Small interfering RNA

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, this is the RNA interference pathway (RNAi) where the siRNA interferes with the expression of a specific gene. While this article largely deals with siRNAs in the RNAi pathway, it should be noted that siRNAs play additional roles 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. Shortly thereafter, in 2001, synthetic siRNAs were then shown to able to induce RNAi in mammalian cells by Thomas Tuschl and colleagues. 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:

Schematic representation of a siRNA molecule: a ~19-21bacepair RNA core duplex that is followed by a 2 nucleotide 3' overhang on each strand. OH: 3' hydroxyl; P: 5' phosphate.

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. 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

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 inadvertantly downregulated by the siRNA (effectively, the siRNA acts as an miRNA), leading to problems in data interpretation and potentially 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-targeting genes.

The Future

(Opinion 1) 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 by turning off their activity with siRNAs or their biosynthetic precursors, therapeutic benefit can be derived directly via RNAi. Indeed, 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.

(Opinion 2) Using siRNA's/shRNA's to knockdown specific genes is certainly a valuable tool in the laboratory. However, there are a great deal of challenges when it comes to taking a laboratory technique and applying it to living animals, especially humans. Firstly, siRNA's show different effectiveness in different cell types, apparently indiscriminately - some cells respond well to siRNA's and show a robust knockdown, others show no such knockdown (even despite efficient transfection). Secondly, and most importantly, the non-specific responses of si/shRNA's are still relatively poorly understood. Until these responses can be understood and overcome, the chances of using si/shRNA's outside of the lab, e.g. as an effective new class of drug, remain slim. (As such, Ian McEwan's novel "Saturday" suggests false promise in the hope of an siRNA-based treatment for Huntington's Disease).

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