Guide Strand 3′‐End Modifications Regulate siRNA Specificity

Abstract

Short interfering RNA (siRNA)-triggered gene knockdown via the RNA interference (RNAi) pathway is widely used to study gene function and siRNA-based therapeutics are in development. However, since the guide strand of an siRNA can function like a natural microRNA (miRNA), siRNAs often repress hundreds of off-target transcripts with complementary only to the seed region (nucleotides 2–8) of the guide strand. Here we describe novel guide strand 3′ end modifications derived from 1-ethynylribose and copper-catalyzed azide/alkyne cycloaddition reactions and evaluate their impact on target vs miRNA-like off-target knockdown. Surprisingly, when positioned at the guide strand 3′ end the parent 1-ethynylribose modification substantially reduced off-target knockdown while having no measurable effect on on-target knockdown potency. In addition, these modifications are shown to modulate siRNA affinity for the hAgo2 PAZ domain. However, the change in PAZ domain binding affinity is not sufficient to predict a modification’s effect on miRNA-like off targeting.

Comparison of thermally actuated retro-diels-alder release groups for nanoparticle based nucleic acid delivery

“…Additionally, the presence of the maleimide on the 3’ terminal of the siRNA sequence may impact the activity of the siRNA. Previous studies indicate that siRNA retain RNAi activity after 3’ modification with several different chemical moieties [68] [69] [70], but at this time, it is not clear what effect, if any, the 3’ maleimide modification has on the siRNA’s ability to interact with the 3’ UTR region of mRNA. Further studies are required to address this concern.…”

Exploring PAZ/3′-overhang interaction to improve siRNA specificity. A combined experimental and modeling study

“… 6 , 7 The development of chemical modifications has been the focus of recent research, in which attention has been directed towards RNA modifications that enable the avoidance of sense strand-mediated and miRNA-like off-targeting effects. 8 – 12 …”

Effective carrier-free gene-silencing activity of cholesterol-modified siRNAs

“…Previous studies have reported that the 3 0 -end of the sense strand is able to accommodate bulky groups. 25 The wild-type siRNA (wt), which lacks a cholesterol modication, did not display any gene-silencing activity in our carrierfree study. This was expected as unmodied siRNAs are known to have difficulties in crossing the cellular membrane unassisted.…”