Controlling miRNA-like off-target effects of an siRNA with nucleobase modifications.

ABSTRACT: SiRNAs can cause unintended gene silencing due to miRNA-like effects because of the similarity in function of an siRNA guide strand and a miRNA. Here we evaluate the effect on miRNA-like off targeting of introducing the adenosine derivative 7-EAA and triazoles prepared from 7-EAA at different positions in an siRNA guide strand. We find that a sterically demanding triazole placed in the RNA duplex major groove at position six of the guide strand dramatically reduces miRNA-like off targeting potency. A high-resolution structure of an RNA duplex bearing a novel, major-groove localized triazole is reported, which suggests that modified triazoles could be disrupting the hAgo2-guide-target RNA ternary complex. Five different triazole modifications were tested at the guide strand 6-position for effects on on-target and miRNA-like off target knockdown potency. A 7-EAA triazole bearing a benzylamine substituent displayed on-target knockdown activity as potent as the native siRNA, while having an IC50 against a miRNA-like off target >100-fold higher. Melting temperature studies revealed no obvious correlation between potency in knockdown assays and a modification's effect on duplex stability. These results, along with known structures of hAgo2-guide-target ternary complexes, are used to rationalize the effect of 7-EAA triazoles on miRNA-like off target effects.

SUBMITTER: Suter SR 

PROVIDER: S-EPMC5734094 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature