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siRNA

siRNA Duplexes
 

The easiest and most efficient way to achieve RNAi is to use synthetic small interfering RNAs (siRNA). Standard siRNAs are duplexes of short mixed oligonucleotides. These are typically made of 19 RNA nucleotides with symmetric 2 nucleotides (usually DNA bases) 3’ overhangs. 

Until recently, in vitro knock-down of genes was technically troublesome and time-consuming for many species. In this context, the discovery of siRNA-mediated RNA interference (RNAi) appeared as a ray of sun in a gloomy Sunday. RNAi is a new strategy of sequence-specific gene silencing at the post-transcriptional level. This phenomenon occurs when double-stranded RNA (dsRNA) are introduced into a cell. RNAi has been first successfully applied for disruption of gene transcripts in C. elegans and D. melanogaster. 

In these organisms, RNAi is induced by introducing long dsRNA complementary to the target mRNA to be degraded. dsRNA are cleaved into short 21-25 nucleotides small interfering RNAs (siRNAs), by a ribonuclease known as Dicer. The siRNAs then interact with protein components to form an RNA-induced silencing complex (RISC). Activated RISC binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA to cleave it, so promoting mRNA degradation and gene silencing. In mammalian cells and organisms, however, introducing dsRNA longer than 30 base pairs (bp) also activates a potent antiviral response (bystander activation of DNA dependent protein-kinase (PKR) that shuts down all protein expression (1). The introduction of 19 or 21 bp duplex RNA with 2 bases (preferably DNA) 3’ overhangs, namely, small interfering RNA (siRNA), into mammalian cells have been shown by various authors to effectively bypass the antiviral response and specifically silence gene expression sequence (2,3,4,5). An artificial RNAi method is therefore suitable for mammals. 

Virtually, any gene can be targeted by RNA interference! RNAi methods based on synthetic siRNA require minimal time and labor; therefore, mammalian gene knockdown by RNAi becomes more and more popular.


References
 (1) Gil, J., Esteban, M. Review: Induction of Apoptosis by the dsRNAdependent protein kinase (PKR): mechanism of action. Apoptosis, 2000, 5(2), 107-14. 
(2) ELBASHIR S.M., HARBORTH J., LENDECKEL W., YALCIN A, WEBER K., TUSCHL T., «Duplexes of 21 nucleotide RNAs mediate RNA interference in cultured mammalian cells», Nature, 2000, 411, 494-498
(3) Bosher, J.M., Labouesse, M. Review: RNA interference: genetic wand and genetic watchdog. Nat. Cell. Biol., 2000, 2(2), 31-6. 
(4) Nishikura, K. Review: A short primer on RNAi: RNA directed RNA polymerase acts as a key catalyst. Cell, 2001, 107, 415-418. 
(5) Mc Manus, M.T., Sharp, P.A. Review: Gene silencing in mammals by small interfering RNA. Nat. Rev. Genet., 2002, 3(10), 737-747.

 


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