Short interfering RNA: differenze tra le versioni

Versione delle 21:43, 2 ago 2006

Versione delle 22:50, 2 ago 2006

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Uno '''small interfering RNA''' (o '''short interfering RNA''', traducibile come ''breve RNA interferente''), comunemente conosciuto come '''siRNA''', è una molecola di [[RNA]] lunga tra i 20 ed i 25 [[nucleotide|nucleotidi]] in grado di svolgere numerosi ruoli biologici.

<!--Most notably, this is 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[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10542148&query_hl=1&itool=pubmed_DocSum]. Shortly thereafter, in 2001, synthetic siRNAs were then shown to able to induce RNAi in mammalian cells by Thomas Tuschl and colleagues[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11373684&query_hl=3&itool=pubmed_docsum]. This discovery led to a surge in interest in harnessing RNAi for biomedical research and drug development.

Più precisamente, gli siRNA sono coinvolti anzitutto nel ''pathway'' della [[RNA interference]], che conduce alla inibizione dell'[[espressione genica|espressione]] di singoli [[gene|geni]]. Hanno un ruolo importante anche in altri processi legati alla RNAi, come alcuni meccanismi antivirali o nel modellamento della struttura della [[cromatina]].

==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:

Il meccanismo esatto di tutti questi processi sta avendo solo in questi ultimi anni una caratterizzazione completa ed esaustiva. Gli siRNA furono inizialmente individuati dal gruppo di ricerca di David Baulcombe a [[Norwich]], come attori principali nel cosiddetto ''silenziamento genico post-trascrizionale'' nelle piante <ref> {{en}} [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10542148&query_hl=1&itool=pubmed_DocSum Hamilton AJ, Baulcombe DC, A species of small antisense RNA in posttranscriptional gene silencing in plants, Science. 1999 Oct 29;286(5441):950-2]</ref>. Successivamente, nel [[2001]], siRNA sintetici sono stati utilizzati per indurre RNAi in cellule di mammifero da parte del gruppo di ricerca di Thomas Tuschl<ref> {{en}} [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11373684&query_hl=3&itool=pubmed_docsum Elbashir SM ''et al'', Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells, Nature. 2001 May 24;411(6836):494-8]</ref>. Queste scoperte hanno portato ad un interesse crescente per la RNAi e le sue possibili applicazioni in ricerca ed in clinica.

==Struttura==

<!--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 hairpin RNAs into siRNAs[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=11201747&query_hl=6&itool=pubmed_docsum]. 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.-->

==Induzione di RNAi attraverso siRNA o suoi precursori==

==RNAi induction using siRNAs or their biosynthetic precursors==

<!--[[Image:DICER.jpg|thumb|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 [[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]].-->

==Specificità degli siRNA==

==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]], unintended off-targeting may be effected by the introduction of an siRNA.

===InnateImmunità Immunityinnata===

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 [[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 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 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.-->

==TheProspettive Futurefuture==

<!--(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.

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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 (novel)|Saturday]]" suggests false promise in the hope of an siRNA-based treatment for [[Huntington's Disease]]).

==SeeVoci alsocorrelate==

[[miRNA|MicroRNA (miRNA)]] are small ribonucleaic acid chains, ~22nt long. They have been implicated in cell growth and apoptosis, embrionic development, neuronal plasticity and remodeling, and even insulin secretion. An overabundance of miRNA has been reported in cases of Fragile X Mental Retardation while some cancers have been reported to have downregulated miRNA genes.

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Check out an illustrated tutorial on miRNA.[http://www.ambion.com/main/explorations/mirna.html]

==ReferencesBibliografia==

<references/>

<!--General background:

*[http://www.nature.com/nature/journal/v431/n7006/full/nature02870.html Unlocking the potential of the human genome with RNA interference - a good introductory review article]

*[http://www.nature.com/nature/journal/v391/n6669/abs/391806a0_fs.html Potent and specific genetic interference by double-stranded RNA in ''Caenorhabditis elegans'' - the original paper by Fire et. al. describing RNAi]

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*[http://www.blackwell-synergy.com/doi/abs/10.1111/j.1432-0436.2004.07202001.x Determinants of interferon-stimulated gene induction by RNAi vectors]

*[http://www.nature.com/ncb/journal/v5/n9/abs/ncb1038.html Activation of the interferon system by short-interfering RNAs]

*[http://www.nature.com/nmeth/journal/v3/n3/abs/nmeth854.html 3' UTR seed matches, but not overall identity, are associated with RNAi off-targets]-->

==External links==

*[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10542148&query_hl=3 First description of siRNA's (1999).]

*[http://design.rnai.jp/ siDirect: a web-based online software system for computing siRNA sequences]

*[http://nar.oupjournals.org/cgi/content/full/32/suppl_2/W124 Paper describing siDirect]

*[http://nar.oupjournals.org/cgi/content/full/32/3/936 Paper describing effectivity of siDirect]

*[http://www.human-siRNA-database.net HuSiDa: Human siRNA Database]

*[http://siRNA.cgb.ki.se siRNAdb: a database of 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://miracle.igib.res.in/miracle/ miRacle: tool fro prediction of siRNA and microRNA targets using an algorithm which incorporates RNA secondary structure]

==Collegamenti esterni==

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* {{en}} [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=10542148&query_hl=3 FirstPrima descriptiondescrizione ofdegli siRNA's (1999).]

* {{en}} [http://design.rnai.jp/ ''siDirect'': un software ''on-line'' per individuare sequenze di siRNA]

* {{en}} [http://nar.oupjournals.org/cgi/content/full/32/suppl_2/W124 PaperArticolo di presentazione describingdi ''siDirect'']

* {{en}} [http://nar.oupjournals.org/cgi/content/full/32/3/936 PaperArticolo describingsulla effectivityefficacia ofdi ''siDirect'']

* {{en}} [http://www.human-siRNA-database.net ''HuSiDa'': Human siRNA Database]

* {{en}} [http://siRNA.cgb.ki.se ''siRNAdb'': aun database ofdelle siRNAsequenze sequencesdi siRNA]

* {{en}} [http://sisearch.cgb.ki.se ''siSearch'': uno strumento per il ''design'' delle sequenze di siRNA]

* {{en}} [http://sonnhammer.cgb.ki.se/SpecificityServer/ ''SpecificityServer'': auno toolstrumento forper checkingtestare thela specificityspecificità ofdi aun givendeterminato siRNA]

* {{en}} [http://miracle.igib.res.in/miracle/ ''miRacle'': strumento per la predizione di bersagli molecolari per siRNA e microRNA attraverso un algoritmo che analizza le strutture secondarie dell'RNA]

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