Chirality matters: stereo-defined phosphorothioate linkages at the termini of small interfering RNAs improve pharmacology in vivo

Abstract

A critical challenge for the successful development of RNA interference-based therapeutics therapeutics has been the enhancement of their in vivo metabolic stability. In therapeutically relevant, fully chemically modified small interfering RNAs (siRNAs), modification of the two terminal phosphodiester linkages in each strand of the siRNA duplex with phosphorothioate (PS) is generally sufficient to protect against exonuclease degradation in vivo. Since PS linkages are chiral, we systematically studied the properties of siRNAs containing single chiral PS linkages at each strand terminus. We report an efficient and simple method to introduce chiral PS linkages and demonstrate that Rp diastereomers at the 5′ end and Sp diastereomers at the 3′ end of the antisense siRNA strand improved pharmacokinetic and pharmacodynamic properties in a mouse model. In silico modeling studies provide mechanistic insights into how the Rp isomer at the 5′ end and Sp isomer at the 3′ end of the antisense siRNA enhance Argonaute 2 (Ago2) loading and metabolic stability of siRNAs in a concerted manner.

Interfering with nucleotide excision by the coronavirus 3’-to-5’ exoribonuclease

“…The most efficient strategy to interfere with nucleotide excision is to introduce a nonhydrolyzable scissile bond. Phosphorothioate (P-S) substitutions at the scissile phosphodiester bond inhibit excision by DNA exonucleases, usually requiring a specific stereoisomer (SP or RP) (64)(65)(66)(67)(68)76). Here we show that the presence of a phosphorothioate in the RP configuration of the scissile phosphodiester bond inhibits hydrolysis by ExoN (Figs.…”

“…Introduction of the P-S-substituted bond at ultimate, penultimate, and/or antepenultimate position will create substrates to facilitate elucidation of the details of ExoN-catalyzed hydrolysis by limiting the extent to which a product can be consumed. Current strategies to synthesize P-S-substituted oligonucleotides yield diastereomeric mixtures at phosphorous, but stereoisomer-specific solutions are on the horizon (76).…”

Interfering with nucleotide excision by the coronavirus 3′-to-5′ exoribonuclease

“…The most efficient strategy to interfere with nucleotide excision is to introduce a non-hydrolyzable scissile bond. Phosphorothioate (P-S) substitutions at the scissile phosphodiester bond inhibit excision by DNA exonucleases, usually requiring a specific stereoisomer (S P or R P ) ( 65–69 , 77 ). Here we show that the presence of a phosphorothioate in the R P configuration of the scissile phosphodiester bond inhibits hydrolysis by ExoN (Figures 5 and 6 and Table 2 ).…”

“…Introduction of the P-S-substituted bond at ultimate, penultimate, and/or antepenultimate position will create substrates to facilitate elucidation of the details of ExoN-catalyzed hydrolysis by limiting the extent to which a product can be consumed. Current strategies to synthesize P-S-substituted oligonucleotides yield diastereomeric mixtures at phosphorous, but stereoisomer-specific solutions are on the horizon ( 77 ).…”

Chemical Modifications in Nucleic Acids for Therapeutic and Diagnostic Applications

“…Recently, an interesting observation has been described regarding the effect produced by the presence of a single chiral phosphorothioate at the ends of siRNAs [66] . The siRNA that combine the presence of the R p isomer at the 5’‐end and the S p isomer at the 3’‐end of the guide strand was found to be practically one order of magnitude more potent in a mouse model.…”