Programmable Recognition of Organyl 5′-Phosphates by Human Argonaute-2

Abstract To effectuate RNA interference (RNAi), Argonaute (AGO) proteins recognize and load RNA guide strands by their requisite 5′-phosphate (5′-P). However, the structural features that govern AGO2 tolerance for chemically modified 5′-P architectures remain poorly defined, despite their importance for small interfering RNA (siRNA) drug design. Moreover, degradative enzymes also recognize 5′-P, yet few stereoelectronic features are known to differentiate AGO2 recognition from competing degradation pathways. Here, we show that AGO2 accommodates a substantially broader range of 5′-P derivatives than previously recognized, and that select 5′-P mimics can exploit novel interactions with the protein to reinforce guide anchoring. Through systematic diversification of 35 organic substituents, we interrogated structural determinants of guide anchoring and enzymatic susceptibility. Our analysis identified previously uncharacterized 5′-P mimics, including methyl and phenylpropargyl 5′-phosphates (5′-PO-Me, 5′-PO-PhPrp), phenylpropargyl 5′-phosphorothioate (5′-PS-PhPrp), and 5′-mesylphosphoramidate (5′-MsPA), that preserve efficient AGO2 loading in cells while inhibiting phosphatase-mediated degradation. We further found that 5′-exonuclease susceptibility depends strongly on canonical 5′ phosphate chemistry rather than steric accessibility. Structural analysis of a 5′-PO-PhPrp guide:AGO2 complex shows that the phenylpropargyl substituent engages a π-stacking network within a cluster of aromatic side chains (Y529/F811/Y815), while extended 5′-P substituents are accommodated through rotation of the 5′-P and coordinated rearrangement of water molecules within the 5′-P binding pocket. Collectively, these findings reveal the structural basis for plasticity of guide anchoring in AGO2 and establish chemically programmable 5′-end interactions as a general strategy for modulating oligonucleotide stability and activity.