Unexpected origins of the enhanced pairing affinity of 2′-fluoro-modified RNA

TLDR

Various chemical modifications, especially 2′‑fluoro‑RNA, are being evaluated to improve siRNA efficacy, with 2′‑F‑RNA being well tolerated, non‑immunogenic, nuclease resistant, and more effective in vitro and in vivo. The study aims to uncover the origins of 2′‑F‑RNA’s distinct behavior by performing thermodynamic and crystallographic analyses of fully and partially modified RNAs. Thermodynamic measurements and high‑resolution X‑ray crystallography were used to analyze fully and partially 2′‑F‑modified RNA duplexes. The enhanced pairing affinity of 2′‑F‑RNA is driven primarily by enthalpy rather than entropic pre‑organization, as shown by thermodynamics and crystal structures revealing reduced hydration and suggesting the fluorine substituent strengthens base pairing and π‑π stacking.

Abstract

Various chemical modifications are currently being evaluated for improving the efficacy of short interfering RNA (siRNA) duplexes as antisense agents for gene silencing in vivo . Among the 2′-ribose modifications assessed to date, 2′deoxy-2′-fluoro-RNA (2′-F-RNA) has unique properties for RNA interference (RNAi) applications. Thus, 2′-F-modified nucleotides are well tolerated in the guide (antisense) and passenger (sense) siRNA strands and the corresponding duplexes lack immunostimulatory effects, enhance nuclease resistance and display improved efficacy in vitro and in vivo compared with unmodified siRNAs. To identify potential origins of the distinct behaviors of RNA and 2′-F-RNA we carried out thermodynamic and X-ray crystallographic analyses of fully and partially 2′-F-modified RNAs. Surprisingly, we found that the increased pairing affinity of 2′-F-RNA relative to RNA is not, as commonly assumed, the result of a favorable entropic contribution ('conformational preorganization'), but instead primarily based on enthalpy. Crystal structures at high resolution and osmotic stress demonstrate that the 2′-F-RNA duplex is less hydrated than the RNA duplex. The enthalpy-driven, higher stability of the former hints at the possibility that the 2′-substituent, in addition to its important function in sculpting RNA conformation, plays an underappreciated role in modulating Watson–Crick base pairing strength and potentially π–π stacking interactions.