Enzymatic PCR Replication of Fully Orthogonal Nucleic Acids as Synthetic Model Genomes

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The detailed investigation of biological systems represents one of the most important tasks, which are of interest for scientific fields like medicine, biology, parts of chemistry and the respective overlapping areas. By introducing of unnatural and metabolically stable nucleic acids, additional genetic information can be introduced into the microorganisms or cells, which shall be examined. This manipulation allows a deeper understanding of the cellular processes and represents the central scientific task of synthetic biology, which follows the aim to generate and design a working biological system artificially. In this project two unnatural nucleotide base pairs shall be synthesized, which differ in their hydrogen bonding pattern from the natural Watson Crick model. To ensure the hydrolytic and metabolic stability of these building blocks, they shall be designed as so-called C-nucleosides, whose nucleosidic linkage is bound to a carbon atom in the nucleobase. The aim of this work is to build complete DNA sections from these artificial building blocks and to amplify them in vivo by cellular replication. Therefore, suitable enzymes (DNA polymerases, reverse transcriptases) have to be found, which recognize and efficiently use the unnatural nucleotides for enzymatic DNA synthesis. This method shall be applied to shorter oligonucleotides (20 - 30 base pairs), as well as longer DNA sequences (500 bp up to 2 kbp). Here, also the possibility of artificial DNA amplification by polymerase chain reaction (PCR) shall be demonstrated. Finally, the above described, unnatural DNA fragments shall be enzymatically cloned into plasmids and transformed into microorganisms (e.g.: E. coli). Thereby can be demonstrated, that the developed unnatural nucleotides also form in vivo a stabile base recognition and that the corresponding DNA strands can be amplified by cellular replication. By means of these experiments, the synthesized nucleosides shall be used in future to code genetic information. They can be used to equip microorganisms with full synthetic, orthogonal genomes and thus with additional properties, whereby a variety of new applications (e.g.: expression of unnatural metabolic enzymes for catalyzing useful reactions) becomes possible.