Advances in DNAzyme Selection, Molecular Engineering and Biomedical Applications.

DNAzymes are catalytically active single-stranded DNAs that fold into metal-ion-assisted architectures to mediate diverse reactions. Addressing the performance gap in biological settings, we establish a novel conceptual framework based on a continuous iteration workflow of selection, enhancement, and application. This paradigm integrates selection constraints, molecular engineering, and clinical context into a unified cycle. We summarize the evolution of SELEX toward application-driven selection incorporating functional/environmental constraints, deep-sequencing-enabled high-throughput activity readouts, droplet compartmentalization and structure- and computation-guided design. We further consolidate engineering strategies to improve stability, kinetics and controllability, including 2'-sugar modifications and XNA substitution, backbone and nucleobase functionalization, arm and secondary-structure engineering for switchable or split architectures and multivalent organization on nanocarriers or nucleic acid scaffolds to enhance local concentration, protection and targeted delivery. Finally, we survey applications in ultrasensitive biosensing and portable diagnostics, activatable and multimodal in vivo imaging, and therapies for cancer, inflammatory diseases and airway disorders, and outline translational priorities: data-driven design, next-generation delivery, standardized safety/PK-PD evaluation and scalable manufacturing, ultimately for clinical and point-of-care deployment.