Abstract
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.
Highlights
Several DNAzymes are known, their utility is limited by a narrow range of substrate specificity
DNAzymes have various catalytic activities, such as RNA cleavage, DNA ligation[16,17], DNA cleavage[18,19,20,21,22], and phosphorylation[23]. Because of their high substrate sequence specificity, metal ion selectivity, high stability, and low cost, RNA-cleaving DNAzymes have been used in various applications, including gene therapy[24,25], metal sensors[26], nanodevices[27], diagnosis[28], and gene computing[29]
Efforts to engineer DNAzyme 8–17 led to an enzyme that cleaves the phosphodiester bond between any nucleotide combination (5′-rNrN-3′) except between pyrimidine-pyrimidine nucleotides (5′-rPyrPy-3′)
Summary
Several DNAzymes are known, their utility is limited by a narrow range of substrate specificity. 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. DNAzymes have various catalytic activities, such as RNA cleavage, DNA ligation[16,17], DNA cleavage[18,19,20,21,22], and phosphorylation[23] Because of their high substrate sequence specificity, metal ion selectivity, high stability, and low cost, RNA-cleaving DNAzymes have been used in various applications, including gene therapy[24,25], metal sensors[26], nanodevices[27], diagnosis[28], and gene computing[29]. We aimed to obtain more compact DNAzymes that can universally cleave any sequence junctions (5′-rNrN-3′) in a short core substrate sequence
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