Mechanistic understanding of DNA-mediated catalysis

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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.