Prokaryotic Argonaute proteins (pAgos) are nucleic acid-guided endonucleases with diverse functions. Mucilaginibacter paludis Argonaute (MbpAgo) is unusual in using guide DNA (gDNA) to cleave target RNA (tgRNA), but the structural basis for this activity has been unclear. Here we present cryo-electron microscopy structures of MbpAgo in apo, binary, and ternary states at up to 2.55 Å resolution. The apo structure reveals a conserved bilobal scaffold with unique insertions in the PIWI and MID domains that stabilize the catalytic conformation. Upon gDNA binding, MbpAgo forms a dimer stabilized by multiple protein-protein interfaces and an auxiliary nucleic acid-like density bridging the PAZ-MID lobes. The auxiliary nucleic acid interactions coordinate gDNA binding and dimer stabilization to support MbpAgo activity, with dimerization becoming particularly important for efficient cleavage with double-stranded DNA (dsDNA) guides. Binding of tgRNA induces a DNA-RNA hybrid duplex and conformational changes that destabilize the dimer, reverting MbpAgo to an active monomer capable of cleaving structured viral RNAs such as the SARS-CoV-2 5'UTR and HIV-1 CES. These findings suggest a dynamic monomer-dimer transition as both the regulatory mechanism of MbpAgo and an evolutionary adaptation for processing dsDNA-derived guides, providing a structural framework for programmable RNA targeting.
Zhao et al. (2026) studied this question.