Mechanistic understanding of DNA-mediated catalysis

DNA 介导的催化机制的理解

• 批准号: 401425239
• 负责人: Dr. Manuel Etzkorn
• 金额: --
• 依托单位: Institut für Physikalische Biologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/401425239?language=en
• 关键词: Mechanistic; understanding; DNA; mediated; catalysis

The objective of this proposal is to generate a comprehensive mechanistic understanding of RNA-cleavage via DNA-mediated catalysis. The respective so-called DNAzymes could so far not fulfil their great therapeutic potential. This dilemma originates from an unexpected and still not sufficiently understood reduced in vivo activity of DNAzymes, which coincides with a lack of their mechanistic understanding. During the first funding period we could successfully determine a DNAzyme structure, its metal-ion binding sites, and follow its catalytic reaction in real-time. Following the success of this approach, we now aim to apply a similar strategy to other DNAzyme systems, focusing in particular on the effects of the type and sequence variations of the catalytic loop, as well as the substrate-cleavage site. Using this strategy, we aim to generate a coherent mechanistic understanding of RNA-cleaving DNAzymes in vitro. In addition, the pressing question regarding the effects that the limit DNAzyme activity in a cellular context should be answered using suitable in-cell NMR and complementary approaches. The anticipated holistic picture of DNAzyme function and limitations should answer long standing central research questions about catalytic capabilities of specific DNA sequences and serve as basis for a rational-design strategy to develop next-generation DNAzymes with increased in vivo activity.

该提案的目的是通过 DNA 介导的催化对 RNA 切割产生全面的机制理解。迄今为止，各自所谓的 DNAzyme 还无法发挥其巨大的治疗潜力。这种困境源于出乎意料且尚未充分理解的 DNAzyme 体内活性降低，这与缺乏对其机制的了解相一致。在第一个资助期间，我们可以成功确定 DNAzyme 结构、其金属离子结合位点，并实时跟踪其催化反应。随着这种方法的成功，我们现在的目标是将类似的策略应用于其他 DNAzyme 系统，特别关注催化环的类型和序列变化以及底物切割位点的影响。利用这一策略，我们的目标是对体外 RNA 切割 DNAzyme 产生连贯的机制理解。此外，关于限制 DNAzyme 活性在细胞环境中的影响的紧迫问题应该使用合适的细胞内 NMR 和补充方法来回答。预期的 DNAzyme 功能和局限性的整体图景应能回答有关特定 DNA 序列催化能力的长期存在的核心研究问题，并作为合理设计策略的基础，以开发体内活性增强的下一代 DNAzyme。