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

Abstract

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 C2ʹ/O2ʹ atoms are the most common sites for modification. Compared to 2ʹ-O-substituents, ribose 4ʹ-C-substituents lie in proximity of both the 3ʹ- and 5ʹ-adjacent phosphates. To investigate potentially beneficial effects on nuclease resistance we combined 2ʹ-F and 2ʹ-OMe with 4ʹ-Cα- and 4ʹ-Cβ-OMe, and 2ʹ-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 4ʹα-epimers affect thermal stability only minimally and show increased nuclease stability irrespective of the 2ʹ-substituent (H, F, OMe). The 4ʹβ-epimers are strongly destabilizing, but afford complete resistance against an exonuclease with the phosphate or phosphorothioate backbones. Crystal structures of RNA octamers containing 2ʹ-F,4ʹ-Cα-OMe-U, 2ʹ-F,4ʹ-Cβ-OMe-U, 2ʹ-OMe,4ʹ-Cα-OMe-U, 2ʹ-OMe,4ʹ-Cβ-OMe-U or 2ʹ-F,4ʹ-Cα-Me-U help rationalize these observations and point to steric and electrostatic origins of the unprecedented nuclease resistance seen with the chain-inverted 4ʹβ-U epimer. We used structural models of human Argonaute 2 in complex with guide siRNA featuring 2ʹ-F,4ʹ-Cα-OMe-U or 2ʹ-F,4ʹ-Cβ-OMe-U at various sites in the seed region to interpret in vitro activities of siRNAs with the corresponding 2ʹ-/4ʹ-C-modifications.