ABSTRACT
Common features of RNA silencing are production of small (21-23 nucleotide) double-stranded RNAs (Ryther et al. 2005). These short double-stranded RNAs are recognized by the endonuclease, Dicer, and cleaved into two fragments called siRNA. The antisense strand of the two strands becomes associated with a complex of proteins, designated as the RNA-Induced Silencing Complex (RISC), which targets mRNA. Next, Argonaute 2 (Ago2), a RNA endonuclease within the complex cleaves the target mRNA and leads to its degradation, shutting down protein expression. The introduction of a 18-26 base pairs siRNA triggers gene silencing in cells and is expected to be more selective and more effective than longer fragments especially in tumors that have low Dicer levels. For therapeutic applications, siRNA may be preferable over long fragments such as shRNA because cells with low Dicer levels can have impaired gene silencing with shRNA, but not with siRNA. In addition, longer RNA fragments may result in greater potential for toxic effects, especially in the liver (Brummelkamp et al. 2002; Ozpolat et al. 2009). Careful selection of siRNA sequences to avoid off-target effects is an important issue and can be minimized or eliminated by avoiding certain sequence motifs, and validation of the siRNA sequences. Exclusion of partially complementary sequences and certain motifs that induce immune response and the use of the minimum effective dose of siRNA can further reduce toxicities and eliminate off-target effects. One of the most important advantages of using siRNA is that compared to antisense oligos, siRNA is 10 to 100-fold more potent for gene silencing (Ozpolat et al. 2009).
