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

对生物系统的详细研究是最重要的任务之一，这对医学，生物学，化学部分以及各自的重叠领域等科学领域都很重要。通过引入非天然的和代谢稳定的核酸，可以将额外的遗传信息引入微生物或细胞中，这将被检查。这种操作可以更深入地了解细胞过程，并代表合成生物学的核心科学任务，其目标是人工生成和设计一个工作的生物系统。在这个项目中，将合成两个非天然的核苷酸碱基对，它们的氢键模式不同于天然的沃森克里克模型。为了确保这些结构单元的水解和代谢稳定性，它们应被设计为所谓的C-核苷，其核苷键与核碱基中的碳原子结合。这项工作的目的是从这些人工构建模块构建完整的DNA片段，并通过细胞复制在体内扩增它们。因此，必须找到合适的酶（DNA聚合酶、逆转录酶），其识别并有效地使用非天然核苷酸用于酶促DNA合成。该方法应适用于较短的寡核苷酸（20 - 30个碱基对）以及较长的DNA序列（500 bp至2 kbp）。在此，还应证明通过聚合酶链式反应（PCR）进行人工DNA扩增的可能性。 最后，将上述非天然DNA片段酶促克隆到质粒中并转化到微生物中（例如：E. coli）。由此可以证明，开发的非天然核苷酸也在体内形成稳定的碱基识别，并且相应的DNA链可以通过细胞复制扩增。通过这些实验，合成的核苷将在未来用于编码遗传信息。它们可用于使微生物具有完整的合成正交基因组，从而具有额外的特性，从而产生各种新的应用（例如：用于催化有用反应的非天然代谢酶的表达）成为可能。

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.