A TtAgo‐Driven Autocatalytic Circuit with Thermal‐Enhanced Kinetics for One‐Pot Nucleic Acid Detection

Abstract Catalytic DNA circuits hold significant promise for nucleic‐acid‐based diagnostics, yet they remain hindered by slow reaction kinetics and the non‐universal nature of one‐pot approaches. Here, a universal catalytic DNA circuits termed TACTIC ( T hermus thermophilus A rgonaute (TtAgo) protein‐driven auto c ataly ti c c ircuit) is developed for one‐pot detection of DNA and RNA in multiple clinical samples. TACTIC employs the heat‐activated cleavage activity of TtAgo to accelerate reaction kinetics of rolling circle amplification (RCA) by producing an efficient circular template with Gibbs free energy approaching zero at 70. Combined with TtAgo cleavage‐mediated explosive regeneration and accumulation of target mimics, an autocatalytic positive‐feedback circuit is successfully constructed for universal and sensitive detection of nucleic acid biomarkers. Efficient TACTIC is developed by increasing 381% amplification efficiency and achieved detection sensitivity at the attomolar (aM) level. TACTIC enables rapid one‐pot detection of bacterial DNA, mutant mRNA, and four extracellular vesicle‐derived miRNAs (EV miRNAs) within 30 min. Integrated with machine learning, the distinct expression patterns of four EV miRNAs across different biofluids are accurately profiled, and machine learning‐driven diagnostic and staging models for breast cancer are established within a clinical cohort. TACTIC offers new insights for advancing higher‐order catalytic circuits and expanding the toolbox for accurate nucleic acid detection.