Exploring the catalytic mechanism of the 10–23 DNAzyme: insights from pH–rate profiles

Abstract

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.