Small interfering RNA
584617
224135559
2008-07-07T13:55:39Z
GrahamHardy
2956291
/* siRNAs in fiction */ italicisation
[[Image:SiRNAvitro.gif|thumb|300px|right|Mediating RNA interference in cultured mammalian cells.]]
'''Small interfering RNA''' ('''siRNA'''), sometimes known as '''short interfering RNA''' or '''silencing RNA''', are a class of 20-25 [[nucleotide]]-long [[RNA#Double-stranded RNA|double-stranded RNA]] [[molecule]]s that play a variety of roles in biology. Most notably, siRNA is involved in the [[RNA interference]] (RNAi) pathway where the siRNA interferes with the [[gene expression|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 anti[[viral]] 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-[[Transcription (genetics)|transcriptional]] [[gene silencing]] (PTGS) in plants. The group published their findings in ''[[Science (journal)|Science]]'' in a paper titled "A species of small antisense RNA in posttranscriptional gene silencing in plants".<ref>{{cite journal |author=Hamilton A, Baulcombe D |title=A species of small antisense RNA in posttranscriptional gene silencing in plants |journal=Science |volume=286 |issue=5441 |pages=950–2 |year=1999 |pmid=10542148 |doi=10.1126/science.286.5441.950}}</ref> Shortly thereafter, in [[2001]], synthetic siRNAs were then shown to be able to induce RNAi in mammalian cells by [[Thomas Tuschl]] and colleagues in a paper published in ''[[Nature (journal)|Nature]]''.<ref>{{cite journal |author=Elbashir S, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T |title=Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells |journal=Nature |volume=411 |issue=6836 |pages=494–8 |year=2001 |pmid=11373684 |doi=10.1038/35078107}}</ref> 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-[[nucleotide|nt]]) double-strand of [[RNA]] (dsRNA) with 2-[[nucleotide|nt]] 3' overhangs on either end:
[[Image:SiRNA structure2.jpg]]
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 [[small hairpin RNA]]s into siRNAs.<ref>{{cite journal |author=Bernstein E, Caudy A, Hammond S, Hannon G |title=Role for a bidentate ribonuclease in the initiation step of RNA interference |journal=Nature |volume=409 |issue=6818 |pages=363–6 |year=2001 |pmid=11201747 |doi=10.1038/35053110}}</ref> 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==
[[Image:2ffl-by-domain.png|thumb|Dicer protein colored by [[protein domain]].]]
[[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 [[Gene expression|expressed]] by an appropriate [[Vector (biology)|vector]], e.g. a [[plasmid]]. This is done by the introduction of a loop between the two strands, thus producing a single [[Transcription (genetics)|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 [[Splicing (genetics)|gene splicing]]; H1 is the [[Ribonuclease|RNase]] component of human RNase P). It is assumed (although not known for certain) that the resulting siRNA transcript is then processed by [[Dicer]].
==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 ([[saRNA]]s). It is currently unknown if RNAa is conserved in other organisms.<ref name= LiLC>{{cite book |chapterurl=http://www.horizonpress.com/rnareg|author= Li LC|year=2008|chapter=Small RNA-Mediated Gene Activation|title=RNA and the Regulation of Gene Expression: A Hidden Layer of Complexity|publisher=Caister Academic Press|id=[http://www.horizonpress.com/rnareg ISBN 978-1-904455-25-7]}}</ref>
==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 [[miRNA|microRNAs]] modulate gene expression largely via incomplete complementarity with a target [[mRNA]], the introduction of an siRNA may cause unintended off-targeting.
===Innate Immunity===
Introduction of too much siRNA can result in non-specific events due to activation of innate immune responses. Most evidence to date 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. The induction of cytokines via Toll-like receptor 7 (TLR7) has also been described. One promising method of reducing the non-specific effects is to convert the siRNA into a [[miRNA|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 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.<ref>{{cite journal |author=Birmingham A, Anderson E, Reynolds A, Ilsley-Tyree D, Leake D, Fedorov Y, Baskerville S, Maksimova E, Robinson K, Karpilow J, Marshall W, Khvorova A |title=3' UTR seed matches, but not overall identity, are associated with RNAi off-targets |journal=Nat Methods |volume=3 |issue=3 |pages=199–204 |year=2006 |doi= 10.1038/nmeth854 |pmid=16489337}}</ref>
==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 are 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. Experimentally 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, while 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.<ref>{{cite news | url=http://www.sfgate.com/cgi-bin/article.cgi?f=/c/a/2006/08/11/BUGPNKFU6T1.DTL | publisher=San Francisco Chronicle | author=Tansey B | title=Macular degeneration treatment interferes with RNA messages | date=11 August 2006}}</ref> siRNAs and related RNAi induction methods therefore stand to become an important new class of drugs in the foreseeable future.{{Fact|date=April 2008}}
== siRNAs in fiction ==
[[Ian McEwan]]'s novel ''[[Saturday (novel)|Saturday]]'' suggests an siRNA-based treatment for [[Huntington's disease]].
==See also==
[[miRNA|MicroRNA (miRNA)]] are a related class of gene regulatory small RNAs, typically 21-23nt in length. They typically differ from siRNA because they are processed from single stranded RNA precursors and show only partial [[complementarity]] to mRNA targets. 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 syndrome|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 in a manner 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 (protein)|Pasha]]/DGCR8 (=Microprocessor complex) into pre-miRNAs. The ~75nt pre-miRNA is then exported to the cytoplasm by [[Exportin|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.
==References==
{{reflist}}
===General background===
*{{cite journal |author=Hannon G, Rossi J |title=Unlocking the potential of the human genome with RNA interference |journal=Nature |volume=431 |issue=7006 |pages=371–8 |year=2004 |url=http://www.nature.com/nature/journal/v431/n7006/full/nature02870.html |doi =10.1038/nature02870 |pmid=15372045}}
*{{cite journal |author=Fire A, Xu S, Montgomery M, Kostas S, Driver S, Mello C |title=Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans |journal=Nature |volume=391 |issue=6669 |pages=806–11 |year=1998 |pmid=9486653 |doi=10.1038/35888}} The original paper by Fire et al. describing RNAi
*{{cite journal |author=Elbashir S, Lendeckel W, Tuschl T |title=RNA interference is mediated by 21- and 22-nucleotide RNAs |journal=Genes Dev |volume=15 |issue=2 |pages=188–200 |year=2001 |url=http://www.genesdev.org/cgi/content/full/15/2/188 |pmid=11157775 |doi=10.1101/gad.862301}}
*{{cite journal |author=Zamore P, Tuschl T, Sharp P, Bartel D |title=RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals |journal=Cell |volume=101 |issue=1 |pages=25–33 |year=2000 |url=http://www.cell.com/content/article/abstract?uid=PIIS0092867400806200 |pmid=10778853 |doi=10.1016/S0092-8674(00)80620-0}}
*{{cite journal |author=Hamilton A, Baulcombe D |title=A species of small antisense RNA in posttranscriptional gene silencing in plants |journal=Science |volume=286 |issue=5441 |pages=950–2 |year=1999 |pmid=10542148 |doi=10.1126/science.286.5441.950}} First description of siRNAs.
*{{cite journal | author = Gartel, A.L. | coauthors = Kandel, E.S. | year = 2006 | title = RNA interference in cancer | journal = Biomolecular Engineering | volume = 23 | issue = 1 | pages = 17–34 | doi = 10.1016/j.bioeng.2006.01.002}}
===Non-specific effects===
*{{cite journal |author=Zhang Z, Weinschenk T, Guo K, Schluesener H |title=siRNA binding proteins of microglial cells: PKR is an unanticipated ligand |journal=J Cell Biochem |volume=97 |issue=6 |pages=1217–29 |year=2006 |url=http://www3.interscience.wiley.com/cgi-bin/abstract/112161228/ABSTRACT?CRETRY=1&SRETRY=0 |pmid=16315288 |doi=10.1002/jcb.20716}}
*{{cite journal |author=Kim D, Behlke M, Rose S, Chang M, Choi S, Rossi J |title=Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy |journal=Nat Biotechnol |volume=23 |issue=2 |pages=222–6 |year=2005 |doi= 10.1038/nbt1051 |pmid=15619617}}
*{{cite journal |author=Sledz C, Williams B |title=RNA interference and double-stranded-RNA-activated pathways |journal=Biochem Soc Trans |volume=32 |issue=Pt 6 |pages=952–6 |year=2004 |url=http://www.biochemsoctrans.org/bst/032/0952/bst0320952.htm |pmid=15506933 |doi=10.1042/BST0320952}}
*{{cite journal |author=Pebernard S, Iggo R |title=Determinants of interferon-stimulated gene induction by RNAi vectors |journal=Differentiation |volume=72 |issue=2-3 |pages=103–11 |year=2004 |doi= 10.1111/j.1432-0436.2004.07202001.x |pmid=15066190}}
*{{cite journal |author=Sledz C, Holko M, de Veer M, Silverman R, Williams B |title=Activation of the interferon system by short-interfering RNAs |journal=Nat Cell Biol |volume=5 |issue=9 |pages=834–9 |year=2003 |doi= 10.1038/ncb1038 |pmid=12942087}}
==External links==
Tools for design and quality:
*[http://jura.wi.mit.edu/bioc/siRNAext/ siRNA at Whitehead: A web-based siRNA Selection Program]
*[http://cluster-1.mpi-cbg.de/Deqor/deqor.html Deqor: A web-based tool for the design and quality control of siRNAs]
*[http://genomics.jp/sidirect siDirect: a web-based online software system for designing siRNA sequences]
*[http://sisearch.cgb.ki.se siSearch: a siRNA design tool using a number of popular design methods]
*[http://sonnhammer.cgb.ki.se/SpecificityServer/ SpecificityServer: a tool for checking the specificity of a given siRNA]
*[http://sourceforge.net/projects/seq2svm/ SEQ2SVM: software for designing siRNA seqeunces]
*[http://miracle.igib.res.in/miracle/ miRacle: tool for prediction of siRNA and microRNA targets using an algorithm which incorporates RNA secondary structure]
*[http://www.sirnawizard.com/ InvivoGen's siRNA Wizard: A web-based tool for designing siRNA and scrambled siRNA sequences]
Databases:
*[http://www.human-siRNA-database.net HuSiDa: Human siRNA Database]
*[http://siRNA.cgb.ki.se siRNAdb: a database of siRNA sequences]
siRNA focused Companies:
*[http://www.alnylam.com Alnylam Pharmaceuticals]
*[http://www.anadyspharma.com Anadys Pharmaceuticals]
*[http://www.armagen.com ArmaGen Technologies,Inc]
*[http://www.bdbiosciences.com BD Biosciences]
*[http://www.benitec.com Benitec]
*[http://www.bionomics.com.au Bionomics]
*[http://www.biospring.de BioSpring]
*[http://www.calandopharma.com Calando Pharmaceuticals]
*[http://www.celera.com Celera Genomics]
*[http://www.cenix-bioscience.com Cenix BioScience]
*[http://www.ceptyr.com Ceptyr]
*[http://www.ingenium-ag.com Ingenium Pharmaceuticals]
*[http://www.idtdna.com/Scitools/Applications/Predesign/ Integrated DNA Technologies, Inc.]
*[http://www.invivogen.com InvivoGen]
*[http://www.isispharm.com Isis Pharmaceuticals]
*[http://www.nucleonicsinc.com Nucleonics Inc]
*[http://www.pfizer.com Pfizer Inc]
*[http://www.rnai.co.jp RNAi Co.Ltd]
*[http://www.roche-kulmbach.de/portal/eipf/de/RocheKulmbach/RocheKulmbach Roche Kulmbach GmbH]
*[http://www.sigmaaldrich.com Sigma-Aldrich]
*[http://www.sirna.com Sirna Therapeutics, Inc.]
{{Nucleic acids}}
[[Category:RNA]]
[[Category:RNA interference]]
[[ca:SiRNA]]
[[de:Kleine RNA]]
[[es:ARN interferente pequeño]]
[[fr:ARN interférent]]
[[it:Short interfering RNA]]
[[lt:SiRNR]]
[[nl:SiRNA]]
[[pl:SiRNA]]
[[ur:بسیط متداخل ارنا]]
[[zh:小干擾RNA]]