Scripps researchers have found siRNAs in human cells will localize to cytoplasm or nucleus, depending on where target is located. Also Rosetta researchers have shown off-target effects are 'miRNA-like' and cannot be dose-titrated...but, positive news is they published in a separate article that the 2'-O-methyl ribose (only at position 2) of the guide strand reduces off-target effects, without any effect on primary target.
Another article in the May 8 issue of RNA addresses this off-target side effects which can be reduced by better-designed siRNA. Just an abstract below:
<Off-target effects by siRNA can induce toxic phenotype>
Fedorov Y, Anderson EM, Birmingham A, Reynolds A, Karpilow J, Robinson K, Leake D, Marshall WS, Khvorova A.
Dharmacon Research, Lafayette, Colorado 80026, USA.
Although recent microarray studies have provided evidence of RNA interference (RNAi)-mediated off-target gene modulation, little is known about whether these changes induce observable phenotypic outcomes. Here we show that a fraction of randomly selected small inhibitory RNAs (siRNAs) can induce changes in cell viability in a target-independent fashion. The observed toxicity requires an intact RNAi pathway and can be eliminated by the addition of chemical modifications that reduce off-target effects. Furthermore, an analysis of toxic and nontoxic duplexes identifies a strong correlation between the toxicity and the presence of a 4-base-pair motif (UGGC) in the RISC-entering strand of toxic siRNA. This article provides further evidence of siRNA-induced off-target effects generating a measurable phenotype and also provides an example of how such undesirable phenotypes can be mitigated by addition of chemical modifications to the siRNA.
The Scripps work shows that siRNA can target either RNA found in the nucleus of the cell, or in the cytoplasm, depending on where the target is...the nuclear targets could expand the usefulness of this technology. The Rosetta work is not as positive, in that they show that when you target a certain mRNA with complete sequence identity siRNA, you really can't avoid inhibiting some other (non-target) mRNA when the sequence matches to a limited extent. Even if you drop the dose of siRNA, the effect persists. The modification of the nucleotide at pstn 2 reduces the off-target effects by 66%, while maintaining inhibition completely at the target mRNA. Take home message? Pay attention to non-targets, see what company has IP (chemical modification) to minimize it.