Functions and applications of enzymes in nucleic acid nanotechnology.

Nucleic acid nanotechnology (NAN) has emerged as a powerful platform for constructing programmable molecular architectures, with broad applications spanning biomedicine, materials science, and molecular computing. While numerous reviews have covered the design principles and functional modalities of enzyme-free NAN systems, a comprehensive framework for understanding enzyme-involved NAN remains conspicuously absent. Given the growing interest in enzyme-involved systems, enzymes offer distinct advantages to NAN, including enhanced programmability, dynamic control, and expanded functional versatility. This review addresses that gap by systematically categorizing the enzymatic toolkit-including polymerases, modifying enzymes, endo-/exonucleases, ligases, other protein-based enzymes, as well as nucleic acid enzymes such as DNAzymes, ribozymes, and XNAzymes-and elucidating their roles in enabling structural assembly and dynamic control at the molecular level. Enzymes function not only as assembly agents for nucleic acid nanostructures, facilitating strand extension, ligation, cleavage, and chemical modification, but also as essential driver tools mediating degradation, release, and template-guided polymerization. Their integration has led to highly adaptive and reconfigurable systems with capabilities far surpassing enzyme-free counterparts. We further highlight key advances in enzyme-powered NAN across diverse frontiers, including in vitro diagnostics (IVD), cellular and in vivo imaging, DNA data storage and computing, biomimetic material synthesis, and drug delivery. By mapping catalytic mechanisms to functional outputs, and identifying current bottlenecks in specificity, modularity, and integration, this review establishes a unified conceptual foundation and design roadmap for the next generation of enzyme-driven nucleic acid nanodevices.