Competition for RISC binding predicts in vitro potency of siRNA

Abstract

Short interfering RNAs (siRNA) guide degradation of target RNA by the RNA-induced silencing complex (RISC). The use of siRNA in animals is limited partially due to the short half-life of siRNAs in tissues. Chemically modified siRNAs are necessary that maintain mRNA degradation activity, but are more stable to nucleases. In this study, we utilized alternating 2′-O-methyl and 2′-deoxy-2′-fluoro (OMe/F) chemically modified siRNA targeting PTEN and Eg5. OMe/F-modified siRNA consistently reduced mRNA and protein levels with equal or greater potency and efficacy than unmodified siRNA. We showed that modified siRNAs use the RISC mechanism and lead to cleavage of target mRNA at the same position as unmodified siRNA. We further demonstrated that siRNAs can compete with each other, where highly potent siRNAs can compete with less potent siRNAs, thus limiting the ability of siRNAs with lower potency to mediate mRNA degradation. In contrast, a siRNA with low potency cannot compete with a highly efficient siRNA. We established a correlation between siRNA potency and ability to compete with other siRNAs. Thus, siRNAs that are more potent inhibitors for mRNA destruction have the potential to out-compete less potent siRNAs indicating that the amount of a cellular component, perhaps RISC, limits siRNA activity.

Argonaute proteins are key determinants of RNAi efficacy, toxicity, and persistence in the adult mouse liver

“…Evidence (Figures 2 and 5, and Supplemental Figure 7) was that (a) transient or stable Ago-2 overexpression increased siRNA (and shRNA) activity in cells and relieved si-siRNA or sishRNA competition; (b) miRNAs were also enhanced by Ago-2 (at least when directed against a perfect target); and (c) siRNAs and shRNAs were inactive in Ago-2-knockout cells and rescued only by Ago-2, but not any other Ago protein. Together, these data not only confirm, extend, and help to explain prior notions of, for example, si-si/shRNA competition in cells (12)(13)(14), but also validate that Ago-2 is vital for and saturable by all classes of RNAi triggers. Hence, as with shRNAs, care must likewise be taken especially with the latest proficient siRNA formulations (59,60) to avoid in vivo Ago-2 saturation and associated adverse effects in clinical settings.…”

“…We had indeed noted substantial changes in hepatic miRNA levels in livers of shRNAtreated ailing mice (3,4), and miRNA activities were consistently disturbed in shRNA-transfected cells (3,4,6,12). The fact that such adverse effects on miRNAs are seen with transfected siRNAs as well (12)(13)(14)(15)(16) implies that one or more key factors in the RNAi pathway are rate-limiting in cell culture. Indeed, a number of suspects have been controversially discussed in the past, such as Dicer, TRBP and components of RNA-induced silencing complex (RISC) (5,12,(17)(18)(19)(20), including Argonaute-2 (Ago-2) (13,(21)(22)(23).…”

Disruption of small RNA signaling caused by competition for Hfq

“…This type of competition between different sRNA and target mRNA substrates is not unique to Hfq. For example, in human cells, there is in vivo and in vitro evidence that competition occurs between sRNAs for a key mediator of sRNA silencing (RNA-induced silencing complex) (40)(41)(42).…”

Inhibition of Coxsackievirus A24 in permissive Hep2 cells by small interfering RNA (siRNA)

“…To check the synergistic effect of our designed siRNA, we tested them alone and in combinations but no significant difference was observed in their antiviral potency (data not shown). The reason for the failure of synergistic effect may be competition of processed siRNA for RISC by which their independent activity reduced [31].…”