Towards Enzymatic Synthesis of Uncharged Xeno Nucleic Acids

Change log
Duffy, Karen 

A genetic material is a defining requirement for life. The many practical uses of DNA and RNA have also made these molecules integral tools for human manipulation of biological systems. However, high susceptibility to nuclease degradation and limited chemical diversity constrain their use in therapeutic applications and beyond. Chemically modified analogues of DNA, referred to collectively as xeno nucleic acids (XNAs), can address some such limitations. The polyanionic backbone of DNA dominates its physicochemical properties and decouples them from its information content. In this thesis, I explore two XNA chemistries with neutrally charged backbones: phosphonate nucleic acids, in which an alkyl group replaces one of the non-bridging backbone oxygens, and morpholino nucleic acids, which carry a six-membered morpholine ring as well as dimethylamino or alkyl substituents on the backbone. The choice of these two chemistries is motivated by the hypothesis that such uncharged genetic polymers should display increased chemical, structural, and functional diversity as compared to natural nucleic acids. Furthermore, morpholinos have been evaluated in patients and found to be non-toxic and are therefore a promising chemistry for further development. Specifically, I aim to develop strategies that enable enzymatic synthesis of these uncharged XNAs. I seek to engineer polymerases to accept the requisite non-natural nucleotide substrates, thereby enabling their enzymatic synthesis and in vitro evolution. To identify polymerase mutants with improved activity with these XNA nucleotides, I have leveraged and further developed methods of directed polymerase evolution. I have developed a new method for polymerase evolution, which allowed the discovery of an improved polymerase for the synthesis of phosphonate nucleic acid. Furthermore, I have addressed significant hurdles in evolving polymerases with low basal levels of XNA incorporation. Through careful, iterative optimization of mutations and reaction conditions and some first attempts at evolution, I for the first time achieve enzymatic activity polymerizing morpholino substrates. Overall, my results show that it is possible to encode information in multiple different uncharged XNA polymers by enzymatic copying from a DNA template. This should enable further functional exploration and evolution of these uncharged nucleic acids.

Holliger, Philipp
polymerase, xeno nucleic acids, directed evolution
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
This work was supported by a Gates Cambridge scholarship