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How does RNAi work?
RNAi represents the harnessing of a conserved, powerful gene regulation
pathway, whereby double stranded RNA (dsRNA) molecules are used to
trigger the catalytic degradation of the targeted gene’s mRNA, thus
effectively silencing its expression. From the experimental point of
view, the underlying process can be briefly summarized as follows:
(1) Delivery of trigger dsRNA
A “trigger“ dsRNA is introduced into the cell’s cytoplasm.
(2) Generation of siRNA pool
The Dicer enzyme (and associated co-factors) processes the trigger
dsRNA, forming a pool of small interfering RNAs (siRNAs): these
are approx. 21 base pairs in length, including 2 nucleotide
overhangs at both 3’ ends.
(3) Capture, unwinding of siRNA by RISC
The processed siRNAs are then delivered to an Argonaut-containing
RNA-Induced Silencing Complex (RISC), which unwinds the two siRNA
strands, retaining one strand to act as a RISC-targeting co-factor.
(4) Binding of siRNA-associated RISC to target mRNA
The siRNA’s bound strand confers sequence-based specificity to its
associated RISC complex, allowing recognition and base-pairing with the
complementary target mRNA.
(5) Destruction of target mRNA
The RISC complex contains an endonuclease activity, now ascribed to
the Argonaut subunit, which causes a single-site cleavage of the target
mRNA approximately in the middle of the siRNA binding region. The
resulting fragments of target mRNA are thereby destabilized and
subsequently get fully degraded through natural endogenous mechanisms.
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