Modification of Nucleic Acid Analogs with Protein Domains and Photoswitches in Order to Regulate Gene Function

用蛋白质结构域和光开关修饰核酸类似物以调节基因功能

• 批准号: 196589664
• 负责人: Professor Dr. Thorsten Stafforst
• 金额: --
• 依托单位: Interfakultäres Institut für Biochemie (IFIB)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2011
• 资助国家: 德国
• 起止时间: 2010-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/196589664?language=en
• 关键词: Modification; Nucleic; Acid; Analogs; Protein

Recently, we have been demonstrating the feasibility of applying adenosine to inosine (A-to-I) RNA editing to repair point mutations on the RNA-level. For this the catalytic deaminase domain of hADAR1 was engineered into an artificial guideRNA-dependent enzyme. This allowed for the efficient introduction of single point mutations at user-defined sites into mRNAs in a highly rational and simple way. Since A-to-I editing changes the sense of codons and the meaning of RNA processing signals, directed RNA-editing has immense potential as a tool in basic biology research and for application in medicine.In this proposal, we aim to further develop our directed editing strategy into a practical tool for the manipulation of protein and RNA function. Specifically, we want to establish our successful in-vitro-approach in cell culture and aim to apply it in the repair of disease-related point mutations on the RNA-level. To overcome a potential limitation of our initial approach, we aim to further develop a complementary approach which benefits from the possibility that all components are genetically encodable, and that endogenous hADAR can be harnessed for directed editing. Specifically, we want to further elaborate and understand this tool in vitro, but we also aim to establish its functioning inside the cell. If successful, the proposed work will pave the way for the application of directed RNA editing in basic biology and life sciences.

最近，我们一直在证明应用腺苷到肌苷（A-to-I）RNA编辑来修复RNA水平上的点突变的可行性。为此，hADAR1 的催化脱氨酶结构域被设计成人工引导 RNA 依赖性酶。这允许以高度合理和简单的方式将用户定义位点的单点突变有效地引入到 mRNA 中。由于A-to-I编辑改变了密码子的意义和RNA加工信号的含义，因此定向RNA编辑作为基础生物学研究和医学应用的工具具有巨大的潜力。在本提案中，我们的目标是进一步将我们的定向编辑策略发展成为操纵蛋白质和RNA功能的实用工具。具体来说，我们希望在细胞培养中建立成功的体外方法，并旨在将其应用于 RNA 水平上疾病相关点突变的修复。为了克服我们最初方法的潜在局限性，我们的目标是进一步开发一种补充方法，该方法受益于所有组件都是可遗传编码的可能性，并且可以利用内源 hADAR 进行定向编辑。具体来说，我们希望在体外进一步详细阐述和理解这个工具，但我们也致力于确定其在细胞内的功能。如果成功，拟议的工作将为定向RNA编辑在基础生物学和生命科学中的应用铺平道路。