Amides and Other Nonionic Backbone Modifications in RNA

The development of RNA interference (RNAi) and CRISPR as powerful research tools and novel therapeutic approaches has reinvigorated interest in chemical modifications of RNA. Short interfering RNAs (siRNAs) and CRISPR-associated RNAs are the key components driving the endonuclease activity and specificity of Ago2 and Cas proteins, the main players in RNAi and CRISPR. Consequently, chemical modification of these RNAs is one of the most straightforward and flexible ways to improve these technologies. The present chapter focuses on nonionic linkages as replacements for phosphodiesters, which are underexplored modifications of RNA with significant potential applications in RNAi and CRISPR. Amide linkages are the most extensively studied nonionic backbones in DNA and RNA. However, other linkages, for example, formacetals, have shown intriguing biophysical properties but have not yet been tested in biological experiments. The present chapter concludes that amides and other nonionic backbone modifications may be tolerated even better in RNA than in the initial studies in DNA. Amides have already been reported to increase the RNAi activity and specificity when placed at specific positions within siRNAs and may have similar potential to optimize CRISPR technology. Furthermore, advances in the structural biology of protein-RNA complexes should enable rational design and testing of other nonionic RNA linkages with the goal of further improving the activity and specificity of RNAi and CRISPR.