Tolerated wobble mutations in siRNAs decrease specificity, but can enhance activity in vivo

Abstract

RNA interference (RNAi) has become an invaluable tool for functional genomics. A critical use of this tool depends on an understanding of the factors that determine the specificity and activity of the active agent, small interfering RNA (siRNA). Several studies have concluded that tolerance of mutations can be considerable and hence lead to off-target effects. In this study, we have investigated in vivo the toleration of wobble (G:U) mutations in high activity siRNAs against Flap Endonuclease 1 (Fen1) and Aquaporin-4 (Aqp4). Mutations in the central part of the antisense strand caused a pronounced decrease in activity, while mutations in the 5′ and 3′ends were tolerated very well. Furthermore, based on analysis of nine different mutated siRNAs with widely differing intrinsic activities, we conclude that siRNA activity can be significantly enhanced by wobble mutations (relative to mRNA), in the 5′ terminal of the antisense strand. These findings should facilitate design of active siRNAs where the target mRNA offers limited choice of siRNA positions.

Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA

“…In agreement with our expectations a threefold suppression of the silencing potency was observed for the sG2/aD19 duplex (4). Similar effects were reported for the siRNA molecules containing single chemical modifications or mismatches, including wobble base pairs, at the 39-and/or 59-terminal positions of the duplexes (Chiu and Rana 2003;Schwarz et al 2003;Holen et al 2005;Dande et al 2006;Dowler et al 2006). …”

“…4). This observation is interesting in light of earlier reported data about modifications of the central part of the antisense strand of siRNA duplex not being well tolerated (Hamada et al 2002;Saxena et al 2003;Du et al 2005;Holen et al 2005). The remaining two duplexes sG2/aD10 (7) and sG2/aC10 (8) exhibited threeto fourfold suppressed silencing activity.…”

Literature review of baseline information to support the risk assessment of RNAi‐based GM plants

“…A:C mismatches were, in addition to the G:U wobble base pairs, surprisingly well tolerated and target sites containing such mismatches were silenced almost as efficiently as with full complementarity (Du et al, 2005). G:U wobble base pairing in the central part of the antisense strand caused a pronounced decrease in activity, while mutations at the 5' and 3'ends were well-tolerated (Holen et al, 2005). Interestingly, analysis of siRNA selectivity suggested that siRNAs with G:U wobble base pairs or a mismatches located in the "seed" are discriminating less between perfect and mismatched target than those in which the mismatch was located 3' to the seed (nucleotides [9][10][11][12][13][14]; this region is critical for target cleavage but not siRNA binding (Schwarz et al, 2006).…”

Diet-delivered RNAi in Helicoverpa armigera – Progresses and challenges

“…For non-target species, the likelihood of off-target effects due to gene homologs has been shown to be affected by both the number and position of mismatches between the siRNA strand(s) and the gene sequence it cross-hybridizes with (Du et al, 2005;Holen et al, 2005). Efforts at minimization of RNAi off-target effects has seen development of software such as siDirect (Naito et al, 2004) and dsCheck (Naito et al, 2005).…”