DNAzymes: a promising tool in gene therapy.

The growing demand for precise and effective gene therapy has driven the extensive exploration of nucleic acid therapeutics. DNAzymes are catalytically active single-stranded DNA molecules with high programmability and substrate-recognition specificity, and they have been considered as promising tools for gene therapy. Nevertheless, their clinical translation remains constrained by several major challenges, including structural instability, limited catalytic activity under physiological conditions, inadequate spatiotemporal controllability, and inefficient in vivo delivery. This review first outlines the fundamental structural features of DNAzymes and summarizes de novo and target-guided screening strategies. It then discusses mechanistic insights to guide the rational design of DNAzymes. Recent advances in controllable activation systems are also reviewed, followed by the design of delivery platforms with an emphasis on cofactor supply and amplification. Additionally, synergistic therapeutic applications are discussed as important strategies for disease intervention. Finally, current challenges are highlighted and future research directions are proposed to guide the development of precise and efficient DNAzyme-based gene therapy systems, thereby facilitating their transition from laboratory research to therapeutic applications.