Abstract

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. © 2025 The Royal Society of Chemistry.