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

• Published in: Nucleic acids research Vol. 46; no. 16; pp. 8090 - 8104
• Main Authors: Harp, Joel M, Guenther, Dale C, Bisbe, Anna, Perkins, Lydia, Matsuda, Shigeo, Bommineni, Gopal R, Zlatev, Ivan, Foster, Donald J, Taneja, Nate, Charisse, Klaus, Maier, Martin A, Rajeev, Kallanthottathil G, Manoharan, Muthiah, Egli, Martin
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 19.09.2018
• Subjects: Chemical Biology and Nucleic Acid Chemistry; Humans; Models, Molecular; Nucleic Acid Conformation; Oligonucleotides - chemistry; Oligonucleotides - genetics; Phosphates - chemistry; Ribonucleases - chemistry; Ribose - chemistry; RNA Interference; RNA Stability - genetics; RNA, Small Interfering - chemistry; Thermodynamics; Uridine - chemistry; Uridine - genetics
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gky703

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.