BIOTECHNOLOGY 45
RNAi and DsiRNA: pathway, mechanism and design
Since its discovery, scientists have co-opted the RNAi mechanism as an experimental tool for studying the effects of gene silencing both in vitro and in vivo. Jaime Sabel and Hans Packer report.
Depuis sa découverte, les scientifiques se sont approprié le mécanisme ARNi en tant qu’outil expérimental pour étudier les effets d’extinction des gènes à la fois in vitro et in vivo. Selon Jaime Sabel et Hans Packer.
Seit seiner Entdeckung nutzen Wissenschaftler den RNAi- Mechanismus sowohl in vitro als auch in vivo als Versuchswerkzeug für die Erforschung der Auswirkungen des Gen-Silencing. Jaime Sabel und Hans Packer berichten.
R
NA interference (RNAi) is a conserved pathway found in most eukaryotes where
short, dsRNAs suppress expression of genes with complementary sequences1-2
.
In mammalian cells, RNAi occurs when long dsRNA molecules are processed by Dicer into siRNAs, which are 21 bp long dsRNAs, with a central 19 bp duplex, and characteristic 2-base, 3’ overhangs. Dicer processing occurs in a multi- protein complex that includes the TAR RNA-binding protein (TRBP). Te nascent siRNA associates with Dicer, TRBP, and Argonaute 2 (Ago2) to form RISC3
.
Once incorporated into RISC, one strand of the siRNA (the passenger strand) is degraded or discarded while the other strand (the guide strand) remains to direct sequence
Once the RISC complex is activated by an siRNA, it can target numerous mRNA transcript copies. Tis multiple targeting by a single siRNA molecule amplifies gene silencing, allowing the effects to persist for 3-7 days in rapidly dividing cells and up to several weeks in non-dividing cells4
.
■ Synthetic RNA Duplexes as siRNA Reagents. Synthetic RNA duplexes that mimic natural siRNAs allow researchers to take advantage of the RNAi mechanism in cells to reduce expression of target genes. However, in mammals, the artificial introduction of long dsRNAs (several hundred bp) activates
■ Dicer-Substrate RNAi technology. Traditional siRNA designs use duplexed, chemically-synthesised 21mers that structurally resemble endogenous siRNAs, the end product of Dicer cleavage. Dicer- substrate RNAs [DsiRNA (IDT)] are an alternative to traditional 21mer siRNAs, with an increased effectiveness of up to 100-fold compared to conventional 21mer designs, and are able to effectively target some sites that 21mers cannot6
.
DsiRNAs are 27mer RNA duplexes with a novel asymmetric design that allows them to be processed by Dicer into the desired, 21mer siRNA
specificity of the silencing complex. Te Ago2 component of RISC is a ribonuclease that cleaves target RNAs under direction of the guide strand.
the innate immune system to trigger interferon (IFN) responses. Terefore, short dsRNAs that are less likely to induce IFN responses, are typically used.
Fig. 1. IDT synthesises DNA/RNA from its facilities in Coralville, IA, San Diego, CA, USA, and Leuven, Belgium.
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