Small interfering RNA (siRNA) function as part of the post-transcriptional gene silencing (PTGS) pathway.  After their discovery in plant cells, scientists started studying the details of gene expression by using siRNAs in vitro.  Further use of RNA interference (RNAi) experiments in invertebrates proved sufficient to degrade target mRNA, and is easy to achieve with a simple transfection of long dsRNA.  However, vertebrate cells elicit a strong antiviral response upon detection of a long dsRNA.  Consequently, the cell signals apoptosis instead of knocking down the target gene, thus, giving entirely undesirable results.

siRNA, also referred to as silencing RNA or short interfering RNA, is used in a gene silencing technique to suppress gene expression.  Typically consisting of 18-22 base pairs, double stranded RNA (dsRNA), siRNA is extensively used in RNAi.  Further structure analysis reveals the siRNA ends with two overhanging nucleotides (i.e. tt) and contains phosphorylated 5′ ends and hydroxylated 3′ ends.  The effects of siRNA last 1 to 7 days when transiently transfected into cells.

Target gene expression knockdown by siRNA enables scientists to understand the complexity of gene pathways and will eventually aid in the development of effective treatments for many diseases such as autoimmune discrepancies, viral infections, cancer, Huntington’s disease and degenerative conditions. Undoubtedly, laboratory use of siRNA for therapeutic uses through the RNAi pathway is gaining traction in the clinics.

siRNA Mechanism of Action

siRNAs can be introduced into cell lines either through transfection or electroporation.  After entry into the host cell, siRNA molecules enter the RNA-induced silencing complex (RISC).  Using the antisense strand of the siRNA as the guide strand, RISC recognizes and degrades the target mRNA that is complimentary to the siRNA; thus, inhibiting its translation.  Assays such as qRT-PCR, RNA-Seq or Western Blots are later performed to quantify RNAi activity of the siRNA.  Positive and negative controls are also transfected so any observed RNAi results can be properly summarized.

siRNA Efficacy

The success of RNAi depends on the delivery of the siRNA (siRNA transfection) to the correct location for maximum expected response.  Precision of delivery to the desired tissue is extremely difficult to accomplish and is the main source of RNAi therapeutic failure.  Often, apparent gene expression down regulation is accompanied by off-target effects that can be cytotoxic and lethal.

Scientists are able to bypass some of the problems of antiviral response with the use of small siRNA created in one of two ways.  Researchers create siRNA from long dsRNA by exposing small hairpin RNAs or long dsRNA sequences to Dicer, which is an RNase III family endoribonuclease enzyme.  Dicer cuts dsRNA into smaller siRNA segments, leaving a two base overhang on each 3′ end.  Appropriately designed and synthesized synthetic siRNA mimics do not produce any significant antiviral response, are potent gene silencers and exhibit remarkable specificity to the target mRNA.

RNAi response induced by synthetic siRNAs has prompted many organizations to manufacture optimized transfection kits and pre-designed siRNAs to aid researchers.  Often, 3-5 siRNAs for every target gene are required to be tested to find a potent siRNA sequence.  Pooling siRNAs is also an effective strategy, but unfortunately it also induces substantial off-target effects.