Structure of siRNA
Small interfering RNA (siRNA) is an oligonucleotide (short nucleotide sequence) of about 21 nucleotides (also 21 bases) in length which is used in RNA interference. The process of RNAi which is thought to take place in the cytoplasm is described.
The process begins with dsRNA (double stranded RNA) which is broken down with the help of Dicer into small fragments approximately 21nt in length. These siRNA fragments have 2 nucleotide overhangs on their 3’ ends. Argonaute2 then helps to incorporate siRNA into RISC (RNA-induced silencing complex). This RISC then binds to and cleaves mRNA, knocking out the corresponding gene.
Tuschl et al created an algorithm for designing siRNA (Elbashir, S.M., Harborth,J., Weber, K. and Tuschl, T. (2002) Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods, 26, 199-213). The basic steps of the algorithm are as follows. First pick a target region from the cDNA and avoid the 5’ or 3’ untranslated regions. Then look for a sequence 5’-AA(N19)UU in the mRNA with between 32 and 79% G/C content. The siRNA sequence should be constructed with the sense strand as 5’-(N19)TT and the antisense strand as 5’-(N’19)TT such that N’19 is the reverse complement of N19. Next use a blast search with the siRNA to try to only target one gene. It may be necessary to develop several siRNAs in order to control for specificity.
After developing potential siRNAs to target genes, it is important to be able to pick the most effective and most specific siRNA. Algorithms have been developed to explore properties of siRNAs which have highest efficacy and least amount of off target effects.