THE "TROJAN HORSE" FOR SUCCESSFUL RNAi IN INSECTS Flashcards
Why is RNAi so environmentally friendly for pest control?
Because it is species specific
How do you orally induce RNAi into insects?
By spraying the plants the host feeds on
What is the biggest struggle of inducing our dsRNA into an insect?
Their gut can degrade the dsRNA. We need to find ways to protect it.
Why do we use dsRNA to produce RNAi?
It works better than with antisense RNA
Which RNAse III enzymes are used for initiating RNAi?
Dicer and Drosha
How are the domains in RNAse III enzymes called?
- helicase domain
- PAZ domain
- the dicer
What does the helices domain do?
It unwinds RNA and DNA during replication
What does the PAZ domain do?
- it probably mediates complex formations of proteins
- it recognises the dsRNA substrates in the dicer
What does the dicer do?
It cleaves the substrate at around 22 nt from the open helicoid end
What are the main sRNA based silencing pathways in insects?
- miRNA
- siRNA
- PIWI interacting RNA
what are PIWI interacting RNAs?
It is the largest of the small non coding RNAs
Why are lepidopteran insects way harder to use RNAi on?
Because they have additional nucleases that can degrade dsRNA before the Dicer can do so
Which mechanisms are used in insects to uptake dsRNA?
- inhibitors of Cathrin dependent endocytis
- SID-1- like transmembrane channels (flies don’t have them)
- clathrin mediated pathways through endosomal transport of dsRNA (not so good)
Why are Cathrin mediated pathways not as good as other ones?
Because the dsRNA can escape via endosomes
What role do fatty acids play in the regulation of RNAi efficiency?
Sometimes, higher exposion to them can change the uptake of dsRNAs for the better
With what does the RNAi response correlate with heavily?
With the enzymatic degradation of dsRNA (in the midgut mainly)
What do the donor and receptients cells need to have in insects in order for cell to cell transport to work?
SID-1 Channels
What can we use instead of SID-1 channels?
Nanotube like structures
What can you tell about nanotube structures for cell to cell transport?
- they consist of actin and tubular
- they associate with parts of the RNAi machinery
With which parts of the RNAi machinery do nanotubes associate?
AGO-2 and dsRNA
How do we form nanoparticles with dsRNA?
dsRNA is anionic, by binding it to the cationic polymer CHITOSAN, we get stable nanoparticles
What can nanoparticles do?
knockdown genes
How to we target genes with nanoparticles?
By aerolizing the nanoparticle
What does aerolisation mean?
We create particles that are so small, that they can be carried via air
On which factor does the position of the nanoparticle inside the organism depend?
Its charge
Where does a positively charged nanoparticle appear?
In the gut or any gastrointestinal tract
Where does a negatively charged nanoparticle appear?
They persist through metamophorsis and appear in adult organs like the head or ovaries
How can we protect dsRNA from degradation?
- with guanidine containing polymers
- by binding the siRNA to perfluocarbons to build nanoparticles
Which bacterium do we use to deliver RNAi via bacterium?
RNase III - deficient “Escheria coli” (HT115)
On which idea does the theory of bacterial RNAi delivery build up on?
That the cloning of the desired gene takes place between two T7 promoters on a RNA plasmid called L4440
How does bacterial RNAi delivery work in theory?
- For the transformation HT115 cells are used
- dsRNA gets produced upon induction of T7 RNA polymerase
- after dsRNA production, we introduce the cells into the insects growth medium
- RNAi happens
How can we bypass the negative charge of the siRNA?
By binding it to the PTB-DRBD domain
Why do we want to bypass the negative siRNA charge?
So they can be uptake by PTD mediated cells
Why do we want our siRNA to be uptake by PTD mediated cells?
Because it doubles the dsRNA efficiency
