Eighteen nucleic acid therapeutics have been approved for treatment of various diseases in the last 25 years. Their modes of action include antisense oligonucleotides (ASOs), splice-switching oligonucleotides (SSOs), RNA interference (RNAi) and an RNA aptamer against a protein. Among the diseases targeted by this new class of drugs are homozygous familial hypercholesterolemia, spinal muscular atrophy, Duchenne muscular dystrophy, hereditary transthyretin-mediated amyloidosis, familial chylomicronemia syndrome, acute hepatic porphyria, and primary hyperoxaluria. Chemical modification of DNA and RNA was central to making drugs out of oligonucleotides. Oligonucleotide therapeutics brought to market thus far contain just a handful of first- and second-generation modifications, among them 2′-fluoro-RNA, 2′-O-methyl RNA and the phosphorothioates that were introduced over 50 years ago. Two other privileged chemistries are 2′-O-(2-methoxyethyl)-RNA (MOE) and the phosphorodiamidate morpholinos (PMO). Given their importance in imparting oligonucleotides with high target affinity, metabolic stability and favorable pharmacokinetic and -dynamic properties, this article provides a review of these chemistries and their use in nucleic acid therapeutics. Breakthroughs in lipid formulation and GalNAc conjugation of modified oligonucleotides have paved the way to efficient delivery and robust, long-lasting silencing of genes. This review provides an account of the state-of-the-art of targeted oligo delivery to hepatocytes.

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.