Variations of the 3′ Protruding Ends in Synthetic Short Interfering RNA (siRNA) Tested by Microinjection in Drosophila Embryos

Abstract

Short interfering RNAs (siRNAs) are the processing product originating from long double-stranded RNAs (dsRNAs) that are cleaved by the RNase III-like ribonuclease Dicer. As siRNAs mediate cleavage of specific single-stranded target RNAs, they are essential intermediates of RNA interference (RNAi). When applied in synthetic form, siRNAs likewise can induce the silencing process in the absence of long dsRNAs. Here, we tested variations of a conventional synthetic siRNA that had been used successfully to silence the Drosophila notch gene. The variants had two 3 ' -terminal deoxynucleotides in their protruding single-stranded ends. In one case, the deoxynulceotides would match to the notch mRNA, whereas the other variant had nonmatching deoxy-T residues, representing a widely used siRNA design. siRNAs with different combinations of sense and antisense strands were injected into Drosophila embryos at two different concentrations. We found that the all-ribonucleotide siRNA gave the best inhibition of notch expression. The combination of two modified strands with 3 ' -terminal deoxynucleotides was effective, but if combined with a sense or antisense ribostrand, the efficacy dropped. The siRNAs with nonmatching 3 ' -terminal TT residues showed a reduced silencing potential, which became evident at low concentration. An siRNA with a nonmatching 3 ' -terminal ribonucleotide in the antisense strand retained most of its silencing potential in accordance with the hypothesis that primer extension for generation of ssRNA from single-stranded mRNA does not operate in Drosophila.

The siRNA sequence and guide strand overhangs are determinants of in vivo duration of silencing

“…These data can be found in Supplementary Table S1. This similarity in knockdown between dT-dT overhangs and rN-rN overhangs is consistent with previously published reports on the activity of siRNA duplexes with deoxyribonucleotides in guide strand overhangs (9) though in disagreement with other published articles which showed a detrimental effect of dT-dT overhangs compared specifically to rN-rN guide strand overhangs (12) or to rU-rU overhangs (13). To compare the data statistically, we converted the knockdown results to log 2 -fold change to obtain a normal distribution of variance across silencing levels allowing us to perform standard parametric statistics.…”

“…However, other reports have demonstrated that the character of the 3′-overhang of the guide strand is a determinant of strand selection (10), and that the 3′-overhang on the guide strand interacts with the PAZ domain of RISC in the RNA-binding pocket (11), suggesting that the overhang does in fact have a possible contribution to siRNA activity. In contradiction to the original observation that deoxythymidine nucleotides in the guide strand overhangs have little negative consequence on silencing, other reports have made the observation that these overhangs can reduce maximal silencing activity of siRNAs (12,13). …”

“…Specifically, the dT-dT overhangs, which are a common industry standard, have a consistently negative effect on the duration of action of siRNAs in tissue culture cells. Interestingly, there are reports in the literature that see no effect of the guide strand overhang at the tested timepoint (9) and those where a difference is clearly evident (12,13). We suggest that both of these results can be explained as the result of the timepoints under which the experiments were carried out.…”

Progress Toward In Vivo Use of siRNAs-II

“…6 It soon became clear that use of DNA bases in the overhangs slightly reduced potency compared with the natural RNA design and offered no real advantages. [66][67][68] Furthermore, a recent report demonstrated that dT overhangs in siRNAs can inhibit thymidylate synthase, an important enzyme for cell growth and metabolism, leading to unwanted OTEs. 69 It is therefore preferred to employ target sequence-matched RNA or RNA derivatives in the overhang, at least on the guide strand.…”

Chemical and Structural Diversity of siRNA Molecules

“…These two overhanging nucleotides could be replaced with DNA units, preferably thymidines [29]. Subsequent studies showed that such modified duplexes were less active at lower concentrations [30]. The family of siRNA duplexes has been extended recently with blunt-ended constructs [31,32].…”