Clickable RNA via 4'-C-Ethynyl Cytidine─A Novel Design for Metabolically Stable Guide RNAs in RNA Editing

Chemical modifications in RNA therapeutics have addressed major challenges by enhancing metabolic stability, cellular uptake, and biological activity─regardless of their mechanism of action. Here, we report on the synthesis of 4'-C-ethynyl cytidine (4'-C-EthC) and its 2'-O-methylated derivative (4'-C-EthC-2'-OMe) as phosphoramidite building blocks and their subsequent incorporation into oligonucleotides. These ribose C4-terminal alkyne modifications provide a click handle directly within oligonucleotides. The novel modification is accessible via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and serves as a universal 4'-C-ribose modifier on the oligonucleotide level. We identified both aromatic and aliphatic triazole residues that increase the thermodynamic stability in A-form RNA duplexes. Furthermore, 4'-C-triazole-modified oligonucleotides exhibit high resistance to nuclease-mediated degradation in metabolic stability assays. Finally, we introduced the novel modification and its substituted triazoles into guide RNAs (gRNAs) for site-directed A-to-I editing in mammalian cells and compared their performance with phosphorothioate-modified gRNAs.