Stereoselective RNA Reaction with Chiral 2'-OH Acylating Agents

The reactivity of RNA 2'-OH groups with acylating agents has recently been investigated for high-yield conjugation of RNA strands. To date, only achiral molecules have been studied for this reaction, despite the complex chiral structure of RNA. Here we prepare a set of chiral acylimidazoles and study their stereoselectivity in RNA reactions. Reactions performed with unfolded and folded RNAs reveal that positional selectivity and reactivity vary widely with local RNA macro-chirality. We further document remarkable effects of chirality on reagent reactivity, identifying an asymmetric reagent with 1000-fold greater reactivity than prior achiral reagents. In addition, we identify a chiral compound with higher RNA structural selectivity than any previously reported RNA-mapping species. Further, azide-containing homologs of a chiral dimethylalanine reagent were synthesized and applied to local RNA labeling, displaying 92% yield and 16:1 diastereoselectivity. The results establish that reagent stereochemistry and chiral RNA structure are critical elements of small molecule-RNA reactions, and demonstrate new chemical strategies for selective RNA modification and probing. Overall design: 10 samples are analyzed, including replicates for each conditions: (1) A-case RNA library treated with 50mM S1; (2) A-case RNA library treated with 50mM R1; (3) A-case RNA library treated with 100µM S2; (4) A-case RNA library treated with 100µM R2;(5) A-case RNA library treated DMSO (as control); (6) U-case RNA library treated with 50mM S1; (7) U-case RNA library treated with 50mM R1; (8) U-case RNA library treated with 100µM S2; (9) U-case RNA library treated with 100µM R2;(10) U-case RNA library treated DMSO (as control);