Zn2+-dependent DNAzymes that cleave all combinations of ribonucleotides

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.

Combating small molecule environmental contaminants: detection and sequestration using functional nucleic acids

“…For example, a zinc dependent DNAzyme capable of RNA cleavage activity was isolated by appending the target RNA strand with a biotin handle, which allowed for easy isolation using streptavidin-coated magnetic beads. 88 …”

“…For example, a zinc dependent DNAzyme capable of RNA cleavage activity was isolated by appending the target RNA strand with a biotin handle, which allowed for easy isolation using streptavidin-coated magnetic beads. 88 While DNAzymes have found use in small molecule environmental contaminant detection, it is very rare that they are acting strictly as the biorecognition element. DNAzyme based biosensors instead are selected to rely on a specic heavy metal for an activity such as nucleic acid cleavage, and this metal-dependent activity is then coupled to a uorescence readout or other amplication and sensing motif.…”

“…96 As mentioned previously, DNAzymes are typically cation-dependent and can be selected for activity in the presence of specic heavy metals. [88][89][90][91][92][93]97,98 This has, in turn, enabled detection of heavy metals in complex environments including food, water, and soil. [99][100][101][102] DNAzymes can also be integrated into aptamer architectures to enable small molecule dependent activity, and this strategy can be especially useful for aptasensors that do not meet sensitivity requirements for challenging small molecule targets.…”

High-Throughput Activity Profiling of RNA-Cleaving DNA Catalysts by Deoxyribozyme Sequencing (DZ-seq)

“…Although deoxyribozymes have not been yet found in nature, in vitro selection procedures allowed the discovery of artificial deoxyribozymes with a vast repertoire of catalytic activities. [1][2][3][4] A typical in vitro selection starts from a combinatorial library of up to ~10 15 unique sequences and consists of repetitive rounds of activity screening, selection and amplification. 5 Next-generation sequencing (NGS) techniques have been increasingly replacing the low-throughput Sanger method in the analysis of in-vitro selection libraries of aptamers [6][7][8][9][10][11] and (deoxy)ribozymes.…”

“…5 Next-generation sequencing (NGS) techniques have been increasingly replacing the low-throughput Sanger method in the analysis of in-vitro selection libraries of aptamers [6][7][8][9][10][11] and (deoxy)ribozymes. [12][13][14][15][16] The candidate catalysts are usually selected from the NGS data using the relative sequence abundance or sequence enrichment as proxies for the catalytic activity. However, due to PCR and other biases, [17][18] the candidate catalysts have to be validated by conventional low-throughput biochemical assays.…”