Structural basis for the synergy of 4'- and 2'-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 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'-C?- and 4'-C?-OMe, and 2'-F with 4'-C?-methyl modification. The ?- and ?-epimers of 4'-C-OMe-uridine and the ?-epimer of 4'-C-Me-uridine monomers were synthesized and incorporated into siRNAs. The 4'?-epimers affect thermal stability only minimally and show increased nuclease stability irrespective of the 2'-substituent (H, F, OMe). The 4'?-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'-C?-OMe-U, 2'-F,4'-C?-OMe-U, 2'-OMe,4'-C?-OMe-U, 2'-OMe,4'-C?-OMe-U or 2'-F,4'-C?-Me-U help rationalize these observations and point to steric and electrostatic origins of the unprecedented nuclease resistance seen with the chain-inverted 4'?-U epimer. We used structural models of human Argonaute 2 in complex with guide siRNA featuring 2'-F,4'-C?-OMe-U or 2'-F,4'-C?-OMe-U at various sites in the seed region to interpret in vitro activities of siRNAs with the corresponding 2'-/4'-C-modifications.

SUBMITTER: Harp JM 

PROVIDER: S-EPMC6144868 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature