DNAzymes (deoxyribozymes) are single-stranded DNA molecules endowed with catalytic activity, obtained by in vitro selection. In the past 25 years, dozens of DNAzymes have been identified and employed for applicative purposes, yet our knowledge of the structural and mechanistic basis of DNA catalysis remains very limited. The RNA-cleaving 8-17 DNAzyme, which depends on divalent metal ions for function, is possibly the most studied catalytic DNA in terms of mechanism. It is very efficient, implying that it adopts a combination of distinct catalytic strategies, but until recently it was uncertain which strategies are at play and how they are implemented. Recently, however, new functional studies and the attainment of high-resolution X-ray structures of an 8-17 construct, have offered a great opportunity for a more detailed understanding of its mechanism. This review examines the functional information gathered on 8-17, in the light of the available crystal structures, pointing out the congruences and possible inconsistencies between the functional and structural data. We will analyze separately three aspects of the DNAzyme function: the structural requirements for catalysis, the role of metal ions and the influence of pH on activity. Ultimately, we will contrast the experimental data with a model for the 8-17 mechanism proposed in the crystallographic study, whereby one specific G residue (G14) acts as a general base and a metal-coordinated water molecule acts as a general acid. Throughout this analysis we will signal the most outstanding mechanistic issues that remain to be addressed, with implications for the broader field of DNA catalysis.

Four synthetic Schiff bases (PSB1 [(E)-2-(((4-aminopyridin-3-yl)imino)methyl)-4,6-dibromophenol], PSB2 [(E)-2-(((4-aminopyridin-3-yl)imino)methyl)-4,6-diiodophenol], PSB3 [(E)-2-(((4-aminopyridin-3-yl)imino)methyl)-4-iodophenol], and PSB4 [(E)-2-(((4-aminopyridin-3-yl)imino)methyl)-4-chloro-6-iodophenol]) were fully characterized. These compounds exhibit an intramolecular hydrogen bond between the hydroxyl group of the phenolic ring and the nitrogen of the azomethine group, contributing to their stability. Their antimicrobial activity was evaluated against various Gram-negative and Gram-positive bacteria, and it was found that the synthetic pyridine Schiff bases, as well as their precursors, showed no discernible antimicrobial effect on Gram-negative bacteria, including Salmonella Typhi (and mutant derivatives), Salmonella Typhimurium, Escherichia coli, and Morganella morganii. In contrast, a more pronounced biocidal effect against Gram-positive bacteria was found, including Bacillus subtilis, Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus haemolyticus. Among the tested compounds, PSB1 and PSB2 were identified as the most effective against Gram-positive bacteria, with PSB2 showing the most potent biocidal effects. Although the presence of reactive oxygen species (ROS) was noted after treatment with PSB2, the primary mode of action for PSB2 does not appear to involve ROS generation. This conclusion is supported by the observation that antioxidant treatment with vitamin C only partially mitigated bacterial inhibition, indicating an alternative biocidal mechanism.

PH-Rate profiles indicate hydrated M²⁺ as a general acid in the catalytic mechanism of the RNA-cleaving 8–17 DNAzyme.

The 10–23 DNAzyme, a catalytic DNA molecule with RNA-cleaving activity, has garnered significant interest for its potential therapeutic applications as a gene-silencing agent. However, the lack of a detailed understanding about its mechanism has hampered progress. A recent structural analysis has revealed a highly organized conformation thanks to the stabilization of specific interactions within the catalytic core of the 10–23 DNAzyme, which facilitate the cleavage of RNA. In this configuration, it has been shown that G14 is in good proximity to the cleavage site which suggests its role as a general base, by activating the 2′-OH nucleophile, in the catalysis of the 10–23 DNAzyme. Also, the possibility of a hydrated metal acting as a general acid has been proposed. In this study, through activity assays, we offer evidence of the involvement of general acid–base catalysis in the mechanism of the 10–23 DNAzyme by analyzing its pH–rate profiles and the role of G14, and metal cofactors like Mg²⁺ and Pb²⁺. By substituting G14 with its analogue 2-aminopurine and examining the resultant pH–rate profiles, we propose the participation of G14 in a catalytically relevant proton transfer event, acting as a general base. Further analysis, using Pb²⁺ as a cofactor, suggests the capability of the hydrated metal ion to act as a general acid. These functional results provide critical insights into the catalytic strategies of RNA-cleaving DNAzymes, revealing common mechanisms among nucleic acid enzymes that cleave RNA.

RNA-cleaving DNAzymes have emerged as promising catalytic nucleic acids with potential applications in biotechnology and therapeutics. Among them, the 10–23 and 8–17 DNAzymes, both derived from the same in vitro selection experiment, have emerged as the most widely studied RNA-cleaving DNAzymes due to their high catalytic efficiency and broad metal ion dependence. Despite their apparent structural differences, recent structural, functional, and computational studies have revealed convergent catalytic strategies in their mechanisms. This review examines the commonalities between the 8–17 and 10–23 DNAzymes, offering a comparative mechanistic perspective on the catalytic strategies underlying their RNA cleavage activity, following the nomenclature proposed by Breaker et al. We discuss recent evidence from functional, crystallographic, NMR-based, and molecular dynamics studies that highlight how conserved guanine residues act as general bases, while hydrated metal ions contribute as general acids in both DNAzymes. By summarizing the latest advancements in the field, this review aims to provide a comprehensive perspective on the fundamental catalytic strategies employed by the 8–17 and the 10–23 DNAzymes and their relevance for future applications in nucleic acid-based catalysis.

Specific role of Na⁺ in the catalytic mechanism of the 8–17 DNAzyme with Pb²⁺.