Organyl 5′‑Phosphates in siRNA Guide Strands: Structure–Function Relationships Governing Anchoring in Argonaute 2 and Metabolic Stability

ABSTRACT Efficient siRNA loading into Argonaute2 (AGO2) requires a 5′-phosphate (5′-P) on the guide strand, yet this group is vulnerable to metabolic degradation in vivo. Although numerous chemical mimics of 5′-P have been reported, structural principles governing AGO2 interactions with organyl substituents on the 5′-P remain unclear. Moreover, structural determinants of 5′-P mimic recognition by known degradative enzymes (principally phosphatases and 5′-exonucleases) are also poorly understood. The 5′-P binding site of the AGO2 MID domain contains a stack of aromatic residues (Y527/F811/Y815), presenting a structural basis for augmenting canonical anchoring interactions. Herein, we systematically synthesized and characterized a diverse panel of organyl 5′-phosphates (5′-POR; R = 35 variable substituents) as guide strand 5′-P mimics designed to engage this unique hydrophobic pocket. Among the compounds evaluated, 5′-POR guide strands bearing methyl (Me) or phenylpropargyl (PhPrp) substituents are well-tolerated by AGO2 in cells. Previously uncharacterized 5′-P mimics, including 5′-phosphorothioate (5′-PS), phenylpropargyl 5′-phosphorothioate (5′-PS-PhPrp), and 5′-mesylphosphoramidate (5′-MsPA), maintain comparable AGO2 compatibility. All examined 5′-P mimics are markedly resistant to phosphatase, while 5′-POR variants and 5′-PS-PhPrp are also resistant to 5′-exonuclease degradation due to masking a negative charge of 5′-P. A crystal structure of a 5′-PO-PhPrp guide strand loaded into AGO2 reveals an unexpected network of π–π interactions between the rigid phenylpropargyl group and the targeted hydrophobic pocket of the MID domain. Collectively, these findings expand the functional chemical space of 5′-P mimics and define new modes for metabolically stabilizing the guide strand 5′-end while augmenting AGO2 MID anchoring. Graphical Abstract