Elucidation of the conformational dynamics and assembly of Argonaute–RNA complexes by distinct yet coordinated actions of the supplementary microRNA

Argonaute (AGO) proteins, the core of RNA-induced silencing complex, are guided by microRNAs (miRNAs) to recognize target RNA for repression. The miRNA–target RNA recognition forms initially through pairing at the seed region while the additional supplementary pairing can enhance target recognition and compensate for seed mismatch. The extension of miRNA lengths can strengthen the target affinity when pairing both in the seed and supplementary regions. However, the mechanism underlying the effect of the supplementary pairing on the conformational dynamics and the assembly of AGO–RNA complex remains poorly understood. To address this, we performed large-scale molecular dynamics simulations of AGO–RNA complexes with different pairing patterns and miRNA lengths. The results reveal that the additional supplementary pairing can not only strengthen the interaction between miRNA and target RNA, but also induce the increased plasticity of the PAZ domain and enhance the domain connectivity among the PAZ, PIWI, N domains of the AGO protein. The strong community network between these domains tightens the mouth of the supplementary chamber of AGO protein, which prevents the escape of target RNA from the complex and shields it from solvent water attack. Importantly, the inner stronger matching pairs between the miRNA and target RNA can compensate for weaker mismatches at the edge of supplementary region. These findings provide guidance for the design of miRNA mimics and anti-miRNAs for both clinical and experimental use and open the way for further engineering of AGO proteins as a new tool in the field of gene regulation.