Polymerases for synthetic nucleic acids.

Besides being information-encoding polymers, nucleic acids have the capacity to assemble into geometrical nanostructures, to bind specifically to diverse ligands (as aptamers) and to catalyze chemical reactions (as aptazymes). Natural nucleic acids by their fysicochemical properties, however, encompass only a limited part of the potential chemical and structural space. Recent developments in nucleic acid chemistry and polymerase structural analyses have shown that enzymatic synthesis and replication of artificial nucleic acids can considerably enlarge nucleic acid chemical diversity. The enzymatic synthesis of artificial nucleic acids is, however, currently limited to nucleic acids with strong similarity to DNA and RNA. This project aims at the development of polymerases for the synthesis of new, orthogonal nucleic acids (termed xeno-nucleic acids or XNA) and derived synthetic aptamers (or syntamers). We opt for candidate nucleic acids with modified sugar backbone, including xylose nucleic acids for which a deviating Z DNA-like helical structure could be predicted, and of which nucleotide incorporation by archaea polymerases, albeit limited, could be demonstrated. The observed promiscuous incorporation activity of archaea polymerases is the starting point for molecular evolution of these polymerases towards enhanced efficiency and specificity for defined xeno nucleotides. Evolution experiments start from libraries of mutant family B polymerases, and from in house synthesized xeno nucleotides and modified primers, and are rationally supported by molecular modelling and christal structure analysis. Evolved polymerases will subsequently be employed to transcribe DNA libraries into XNA libraries for selection of synthamers by means of SELEX technology. Insights obtained form these polymerases and XNA ultimately may form the basis for later development of an in vivo orthogonal information system for applications in synthetic biology.

Keywords:Artificial nucleic acids, Xylose nucleic acids, Aptamer, Orthogonality, Synthetic biology

Disciplines:Biochemistry and metabolism, Medical biochemistry and metabolism, Biomaterials engineering, Biological system engineering, Biomechanical engineering, Other (bio)medical engineering, Environmental engineering and biotechnology, Industrial biotechnology, Other biotechnology, bio-engineering and biosystem engineering