Background
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. Transfection of double stranded RNA (dsRNA) was also found to be relatively easy and efficient. 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, resulting in the death of the cell.
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 of double stranded RNA (dsRNA), siRNA is extensively used in RNAi. Structural analysis has revealed that 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. siRNA can be transfected into cells with various methods, including electroporation, cationic lipid or polymer-based transfection, or modifying the siRNA duplex to allow its uptake by the cell. Each of these methods has advantages and disadvantages, so picking the right transfection protocol is essential in maximizing the success of the given experiment.
Target gene expression knockdown by siRNA enables scientists to understand the complexity of gene pathways and their cellular phenotypes, like apoptosis, insulin signaling, cellular differentiation, and cytokinesis. siRNA screens will also eventually aid in the development of effective treatments for many diseases such as autoimmune discrepancies, viral infections, cancer, Huntington’s disease, and degenerative conditions. siRNA could silence the particular genes responsible for harmful proteins or diseases, like in cancer. However, siRNA is vulnerable to nuclease degradation, and like mentioned before, can cause unintended off-target effects if administered. Research has looked into the usage of nanocarriers as delivery systems for siRNA cancer therapy to bypass these barriers. Undoubtedly, laboratory use of siRNA for therapeutic uses through the RNAi pathway is gaining traction in clinical fields (https://pubmed.ncbi.nlm.nih.gov/31369717/).
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, 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 problems of antiviral response with the use of small siRNA created by special cleavage of dsRNA. Researchers can 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 must be tested to find a potent siRNA sequence. Pooling siRNAs is also an effective strategy, but unfortunately it also induces substantial off-target effects.