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′-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.

Extensive CRISPR RNA modification reveals chemical compatibility and structure-activity relationships for Cas9 biochemical activity

“…Other than 2′F-RNA, and to a small degree 2′,5′-RNA, none of these modifications supported activity. This was surprising given the RNA-like (C3′- endo ) sugar pucker previously reported for 2′,4′-di-Cα- O Me and 2′F-4′-Cα- O Me (48,50,51). Multiple 4′- O -methyl groups may introduce unfavorable steric constraints due to their bulkiness relative to a hydrogen atom.…”

Current Development of siRNA Bioconjugates: From Research to the Clinic

“…Although other positions in ribose, such as 4′ carbon, can be modified [4′S Gore et al, 2012, 4′C-aminomethyl-2′-O-methyl Takahashi et al, 2012, and 4′C-O-methyl-2′-O-methyl Harp et al, 2018 (Table 1)] and such modifications protect siRNAs from nucleases in vitro efficiently, these modifications are not widely used in biomedical research because they significantly inhibit RNAi (Deleavey and Damha, 2012).…”

siRNA Targeting and Treatment of Gastrointestinal Diseases

“…Replacement of sulfur for the nonbridging oxygen blocks exonuclease activity and increases binding to plasma proteins preventing rapid renal clearance . Further research into modifications at sites, such as the ribose 4′‐C, are underway …”