De ontwikkeling van XNA ligases als tool voor de synthetische biologie

Beyond storing the complete genetic blueprint for all life on Earth, DNA and RNA also function as sensors, regulators, and even catalysts. Attempts have been made to expand their functionality towards pharmaceutical and biotechnological applications, but limited (biological) stability makes them ineffective candidates. Xenobiotic nucleic acids (XNAs) are artificial analogues of DNA and RNA with increased chemical and biological stability due to altered chemical moieties. XNA oligonucleotides can be made using the same solid-chemistry methodology as DNA. Due to the limited coupling efficiencies of the phosphoramidites and complex purification, the chemical synthesis of XNA oligonucleotides suffers from size limitations. Enzymatic synthesis methods can be promising alternatives to generate longer, more diverse XNAs in a more sustainable, solvent-free environment. However, the XNA research field was, and still is, primarily focused on the development of XNA polymerases. While tremendous advancements have been achieved, the XNA polymerases still suffer from low efficiency, high error rates and cannot deliver the same chemical diversity as chemical synthesis. The main goal of this thesis was to develop XNA ligases for synthesising chemically diverse and extended XNA fragments, previously unreachable with chemical or polymerase-driven synthesis methods. This will be a new step towards the maturation of the XNA research field, while simultaneously gaining insight into XNA (and by extension DNA) ligation.