Xeno-nucleic acid polymerases by directed evolution.
Artificial or xeno nucleic acids (XNAs) present an alternative to natural genetic polymers by expanding chemical diversity and improving chemical and biological stability, with potential applications in, for example, therapeutics. XNA differs from its natural counterparts by modifications applied to the nucleobases, sugar-phosphate backbone, nucleotide leaving group, or a combination of these. In the long run, XNA could form the basis of an orthogonal genetic system, “invisible” to and replicating independently from the natural world by chemical and enzymatic decoupling. Key to the development and manipulation of these XNAs are suitable polymerases that can incorporate synthetic nucleotides into a growing XNA chain in a template-dependent way. These polymerases can be created starting from natural variants by directed evolution, an in vitro process of repeated mutagenesis and selection. In this project, we propose bacteriophage phi29 DNA polymerase as a new candidate for directed evolution towards XNA polymerase activity. The symmetrical, protein-primed replication mode of this polymerase is an important feature that could enable the establishment of a straightforward in vivo XNA episome. We showed promiscuous activity of phi29 DNA polymerase towards sugar-modified nucleotides, further justifying our choice for this enzyme. A number of mutant libraries was designed and created, guided by structural information to increase the chance of finding interesting variants. The polymerase was successfully displayed on phage and shown to be active. A novel phage display system involving co-display of polymerase and HaloTag was developed to simplify attachment of a primer-template substrate and selection based on modified nucleotide incorporation. The presented co-display system might also be used for the evolution of a wide range of other enzymes, different from polymerases. Preliminary selection experiments exemplified the importance of high-quality libraries, precise control of growth conditions and protein expression, and an optimal selection procedure to maximize enrichment of active variants. Further refinement of selection conditions is needed for the isolation of functional XNA polymerases, to be eventually applied in an orthogonal XNA episome.