RNA interference (RNAi) is now widely used to knockdown gene expression in a sequence-specific manner, making it a powerful tool for studying gene function (1
). The process of RNAi is mediated by double-stranded RNA (dsRNA) that contains a sequence homologous to the target mRNA. Long dsRNA introduced into the cell is cleaved by the enzyme Dicer into short-interfering RNA (siRNA) followed by incorporation into the RNA-induced silencing complex (RISC), which is responsible for target mRNA degradation (4
One of the most serious problems in RNAi is ‘off-target’ silencing effects (5
). Off-target silencing effects are caused by siRNA (introduced directly into cells, or produced in vivo
from long dsRNA) that has sequence similarities with unrelated genes. In Caenorhabditis elegans
or plants, RNAi experiments are usually performed using long dsRNAs. In these cases, there is a high risk of cross-suppression or co-suppression between closely related genes that share a highly conserved region.
To minimize the possibility of off-target effects, it is necessary to perform an off-target search to design dsRNA or siRNA that has limited sequence similarities with unrelated genes. Recently, fast and sensitive off-target search software for siRNA design has been reported (6
), but commonly used siRNA design servers are not useful in performing off-target searches for long dsRNAs. DEQOR server uses BLAST to perform off-target searches for endoribonuclease-prepared siRNAs (8
), although BLAST frequently fails to identify off-targets (6
). Therefore, we have developed a new web-based online software system, dsCheck, to provide fast and accurate off-target searches for long dsRNA sequences. The software ‘dices’ the input sequence into an siRNA cocktail and performs an exhaustive scan for each siRNA to find off-target gene candidates, simulating the biochemical process of dsRNA-mediated RNAi in vivo
. dsCheck also provides efficient design of ‘off-target minimized’ dsRNA by avoiding regions that share a considerable number of diced siRNAs with a specific off-target gene, and monitoring the total number of off-target hits. The software should be especially useful for checking whether previously designed dsRNAs have off-target gene candidates, as well as for designing target-specific dsRNA when off-target effects are suspected.