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

Clostridium butyricum (CbAgo)-based bioassays are popular due to their programmability and directional cleavage capabilities. However, the relatively compact protein structure of CbAgo 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 CbAgo cleavage efficiency. Then, we invented a novel gDNA containing DNAzyme segments (gDNAzyme) that substantially enhanced the CbAgo 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 CbAgo and an increased number of cleavage sites. Moreover, this gDNAzyme 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 CbAgo-based biosensor but also provides unique insight into the protein–DNA interactions.