Circular nucleic acid synthesis: Enzymatic, chemical, and emerging strategies for diagnostic and bioengineering applications

Circular DNA and RNA have found broad application in biosensing, gene therapy, diagnostics, and fundamental nucleic acid research due to their high structural stability. Their covalently closed topology provides strong resistance to exonuclease degradation, enabling applications such as rolling circle amplification or transcription (RCA/RCT), and highly sensitive CRISPR-based biosensing. Various strategies have been developed for the preparation of circular nucleic acids, primarily including chemical and enzymatic ligation, while circular RNA can additionally be generated via ribozyme or self-splicing intron mediated approaches. Each synthesis method has distinct features that influence its suitability for particular applications. In this review, we summarize recent advances in the synthesis of circular DNA, RNA, and RNA-DNA hybrids, discuss key factors affecting circularization, and how these methods can support the development of CRISPR biosensing and help broaden the future landscape of molecular diagnostics. • Circular nucleic acid synthesis governs structural fidelity and downstream enzyme compatibility • Standardized synthesis–purification–QC workflows enable reliable selection of circles for specific applications • Circular nucleic acids unlock advanced biosensing mechanisms including autocatalytic CRISPR signal amplification • Application-driven design bridges nucleic acid engineering with diagnostics, therapeutics, and bioengineering systems