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

Abstract

Nucleotides with carbon substitution for heteroatoms are common in biological and therapeutic RNAs. Important examples include the C-nucleosides pseudouridine and N1-methyl-pseudouridine; these modifications were reported to slow 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 find that carbon substitution markedly reduces strand cleavage rates in RNA by both mechanisms. Studies of nucleophilic acylation reactions of RNAs and of small alcohols of varied pKa suggest that reduced inductive effects resulting from carbon substitution for electronegative atoms results in both higher pKa and lower nucleophilicity. The results provide insight into native transcriptome modifications as well as RNA therapies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/666442v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@73c194org.highwire.dtl.DTLVardef@1aa0af7org.highwire.dtl.DTLVardef@b5f8a0org.highwire.dtl.DTLVardef@d601fa_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOTOC GraphicC_FLOATNO C_FIG SynopsisThis study reveals mechanisms by which modifications found in native and therapeutic RNAs enhance RNA stability. Carbon substitution for nitrogen or oxygen results in reduced 2'-OH nucleophilicity.