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

作为合成模型基因组的完全正交核酸的酶促 PCR 复制

• 批准号: 421699074
• 负责人: Dr. Damian Ploschik
• 金额: --
• 依托单位: Laboratory for Medical Chemistry
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/421699074?language=en
• 关键词: Enzymatic; PCR; Replication; Fully; Orthogonal

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.

对生物系统的详细研究是最重要的任务之一，这是医学、生物学、部分化学及其各自重叠领域等科学领域感兴趣的任务之一。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，并对其进行检查。这种操作允许更深入地了解细胞过程，并代表了合成生物学的中心科学任务，其目标是人工产生和设计一个工作的生物系统。在这个项目中，将合成两个非自然的核苷酸碱基对，它们的氢键模式与自然的Watson Crick模型不同。为了确保这些构件的水解性和代谢稳定性，它们应被设计为所谓的C-核苷，其核苷键与碱基中的碳原子结合。这项工作的目的是从这些人造构件中构建完整的DNA片段，并通过细胞复制在体内放大它们。因此，必须找到合适的酶(DNA聚合酶、逆转录酶)，识别并有效地利用非天然核苷酸进行酶促DNA合成。这种方法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个碱基对至2个碱基对)。在这里，还将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片段用酶法克隆到质粒中，并转化到微生物中(例如：大肠杆菌)。由此可以证明，所开发的非天然核苷酸在体内也形成了稳定的碱基识别，并且相应的DNA链可以通过细胞复制来扩增。通过这些实验，合成的核苷将在未来用于对遗传信息进行编码。它们可以用来为微生物配备完全合成的、正交的基因组，从而具有额外的性质，从而使各种新的应用(例如：表达用于催化有用反应的非天然代谢酶)成为可能。