See also:

• Gene Silencing - RNA interference (RNAi)
  

Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long RNA molecules that play a variety of roles in biology. Most notably, it is involved in the RNA interference pathway (RNAi) where the siRNA interferes with the expression of a specific gene. In addition to their role in the RNAi pathway, siRNAs also act 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 were first discovered by David Baulcombe's group in Norwich, England, as part of post-transcriptional gene silencing (PTGS) in plants[1]. Shortly thereafter, in 2001, synthetic siRNAs were then shown to be able to induce RNAi in mammalian cells by Thomas Tuschl and colleagues[2]. 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 21-nt) double-strand of RNA (dsRNA) with 2-nt 3' overhangs on either end:

Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group. This structure is the result of processing by Dicer, an enzyme that converts either long dsRNAs or hairpin RNAs into siRNAs[3]. SiRNAs can also be exogenously (artificially) introduced into cells by various transfection methods to bring about the specific knockdown of a gene of interest. Essentially any gene of which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA. This has made siRNAs an important tool for gene function and drug target validation studies in the post-genomic era.

RNAi induction using siRNAs or their biosynthetic precursors

DICER protein

Transfection of an exogenous siRNA can be problematic, since the gene knockdown effect is only transient, particularly in rapidly dividing cells. One way of overcoming this challenge is to modify the siRNA in such a way as to allow it to be expressed by an appropriate vector, e.g. a plasmid. This is done by the introduction of a loop between the two strands, thus producing a single transcript, which can be processed into a functional siRNA. Such transcription cassettes typically use an RNA polymerase III promoter (e.g. U6 or H1), which usually 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 assumed (although not known for certain) that the resulting siRNA transcript is then processed by Dicer.

Challenges: Avoiding non-specific effects

RNAi intersects with a number of other pathways, so it is not surprising that on occasion non-specific 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, since structurally related microRNAs modulate gene expression largely via incomplete complementarity with a target mRNA, unintended off-targeting may be effected by the introduction of an siRNA.

Innate Immunity

Introduction of too much siRNA can result in non-specific events due to activation of innate immune responses. Most papers suggest that this is probably due to activation of the dsRNA sensor PKR, although retinoic acid inducible Gene I (RIG-I) may also be involved. One promising method of reducing the non-specific 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 minimise non-specific effects.

Off-targeting

Off-targeting is another challenge facing siRNAs as a gene knockdown tool. Here, genes with incomplete complementarity are inadvertently downregulated by the siRNA (effectively, the siRNA acts as an 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 basepairs long stretches from position 2 onwards in the siRNA matching with 3'UTR regions in off-targeted genes.

Possible therapeutic applications and challenges

Given the ability to knockdown essentially any gene of interest, RNAi via siRNAs has generated a great deal of interest in both basic and applied biology. There is an increasing number of large-scale RNAi screens that are designed to identify the important genes in various biological pathways. As 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 a siRNA could produce a therapeutic benefit.

However, applying RNAi via siRNAs to living animals, especially humans, poses many challenges. siRNAs show different effectiveness in different cell types in a manner yet poorly understood: some cells respond well to siRNAs and show a robust knockdown, 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, demonstrate that siRNAs are well tolerated and have suitable pharmacokinetic properties. siRNAs and related RNAi induction methods therefore stand to become an important new class of drugs in the foreseeable future.

siRNAs in fiction

Ian McEwan's novel "Saturday" suggests an siRNA-based treatment for Huntington's Disease.

See also

MicroRNA (miRNA) are a related class of gene regulatory small RNAs, typically 21-23nt in length. They have been implicated in a wide range of functions such as cell growth and apoptosis, development, neuronal plasticity and remodeling, and even insulin secretion. MiRNAs have also been implicated in disease: e.g. an overabundance of miRNA has been reported in cases of Fragile X Mental Retardation, while some cancers are associated with up- and downregulation of certain miRNA genes.

Initial studies have indicated that miRNAs regulate gene expression post-transcriptionally at the level of translational inhibition at P-Bodies in the cytoplasm. However, miRNAs may also guide mRNA cleavage similar to siRNAs. This is often the case in plants where the target sites are typically highly complementary to the miRNA. While target sites in plant mRNAs can be found in the 5'UTR, open-reading frames and 3'UTR, in animals it is the 3' UTR that is the main target. This difference between plants and animals may be explained by their different modes of gene silencing.

MiRNAs are first transcribed as part of a primary microRNA (pri-miRNA). This is then processed by the Drosha with the help of Pasha/DGCR8 (=Microprocessor complex) into pre-miRNAs. The ~75nt pre-miRNA is then exported to the cytoplasm by exportin-5, where it is then diced into 21-23nt siRNA-like molecules by Dicer. In some cases, multiple miRNAs can be found on the pri-miRNA.

Check out an illustrated tutorial on miRNA.[4]

References

General background:

• Unlocking the potential of the human genome with RNA interference - a good introductory review article
• Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans - the original paper by Fire et. al. describing RNAi
• RNA interference is mediated by 21- and 22-nucleotide RNAs
• RNAi: Double-Stranded RNA Directs the ATP-Dependent Cleavage of mRNA at 21 to 23 Nucleotide Intervals
• First description of siRNAs (1999).

Non-specific effects:

• siRNA binding proteins of microglial cells: PKR is an unanticipated ligand
• Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy
• RNA interference and double-stranded-RNA-activated pathways
• Determinants of interferon-stimulated gene induction by RNAi vectors
• Activation of the interferon system by short-interfering RNAs
• 3' UTR seed matches, but not overall identity, are associated with RNAi off-targets

External links

Tools for design and quality:

• Deqor: A web-based tool for the design and quality control of siRNAs
• siDirect: a web-based online software system for designing siRNA sequences
• siSearch: a siRNA design tool using a number of popular design methods
• SpecificityServer: a tool for checking the specificity of a given siRNA
• miRacle: tool for prediction of siRNA and microRNA targets using an algorithm which incorporates RNA secondary structure

Databases:

• HuSiDa: Human siRNA Database
• siRNAdb: a database of siRNA sequences