RNA interference has come a long way since its early beginnings in 1990s. First observations of it were reported in the early 1990s where various groups reported a “quelling” of gene expression when homologous RNA sequences were introduced. Researchers Craig Mello and Andrew Fire were the first to establish in 1998 that double-stranded RNA was capable of silencing the expression of genes carrying the complementary sequence. Double-stranded-ness was a particular necessity as injection of only sense or antisense RNA strands failed to achieve the same effect. For this they won the Nobel Prize in Physiology/Medicine in 2006.
Since then, much work has gone into elucidating the mechanism of how RNA interference works. It is now known that double-stranded RNA is cleaved into smaller lengths of ~21-25 ribonucleotides by an enzyme called Dicer. These small intermediates carry out the gene knockdown effect, specifically the antisense strand would bind to a complementary RNA sequence from an endogenous target gene, recruiting it to a protein complex called RISC or RNA-induced silencing complex. This complex contains Argonaute proteins which then carry out the “slicing” of the target gene, hence silencing its expression.
The scientific world rejoiced as we now had a cool and relatively easy way of knocking down gene expression. It took a while for us to realise that the short interfering RNA or siRNA approach to knocking down genes came with a major drawback of having rather broad off-target effects. These effects occur as siRNAs recognize their targets via a seed region of 2-8 nucleotides which may occur on thousands of mRNAs. siRNAs also look a lot like naturally occurring short RNAs called microRNAs which do not need total complementarity to the target sequence to knock down a gene. Hence varying degrees of knockdown may occur on several genes that give rise to a positively mixed phenotype! Another thing that happens when you introduce double-stranded RNA is one can stimulate an immune response which may also interfere with the overall phenotype. This did not stop people from performing siRNA screens, or charging money for performing siRNA screens though. Millions if not more dollars must have been spent on screens which yielded data that could not be validated.
Thankfully, several methods are now being used to counter the off-target problem. Many rely on chemical modification (e.g. 2′-O-methylation) of the siRNAs that presumably reduce binding to seed sequences of off-target mRNA. However this does not completely eliminate false positives. A particular method proven quite successful is the administration of a pool of several siRNAs that target the same gene using different seed sequences. The use of a pool (of 30 siRNAs for example) allows one to reduce the concentration of each individual siRNA, minimizing off-target effects. This method of course heavily relies on bioinformatics to design appropriate siRNAs and may be limited by gene length. Because of the well-defined characteristics of each pool, one can not only target specific gene paralogs but also target several genes from the same family based on shared sequences. I have to admit I work for the founders of this technology (www.sitoolsbiotech.com) so perhaps I am biased 🙂 But it is truly cool how the use of bioinformatics is incorporated to control the complexity of these siPools.
I believe this is just the start and more advances will be seen over the next few years that would significantly improve siRNA screening. The increasing role of RNA over DNA is also taking the world by storm and I am sure many more discoveries lie ahead.