Zn²⁺-dependent DNAzymes that cleave all combinations of ribonucleotides

Although several DNAzymes are known, their utility is limited by a narrow range of substrate specificity. Here, we report the isolation of two zinc-dependent DNAzymes, ZincDz1 and ZincDz2, which exhibit compact catalytic core sequences with highly versatile hydrolysis activity. They were selected through in vitro selection followed by deep sequencing analysis. Despite their sequence similarity, each DNAzyme showed different Zn²⁺-concentration and pH-dependent reaction profiles, and cleaved the target RNA sequences at different sites. Using various substrate RNA sequences, we found that the cleavage sequence specificity of ZincDz2 and its highly active mutant ZincDz2-v2 to be 5'-rN↓rNrPu-3'. Furthermore, we demonstrated that the designed ZincDz2 could cut microRNA miR-155 at three different sites. These DNAzymes could be useful in a broad range of applications in the fields of medicine and biotechnology.