Here's a few recent abstracts concerning off-target effects (I've posted older ones before). Also a link to a PNAS article on the subject from earlier this year which is now free.
pnas.org
It would appear that heavy use of the BLAST search engine may be a culprit, and that design of siRNAs with a more enlightened approach may yet confer sufficient specificity. But then there's the abstract (immediately below) concerning how they induce complex signals in the pathways they target . . . still some side effect issues?
>>Biochem Soc Trans. 2004 Dec;32(Pt 6):952-6.
RNA interference and double-stranded-RNA-activated pathways.
Sledz CA, Williams BR.
Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, U.S.A.
RNAi (RNA interference) has become a powerful tool to determine gene function. Different methods of expressing the short ds (double-stranded) RNA intermediates required for interference in mammalian systems have been developed, including the introduction of si (short interfering) RNAs by direct transfection or driven from transfected plasmids or lentiviral vectors encoding sh (short hairpin) RNAs. Although RNAi relies upon a high degree of specificity, recent findings suggest that off-target non-specific effects can be encountered. We found that transfection of siRNAs can results in an interferon-mediated activation of the JAK/STAT (Janus kinase/signal transducer and activator of transcription) pathway and global up-regulation of interferon-stimulated genes. This effect is mediated in part by the dsRNA-dependent protein kinase PKR, as this kinase is activated by the 21 bp siRNA, and is required in response to the siRNAs. However, the transcription factor IRF3 (interferon-regulatory factor 3) is also activated by siRNA as a primary response, resulting in the stimulation of genes independent of an interferon response. In cells deficient in IRF3, this response is blunted, but can be restored by re-introduction of IRF3. Thus siRNAs induce complex signalling responses in target cells, leading to effects beyond the selective silencing of specific genes.<<
>> Biochem Biophys Res Commun. 2004 Jun 18;319(1):256-63.
Many commonly used siRNAs risk off-target activity.
Snove O Jr, Holen T.
Interagon AS, Medisinsk-teknisk senter, NO-7489, Trondheim, Norway.
Using small interfering RNA (siRNA) to induce sequence specific gene silencing is fast becoming a standard tool in functional genomics. As siRNAs in some cases tolerate mismatches with the mRNA target, knockdown of genes other than the intended target could make results difficult to interpret. In an investigation of 359 published siRNA sequences, we have found that about 75% of them have a risk of eliciting non-specific effects. A possible cause for this is the popular BLAST search engine, which is inappropriate for such short oligos as siRNAs. Furthermore, we used new special purpose hardware to do a transcriptome-wide screening of all possible siRNAs, and show that many unique siRNAs exist per target even if several mismatches are allowed. Hence, we argue that the risk of off-target effects is unnecessary and should be avoided in future siRNA design.<<
>>Biochem Biophys Res Commun. 2004 Dec 17;325(3):769-773.
Designing effective siRNAs with off-target control.
Snove O Jr, Nedland M, Fjeldstad SH, Humberset H, Birkeland OR, Grunfeld T, Saetrom P.
Interagon AS, Medisinsk teknisk senter, NO-7489 Trondheim, Norway.
Successful gene silencing by RNA interference requires a potent and specific depletion of the target mRNA. Target candidates must be chosen so that their corresponding short interfering RNAs are likely to be effective against that target and unlikely to accidentally silence other transcripts due to sequence similarity. We show that both effective and unique targets exist in mouse, fruitfly, and worm, and present a new design tool that enables users to make the trade-off between efficacy and uniqueness. The tool lists all targets with partial sequence similarity to the primary target to highlight candidates for negative controls.<<
>>Bioinformatics. 2004 Nov 25; [Epub ahead of print]
Accelerated off-target search algorithm for siRNA.
Yamada T, Morishita S.
Department of Computational Biology, Graduate School of Frontier Sciences, University of Tokyo.
MOTIVATION: Designing highly effective siRNA sequences with maximum target-specificity for mammalian RNA interference (RNAi) is one of the hottest topics in molecular biology. The relationship between siRNA sequences and RNAi activity has been studied extensively to establish rules for selecting highly effective sequences. However, there is a pressing need to compute siRNA sequences that minimize off-target silencing effects efficiently and to match any non-targeted sequences with mismatches. RESULTS: The enumeration of potential cross-hybridization candidates is non-trivial, because siRNA sequences are short, ca. 19 nt in length, and at least three mismatches with non-targets are required. With at least three mismatches, there are typically four or five contiguous matches, so that a BLAST search frequently overlooks off-target candidates. By contrast, existing accurate approaches are expensive to execute; thus we need to develop an accurate, efficient algorithm that uses seed hashing, the pigeonhole principle, and combinatorics to identify mismatch patterns. Tests show that our method can list potential cross-hybridization candidates for any siRNA sequence of selected human gene rapidly, outperforming traditional methods by orders of magnitude in terms of computational performance. AVAILABILITY: design.rnai.jp. <<
Cheers, Tuck |
