**6. Improving specificity of siRNAs**

The specificity of siRNAs is a big issue in siRNA mediated gene silencing experiments. Exogenous siRNAs are reported to have off-target effects arising from either silencing unintended targets or toxic effects arising from their recognition by innate immune system [15].

The recognition of siRNAs by innate immune system can result from interferon response triggered by double stranded siRNA duplex or sequence dependent stimulation of toll like receptors. Avoiding some sequence motifs and a constraint related to the siRNA duplex length can effectively reduce immune response stimulation [16].

siRNAs silence unintended transcripts in mainly two ways: transcripts with near perfect complementarity are cleaved while transcripts with imperfect complementarity are translationally repressed. mRNAs other than intended targets which exhibit near perfect sequence complementarity with the siRNA are likely to be degraded by the siRNA. This kind of off-targets can be avoided by choosing targets sites that do not have a large number of consecutive base homology with any other mRNA.

siRNAs down regulate a set of transcripts with 3' UTR complementarity to the 5' portion of the corresponding siRNA guide strand. These 5' ends of the guide strand resemble the seed region of endogenous microRNA and are responsible for target recognition. Such off-targets are regulated by translational repression like miRNA target regulation. This kind of offtarget cannot be fully avoided but can be reduced by computational design.
