Mechanistic understanding of DNA-mediated catalysis

这项建议的目的是通过DNA介导的催化对RNA切割产生一个全面的机制理解。到目前为止，各自的所谓DNAzyme还不能发挥其巨大的治疗潜力。这一困境源于对DNAzyme体内活性的意外降低，这与缺乏对其机制的理解不谋而合。在第一个资助期，我们可以成功地确定DNAzyme的结构，它的金属离子结合位置，并实时跟踪它的催化反应。在这种方法取得成功后，我们现在的目标是将类似的策略应用于其他DNAzyme系统，特别是关注催化环的类型和序列变化以及底物-裂解位点的影响。使用这一策略，我们的目标是在体外对RNA裂解DNAzyme产生一个连贯的机制理解。此外，关于在细胞环境中限制DNAzyme活性的影响的紧迫问题应该使用适当的细胞内核磁共振和补充方法来回答。预期的DNAzyme功能和局限性的整体图景应该回答长期存在的关于特定DNA序列的催化能力的中心研究问题，并作为合理设计策略的基础，以开发具有更高体内活性的下一代DNAzyme。

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.