Structural basis for the synergy of 4′- and 2′-modifications on siRNA nuclease resistance, thermal stability and RNAi activity

Chemical modification is a prerequisite of oligonucleotide therapeutics for improved metabolic stability, uptake and activity, irrespective of their mode of action, i.e. antisense, RNAi or aptamer. Phosphate moiety and ribose C2′/O2′ atoms are the most common sites for modification. Compared to 2′-O-substituents, ribose 4′-C-substituents lie in proximity of both the 3′- and 5′-adjacent phosphates. To investigate potentially beneficial effects on nuclease resistance we combined 2′-F and 2′-OMe with 4′-Cα- and 4′-Cβ-OMe, and 2′-F with 4′-Cα-methyl modification. The α- and β-epimers of 4′-C-OMe-uridine and the α-epimer of 4′-C-Me-uridine monomers were synthesized and incorporated into siRNAs. The 4′α-epimers affect thermal stability only minimally and show increased nuclease stability irrespective of the 2′-substituent (H, F, OMe). The 4′β-epimers are strongly destabilizing, but afford complete resistance against an exonuclease with the phosphate or phosphorothioate backbones. Crystal structures of RNA octamers containing 2′-F,4′-Cα-OMe-U, 2′-F,4′-Cβ-OMe-U, 2′-OMe,4′-Cα-OMe-U, 2′-OMe,4′-Cβ-OMe-U or 2′-F,4′-Cα-Me-U help rationalize these observations and point to steric and electrostatic origins of the unprecedented nuclease resistance seen with the chain-inverted 4′β-U epimer. We used structural models of human Argonaute 2 in complex with guide siRNA featuring 2′-F,4′-Cα-OMe-U or 2′-F,4′-Cβ-OMe-U at various sites in the seed region to interpret in vitro activities of siRNAs with the corresponding 2′-/4′-C-modifications.