Tolerance for mutations and chemical modifications in a siRNA

Abstract

Short interfering RNA (siRNA), the active agent of RNA interference, shows promise of becoming a valuable tool in both basic and clinical research. We explore the tolerance to mutations and chemical modi®cations in various parts of the two 21-nt strands of a siRNA targeting the blood clotting initiator Tissue Factor. The mutations were G/C transversions. The chemical modi®cations were 2¢-O-methylation, 2¢-O-allylation and phosphorothioates. We found that siRNA generally tolerated mutations in the 5¢ end, while the 3¢ end exhibited low tolerance. This observation may facilitate the design of siRNA for speci®c targeting of transcripts containing single nucleotide polymorphisms. We further demonstrate that in our system the single antisense strand of the wild-type siRNA is almost as effective as the siRNA duplex, while the corresponding methylated M2+4 version of the antisense had reduced activity. Most of the chemically modi®ed versions tested had near-wild-type initial activity, while the long-term activity was increased for certain siRNA species. Our results may improve the design of siRNAs for in vivo experiments.

Regulation and functional role of the Runt-related transcription factor-2 in pancreatic cancer

“…Thus, only a fraction of siRNAs result in a significant reduction of targets (Hamada et al, 2002;Holen et al, 2002), which may be due to several factors. First, the nature of siRNA and the presence of nucleotide mismatch (Elbashir et al, 2001;Holen et al, 2002;Amarzguioui et al, 2003;Czauderna et al, 2003). Second, extensive secondary structure in the mRNA, such as the presence of RNAassociated proteins and the specific subcellular localisation of the target mRNA that render the transcript inaccessible to siRNAincorporated RISC binding (Holen et al, 2002).…”

Pharmaceutical Prospects for RNA Interference

“…Several studies addressed the influence of siRNA nucleotide modifications, which can be introduced to label the siRNA or enhance its stability against degradation. In general, modifications to the 3Ј position are better tolerated than modifications to the 5Ј positions with regard to preserving silencing efficacy (9,10). The phosphorylation of the 5Јend of the siRNA, needed for activity, may explain this difference.…”

“…Because of the high target sequence specificity of RNAi, mismatches between mRNA and the siRNA duplex have been shown to strongly reduce activity (7)(8)(9)(10)(11). Mismatches in the center of the duplex, generally, have stronger effects than mismatches closer to the ends of the strand.…”

“…In addition, mismatches in the mRNA-binding antisense strand of the siRNA duplex generally have stronger effects than mismatches in the sense strand (10). Different regions of the targeted gene can display a different tolerance to mutations in the corresponding siRNA (9). This is probably the explanation for the differences found in effects of mismatch introduction on siRNA activity.…”

“…Generally, the target is chosen downstream of the start codon in the translated region of the transcript. siRNAs that are chemically synthesized, preferably have the general motif AA(N19)TT (N ‫ס‬A, C, G, U) and a G/C content of approximately 50% (9,10,24). When the sequence is based on this motif, both strands of the siRNA duplex can be synthesized with symmetric 3Ј TT overhangs.…”