siRNA Pathway
Explore and order pathway-specific siRNAs, real-time PCR assays, and expression vectors. View pathway information and literature references for your pathway.
  • Click on your proteins of interest in the pathway image or review below
  • Select your genes of interest and click "add selection"
  • When you have finished your gene selection, click "Find Products" to find assays, arrays, or create custom products
Download Image Terms of Use Download PPT
Pathway Navigator
siRNA Pathway
Small interfering RNAs (siRNAs) are 21-23nt dsRNA (double-stranded RNA) molecules that facilitate potent and sequence-specific gene suppression via the mechanism of RNAi (RNA interference). When introduced into cultured mammalian cells, siRNAs facilitate the degradation of mRNA sequences to which they are homologous, thereby silencing the encoding gene. The basic mechanism behind RNAi is the breaking of a dsRNA matching a specific gene sequence into short pieces of siRNA. These siRNAs with symmetric 2-3nt 3 overhangs and 5-phosphate and 3-hydroxyl groups post-transcriptionally silences a gene through mRNA inhibition or degradation. Interference of gene expression by siRNA is now recognized as a naturally occurring biological strategy for silencing alleles during development in plants, invertebrates, and vertebrates (Ref.1). It is believed that the small size of the siRNAs, as compared with dsDNA, prevents activation of the dsRNA-inducible interferon system in mammalian cells. This avoids the non-specific phenotypes normally produced by dsRNA (>30 base pairs).

The mechanism for mRNA silencing by siRNA involves the chopping of long dsRNA into smaller pieces of a defined length corresponding to both sense and antisense strands of the target gene by the Rnase-III (Ribonuclease-III) family member, Dicer in an ATP-dependent reaction. Dicer chops dsRNA into two classes of smaller RNAs-miRNAs (microRNAs) and siRNAs-that are around 21-23nts in length. Dicer delivers these siRNAs to a group of proteins called the RISC (RNA-Inducing Silencing Complex), which uses the antisense strand of the siRNA to bind to and degrade the corresponding mRNA, resulting in gene silencing. There is a strict requirement for the siRNA to be 5 phosphorylated to enter into RISC, and siRNAs that lack a 5 phosphate are rapidly phosphorylated by an endogenous kinase. Although the uptake of siRNAs by RISC is independent of ATP, the unwinding of the siRNA duplex requires ATP. After unwinding the single-stranded antisense strand guides RISC to mRNA that has a complementary sequence and results in the endonucleolytic cleavage of the target mRNA. The target mRNA is cleaved at a single site in the centre of the duplex region between the guide siRNA and the target mRNA, 10nt from the 5 end of the siRNA for degradation (Ref.2). Chemically synthesized siRNAs that are introduced into cells bypass the dicing step and are incorporated into the RISC for targeted mRNA degradation. Perfect duplex hairpin RNA can be cleaved by Dicer into siRNAs. Designer siRNAs are also transcribed as stem-loop RNA precursors, which are encoded by an expression vector. However, after cleavage by Dicer, these appear to be treated exactly like siRNAs, leading to the specific degradation of homologous mRNAs.

siRNAs are associated with silencing triggered by transgenes, microinjected RNA, viruses, and transposons, and hence can be considered intermediaries in host defense pathways against foreign nucleic acids. The convenience of producing and using siRNAs has made them important tools for studying gene function. They can be synthesized in vitro and then introduced into cells directly leading to potent target gene suppression. However, one drawback of using synthetic siRNAs is that they are unable to produce stable gene knockdowns. Several groups have developed vector-based siRNA expression systems that can induce RNAi in living cells (Ref.3). Vectors that express siRNAs within mammalian cells typically use an RNA Polymerase-III promoter to drive expression of a short hairpin RNA that mimics the structure of a siRNA. The insert that encodes this hairpin is designed to have two inverted repeats separated by a short spacer sequence. One inverted repeat is complementary to the mRNA to which the siRNA is targeted. A string of thymidines added to the 3 end serves as a Pol-III transcription termination site. Once inside the cell, the vector constitutively expresses the hairpin RNA, which induces silencing of the target gene. The use of specially designed vector constructs for inducing RNA interference has numerous advantages over oligonucleotide-based techniques (Ref.4). The most significant advantage is stability. Promoter regions in the vector ensure that siRNA transcripts are constantly expressed, maintaining long-term mRNA inhibition, whereas cells which are transfected with exogenous synthetic siRNAs typically recover from mRNA suppression within seven days or ten rounds of cell division. And by using expression constructs instead of oligo injection, it is possible to perform multi-generational studies of gene knockdown because the vector can become a permanent fixture in the cell line.