Development of a software tool and criteria evaluation for efficient design of small interfering RNA

► The developed tool predicted siRNA constructs with better thermodynamic stability and total score based on positional and other criteria. ► Off-target silencing below score 30 were observed for the best siRNA constructs for different genes. ► Immunostimulation and cytotoxicity motifs considered and penalized in the developed tool. ► Both positional and compositional criteria were observed to be important.