Optimal compositions of potent sliced-siRNA with DNA bases substitution and insights of Ago2 slicing actions

Abstract

We have previously characterized the nucleotide compositions of single-stranded ago2-sliced small interfering RNAs (sliced-siRNA, sliRNA), a novel type of potent RNAi trigger designed based on the biogenesis mechanism of miRNA-451. sliRNAs have on par antisense mediated silencing activity but about 1000-fold reduced passenger strand activity compared to siRNA. In the current study, to further characterize sliRNAs to facilitate their in vivo application, we have investigated the effects of substituting RNA nucleotides (nts) in sliRNA with DNA nts (sliDRNAs), which will allow us to study the potential effects on their processing, maturation, and silencing activity from individual or multiple nts at different positions. Our results showed sliDRNAs have distinguished composition from siRNA with DNA nts (siDRNA), indicated that, in addition to their different biogenesis mechanisms, these two types of RNAi triggers may also have adopted additional different silencing mechanisms. Our results also revealed some mechanism related to how Ago2 executes its slicer activity and the effects on sliRNA potency by trimming and tailing during sliRNA maturation. These results have paved the way for using other chemically modified nts in sliRNAs that may enhance their in vivo stability and transportability to facilitate their in vivo applications.