed polymorph selectivity when Mg2+ ions were also present in the crystallisation solution. Finally, Chapter 5 describes an investigation in the use of DNA origami nanostructures to template the formation of calcium carbonate. The study demonstrates that the Mg2+ ions, traditionally used to fold the DNA nanostructures, can be replaced with Ca2+ ions, such that they are better suited to their role as templates for CaCO3 mineralisation. In situ AFM studies demonstrated that mineralisation preferentially occurred on the DNA framework. DNA assemblies with different shapes were successfully folded in high yield, confirming the possibility to alter the shape of the template by changing the functionalisation at the edges of the DNA tile used as building block unit.