Dissecting metal ion-dependent folding and catalysis of a single DNAzyme

Protein metalloenzymes use various modes for functions for which metal-dependent global conformational change is required in some cases but not in others. In contrast, most ribozymes require a global folding that almost always precedes enzyme reactions. Herein we studied metal-dependent folding and cleavage activity of the 8-17 DNAzyme using single-molecule fluorescence resonance energy transfer. Addition of Zn²⁺ and Mg²⁺ induced folding of the DNAzyme into a more compact structure followed by a cleavage reaction, which suggests that the DNAzyme may require metal-dependent global folding for activation. In the presence of Pb²⁺, however, the cleavage reaction occurred without a precedent folding step, which suggests that the DNAzyme may be prearranged to accept Pb²⁺ for the activity. Neither ligation reaction of the cleaved substrates nor dynamic changes between folded and unfolded states was observed. These features may contribute to the unusually fast Pb²⁺-dependent reaction of the DNAzyme. These results suggest that DNAzymes can use all modes of activation that metalloproteins use.