The R-diastereomer of 6′-O-toluoyl-carba-LNA modification in the core region of siRNA leads to 24-times improved RNA silencing potency against the HIV-1 compared to its S-counterpart

Abstract

The modified siRNA with pure [6′(S)-O-(p-toluoyl)-7′(S)-methyl]-carba-LNA [6′(S)-O-toluoyl-jcLNA] at position T¹³ displayed an IC₅₀ of 79.8 nM, which has been found to be nearly 24-times less potent as a HIV-1 RNAi silencing agent against TAR RNA than that of the corresponding pure [6′(R)-O-(p-toluoyl)-7′(S)-methyl]-jcLNA [6′(R)-O-(p-toluoyl)-jcLNA] counterpart [IC₅₀ 3.3 nM]. The later [6′(R)-O-(p-toluoyl)-jcLNA]-modified siRNAs have been found to be nearly 2-fold more efficient as a silencing agent than the corresponding 6′-deoxy-jcLNA modified siRNA [IC₅₀ 8.1 nM], and also nearly 3-fold more effective as a silencing agent than that of LNA-modified siRNA [IC₅₀ 11.7 nM], thereby showing that the 6′-carbon center in the jcLNA-modified siRNA in the core region is relatively more exposed to the Ago protein in the RISC with a clear chirality preference for the siRNA cleavage reaction. It is noteworthy that the IC₅₀ of jcLNA-modified siRNAs are very comparable to that of the native siRNA [1.8 nM]. The jcLNA derivatized siRNAs, however, have a clear advantage of being, in general, considerably more stable in human serum. The main structural difference in duplexes of the antisense strand of the 6′(R or S)-O-(p-toluoyl)-jcLNA modified siRNA and target RNA duplex is found to be the spatial orientation of the 6′(R)-O-toluoyl group, which is exposed towards the edge of the duplex backbone, while the 6′(S) makes the minor groove relatively inaccessible for the Ago protein in the RISC. Clearly, any further C6′-modification in jcLNA-modified siRNAs with any hydrophobic group for tighter binding and cleavage or for cross-linking in the core region should preferably be done in the 6′(R)-stereochemistry.