C-Nucleosides Stabilize RNA by Reducing Nucleophilicity at 2′-OH

Nucleotides with a carbon substitution for heteroatoms are common in biological and therapeutic RNAs. Important examples include the C-nucleosides pseudouridine and <i>N1</i>-methylpseudouridine; these modifications were reported to slow the degradation of large RNAs, but the mechanism is unknown. We measured kinetics of spontaneous and enzymatic cleavage at a single bond of synthetically modified RNAs and found that carbon substitution markedly reduces strand cleavage rates in RNA by both mechanisms. Studies of nucleophilic acylation reactions of RNAs and small alcohols of varied p<i>K</i> _a suggest that reduced inductive effects resulting from carbon substitution for electronegative atoms results in both higher p<i>K</i> _a and lower nucleophilicity. The results provide insight into native transcriptome modifications as well as RNA therapies.