Redesigned Guide DNA Enhanced Clostridium butyricum Argonaute Activity for Amplification-Free and Multiplexed Detection of Pathogens

Clostridium butyricum ( Cb Ago)-based bioassays are popular due to their programmability and directional cleavage capabilities. However, the relatively compact protein structure of Cb Ago limits its cleavage activity (even at the optimal temperature), thus restricting its wider application. Here, we observed that guide DNA (gDNA) with specific structural features significantly enhanced Cb Ago cleavage efficiency. Then, we invented a novel gDNA containing DNAzyme segments (gDNA zyme ) that substantially enhanced the Cb Ago cleavage efficency (by 100%). Using a molecular dynamics simulation system, we found that the augmented cleavage efficiency might be attributed to the large-scale global movement of the PIWI domain of Cb Ago and an increased number of cleavage sites. Moreover, this gDNA zyme feature allowed us to create a biosensor that simultaneously and sensitively detected three pathogenic bacteria without DNA extraction and amplification. Our work not only dramatically expands applications of the Cb Ago-based biosensor but also provides unique insight into the protein–DNA interactions.