How hairpinRNAi works

• In plants a transgene delivers a hairpinRNA molecule which folds back on itself to form a double stranded structure.
• The double-stranded RNA produced by the cleaving of the intron in the hairpinRNAi construct serves as the perfect substrate for the enzyme Dicer – which cleaves it into discrete chunks of approximately 21 base pairs long, known as small interfering RNAs (siRNAs).
• The siRNAs interact with the RNA-Induced Silencing Complex (RISC), whose helicase activity directs siRNAs unwinding.
• RISC uses the unwound single stranded siRNA as a guide to find the target mRNA using base pairing.
• Argonaute 1, the principal component of the RISC complex provides the ‘slicer’ activity resulting in 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 destabilised and subsequently fully degraded through natural endogenous mechanisms.

Although the identities of some of the members of this pathway remain to be uncovered, this model of the RNAi pathway has gained the strongest experimental support to date.

The hairpinRNAs generated by CSIRO’s technology are usually long and produce many siRNAs that bind along the length of the target mRNA cleaving it several times thereby increasing the efficiency of mRNA degradation. HairpinRNAi constructs longer than 300 nucleotides have been shown to be most effective.