Time-resolved structural analysis of an RNA-cleaving DNA catalyst

The 10-23 DNAzyme is one ofthe most prominent catalytically active DNA sequences(1,2). Its ability to cleave a wide range of RNA targets with high selectivity entails a substantial therapeutic and biotechnological potential(2). However, the high expectations have not yet been met, a fact that coincides with the lack of high-resolution and time-resolved information about its mode of action(3). Here we provide high-resolution NMR characterization of all apparent states ofthe prototypic 10-23 DNAzyme and present a comprehensive survey ofthe kinetics and dynamics of its catalytic function. The determined structure and identified metal-ion-binding sites ofthe precatalytic DNAzyme-RNA complex reveal that the basis ofthe DNA-mediated catalysis is an interplay among three factors: an unexpected, yet exciting molecular architecture; distinct conformational plasticity; and dynamic modulation by metal ions. We further identify previously hidden rate-limiting transient intermediate states in the DNA-mediated catalytic processvia real-time NMR measurements. Using a rationally selected single-atom replacement, we could considerably enhance the performance ofthe DNAzyme, demonstrating that the acquired knowledge ofthe molecular structure, its plasticity and the occurrence of long-lived intermediate states constitutes a valuable starting point for the rational design of next-generation DNAzymes.

Automated summary

The 10-23 DNAzyme is a highly selective catalytic DNA sequence with therapeutic and biotechnological potential. Research has provided insights into its structure and catalytic mechanism, revealing the importance of three factors and previously hidden intermediate states through real-time NMR measurements.