Structural Hairpin Anchoring-Mediated TtAgo Activity Regulation for Programmable Biosensing

Thermus thermophilus Argonaute (TtAgo) has shown exceptional promise in biosensing and molecular diagnostics owing to its programmable and DNA-guided specific cleavage activity. However, the potential of regulating TtAgo activity through precise structural modification of guide DNA (gDNA) remains to be fully utilized, thereby limiting the flexible expansion of TtAgo-based biosensing strategies. In this work, TtAgo achieves optimal cleavage activity strictly with a specific linear single-stranded gDNA (linear gDNA) length range (about 15-21 nt), whereas an excessively long linear gDNA fails to stably anchor within the PAZ domain. Through rational structural design, introducing a hairpin structure at 3' terminus of the gDNA can provide an effective physical anchor to ensure binding, thereby efficiently regulating TtAgo cleavage activity, whereas overextending this rigid structure toward the catalytic core induces severe steric hindrance that effectively inhibits cleavage. Herein, such an activation and regulatory mechanism for TtAgo is termed "the structural hairpin anchor regulates endonuclease cleavage activity" (SHARE) effect. To harness this programmable strategy, a TtAgo biosensor was engineered wherein the target enzyme, apurinic/apyrimidinic endonuclease 1, catalyzed the structural transformation of an inactive gDNA containing an intact hairpin structure, enabling sensitive and specific detection of the biomarker. Herein, the SHARE effect may enrich TtAgo regulatory strategies based on gDNA conformational engineering and significantly broaden the applications of TtAgo systems.