Small interfering RNA

See also RNA interference

Mediating RNA interference in cultured mammalian cells.

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 base pairs in length. siRNA plays many roles, but it is most notable in the RNA interference (RNAi) pathway, where it interferes with the expression of specific genes with complementary nucleotide sequences. siRNA functions by causing mRNA to be broken down after transcription, resulting in no translation. siRNA also acts in RNAi-related pathways, e.g., as an antiviral mechanism or in shaping the chromatin structure of a genome. The complexity of these pathways is only now being elucidated.

siRNAs and their role in post-transcriptional gene silencing (PTGS) in plants were first discovered by David Baulcombe's group at the Sainsbury Laboratory in Norwich, England and reported in Science in 1999. Thomas Tuschl and colleagues soon reported in Nature that synthetic siRNAs could induce RNAi in mammalian cells. This discovery led to a surge in interest in harnessing RNAi for biomedical research and drug development.

Structure

siRNAs have a well-defined structure: a short (usually 20 to 24-bp) double-stranded RNA (dsRNA) with phosphorylated 5' ends and hydroxylated 3' ends with two overhanging nucleotides. The Dicer enzyme catalyzes production of siRNAs from long dsRNAs and small hairpin RNAs. siRNAs can also be introduced into cells by transfection. Since in principle any gene can be knocked down by a synthetic siRNA with a complementary sequence, siRNAs are an important tool for validating gene function and drug targeting in the post-genomic era.

RNAi induction using siRNAs or their biosynthetic precursors

Dicer protein colored by protein domain.

Gene knockdown by transfection of exogenous siRNA is often unsatisfactory because the effect is only transient, especially in rapidly dividing cells. This may be overcome by creating an expression vector for the siRNA. The siRNA sequence is modified to introduce a short loop between the two strands. The resulting transcript is a short hairpin RNA (shRNA), which can be processed into a functional siRNA by Dicer in its usual fashion.. Typical transcription cassettes use an RNA polymerase III promoter (e.g., U6 or H1) to direct the transcription of small nuclear RNAs (snRNAs) (U6 is involved in gene splicing; H1 is the RNase component of human RNase P). It is theorized that the resulting siRNA transcript is then processed by Dicer.

The gene knockdown efficiency can also be improved by using cell squeezing.

The activity of siRNAs in RNAi is largely dependent on its binding ability to the RNA-induced silencing complex (RISC). Binding of the duplex siRNA to RISC is followed by unwinding and cleavage of the sense strand with endonucleases. The remaining anti-sense strand-RISC complex can then bind to target mRNAs for initiating transcriptional silencing.

RNA activation

It has recently been found that dsRNA can also activate gene expression, a mechanism that has been termed "small RNA-induced gene activation" or RNAa. It has been shown that dsRNAs targeting gene promoters induce potent transcriptional activation of associated genes. RNAa was demonstrated in human cells using synthetic dsRNAs, termed "small activating RNAs" (saRNAs). It is currently not known whether RNAa is conserved in other organisms.

Challenges: avoiding nonspecific effects

Because RNAi intersects with a number of other pathways, it is not surprising that on occasion nonspecific effects are triggered by the experimental introduction of an siRNA. When a mammalian cell encounters a double-stranded RNA such as an siRNA, it may mistake it as a viral by-product and mount an immune response. Furthermore, because structurally related microRNAs modulate gene expression largely via incomplete complementarity base pair interactions with a target mRNA, the introduction of an siRNA may cause unintended off-targeting.

Innate immunity

Introduction of too much siRNA can result in nonspecific events due to activation of innate immune responses. Most evidence to date suggests that this is probably due to activation of the dsRNA sensor PKR, although retinoic acid-inducible gene I (RIG-I) may also be involved. The induction of cytokines via toll-like receptor 7 (TLR7) has also been described. One promising method of reducing the nonspecific effects is to convert the siRNA into a microRNA. MicroRNAs occur naturally, and by harnessing this endogenous pathway it should be possible to achieve similar gene knockdown at comparatively low concentrations of resulting siRNAs. This should minimize nonspecific effects.

Off-targeting

Off-targeting is another challenge to the use of siRNAs as a gene knockdown tool. Here, genes with incomplete complementarity are inadvertently downregulated by the siRNA (in effect, the siRNA acts as a miRNA), leading to problems in data interpretation and potential toxicity. This, however, can be partly addressed by designing appropriate control experiments, and siRNA design algorithms are currently being developed to produce siRNAs free from off-targeting. Genome-wide expression analysis, e.g., by microarray technology, can then be used to verify this and further refine the algorithms. A 2006 paper from the laboratory of Dr. Khvorova implicates 6- or 7-basepair-long stretches from position 2 onward in the siRNA matching with 3'UTR regions in off-targeted genes.

Therapeutic applications and challenges

Given the ability to knock down, in essence, any gene of interest, RNAi via siRNAs has generated a great deal of interest in both basic and applied biology. There are an increasing number of large-scale RNAi screens that are designed to identify the important genes in various biological pathways. Because disease processes also depend on the activity of multiple genes, it is expected that in some situations turning off the activity of a gene with an siRNA could produce a therapeutic benefit.

However, applying RNAi via siRNAs to living animals, especially humans, poses many challenges. Under experiments, siRNAs show different effectiveness in different cell types in a manner as yet poorly understood: Some cells respond well to siRNAs and show a robust knockdown, whereas others show no such knockdown (even despite efficient transfection).

Phase I results of the first two therapeutic RNAi trials (indicated for age-related macular degeneration, aka AMD) reported at the end of 2005 that siRNAs are well tolerated and have suitable pharmacokinetic properties.

In a phase 1 clinical trial, 41 patients with advanced cancer metastasised to liver were administered with RNAi delivered through lipid nanoparticles. The RNAi targeted two genes encoding key proteins in the growth of the cancer cells, vascular endothelial growth factor, (VEGF), and kinesin spindle protein (KSP). The results showed clinical benefits, with the cancer either stabilized after six months or regression of metastasis in some of the patients. Pharmacodynamics analysis of biopsy samples from the patients revealed the presence of the RNAi constructs in the samples, proving that the molecules reached the intended target.

Proof of concept trials have indicated that Ebola-targeted siRNAs may be effective as post-exposure prophylaxis in humans, with 100% of non-human primates surviving a lethal dose of Zaire Ebolavirus, the most lethal strain.

See also

• gene knockdown
• gene silencing
• Oligonucleotide synthesis
• EsiRNA
• NatsiRNA
• MicroRNA
• Viroid
• RNA interference
• CRISPR
• Dharmacon
• Persomics

External links

• RNAiAtlas: a database of RNAi Libraries and their target analysis
• An animation of the mechanism of siRNA by Nature Reviews Genetics can be found HERE
• DesiRM: Designing of Complementary and Mismatch siRNAs for Silencing a Gene .