3Si: a computer program for the optimal design of short interfering RNA (siRNA) for gene silencing

siRNA is a powerful technique for gene silencing. However, it is still not clear how to accurately determine an effective gene targeting region for siRNA. It is also controversial if the targeted mRNA secondary structure would have a significant impact on the gene silencing by the siRNA. Understanding the relationship between the secondary structure and gene silencing by siRNA could help in high throughput function-based genetic screens and for development of therapeutic tools. Our hypothesis is that the selection of a single-stranded region as the target region should increase the efficiency of siRNA silencing. For the first time, a formula and a computer program were developed to select the single-stranded regions for siRNA silencing. We statistically analyzed quantitative relationships between gene silencing by the siRNA and ten parameters from published siRNA sequences. The four nonlinear significant parameters for designing siRNA are the RNA single-stranded index (SSi), a new parameter that identifies the target position and its related secondary structures, the GC content, ΔG free energy of the siRNA, and the 5´GC content of the first four bases of the antisense siRNA. The nonlinear complexity suggested that one should not use simple criteria such as a GC content of around 30 to 50% or the absence of the sequence pattern GGG for designing siRNA. Our results indicate that the targeted mRNA secondary structure is a statistically significant determinant in gene silencing and should be used to optimize RNAi technology.