Guide DNA Conformation Change-Triggered Activation of Clostridium butyricum Argonautes for Biosensing

Argonautes (Agos) are next-generation programmable nucleases that demonstrate great potential for the detection of nucleic acid targets. However, a contemporary difficulty in the intensely active field of Ago-based biosensing is how to enhance its flexibility to meet the requirement in versatile non-nucleic acid target detection. Herein, we proposed an Ago enzymatic activation state regulation strategy to achieve diverse target detection. The normal linear guide DNA of Ago is confined in a hairpin conformation, termed the hgDNA. This unique configuration could effectively prevent Clostridium butyricum Ago ( Cb Ago) activation. Under the action of enzymatic hydrolysis or small-molecule-mediated attack on specific sites, the hairpin structure of hgDNA is disrupted, leading to the release of active gDNA. Therefore, the activation and inactivation states of Cb Ago can be deliberately regulated. The possible activation mechanism could be ascribed to the strand displacement reaction between unstable hgDNA and the complementary strand. This tunability transforms Cb Ago into a generalizable detection platform suitable for various targets such as nucleases, small molecules, and pathogens. This study advances the dynamic response of Cb Ago to gDNA conformations and introduces the universal concept of Ago-based non-nucleic acid detection to broaden the next-generation biosensing applications.