THE "TROJAN HORSE" FOR SUCCESSFUL RNAi IN INSECTS

Question 1

Why is RNAi so environmentally friendly for pest control?

Answer 1

Because it is species specific

Question 2

How do you orally induce RNAi into insects?

Answer 2

By spraying the plants the host feeds on

Question 3

What is the biggest struggle of inducing our dsRNA into an insect?

Answer 3

Their gut can degrade the dsRNA. We need to find ways to protect it.

Question 4

Why do we use dsRNA to produce RNAi?

Answer 4

It works better than with antisense RNA

Question 5

Which RNAse III enzymes are used for initiating RNAi?

Answer 5

Dicer and Drosha

Question 6

How are the domains in RNAse III enzymes called?

Answer 6

• helicase domain
• PAZ domain
• the dicer

Question 7

What does the helices domain do?

Answer 7

It unwinds RNA and DNA during replication

Question 8

What does the PAZ domain do?

Answer 8

• it probably mediates complex formations of proteins

- it recognises the dsRNA substrates in the dicer

Question 9

What does the dicer do?

Answer 9

It cleaves the substrate at around 22 nt from the open helicoid end

Question 10

What are the main sRNA based silencing pathways in insects?

Answer 10

• miRNA
• siRNA
• PIWI interacting RNA

Question 11

what are PIWI interacting RNAs?

Answer 11

It is the largest of the small non coding RNAs

Question 12

Why are lepidopteran insects way harder to use RNAi on?

Answer 12

Because they have additional nucleases that can degrade dsRNA before the Dicer can do so

Question 13

Which mechanisms are used in insects to uptake dsRNA?

Answer 13

• 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)

Question 14

Why are Cathrin mediated pathways not as good as other ones?

Answer 14

Because the dsRNA can escape via endosomes

Question 15

What role do fatty acids play in the regulation of RNAi efficiency?

Answer 15

Sometimes, higher exposion to them can change the uptake of dsRNAs for the better

Question 16

With what does the RNAi response correlate with heavily?

Answer 16

With the enzymatic degradation of dsRNA (in the midgut mainly)

Question 17

What do the donor and receptients cells need to have in insects in order for cell to cell transport to work?

Answer 17

SID-1 Channels

Question 18

What can we use instead of SID-1 channels?

Answer 18

Nanotube like structures

Question 19

What can you tell about nanotube structures for cell to cell transport?

Answer 19

• they consist of actin and tubular

- they associate with parts of the RNAi machinery

Question 20

With which parts of the RNAi machinery do nanotubes associate?

Answer 20

AGO-2 and dsRNA

Question 21

How do we form nanoparticles with dsRNA?

Answer 21

dsRNA is anionic, by binding it to the cationic polymer CHITOSAN, we get stable nanoparticles

Question 22

What can nanoparticles do?

Answer 22

knockdown genes

Question 23

How to we target genes with nanoparticles?

Answer 23

By aerolizing the nanoparticle

Question 24

What does aerolisation mean?

Answer 24

We create particles that are so small, that they can be carried via air

Question 25

On which factor does the position of the nanoparticle inside the organism depend?

Answer 25

Its charge

Question 26

Where does a positively charged nanoparticle appear?

Answer 26

In the gut or any gastrointestinal tract

Question 27

Where does a negatively charged nanoparticle appear?

Answer 27

They persist through metamophorsis and appear in adult organs like the head or ovaries

Question 28

How can we protect dsRNA from degradation?

Answer 28

• with guanidine containing polymers

- by binding the siRNA to perfluocarbons to build nanoparticles

Question 29

Which bacterium do we use to deliver RNAi via bacterium?

Answer 29

RNase III - deficient “Escheria coli” (HT115)

Question 30

On which idea does the theory of bacterial RNAi delivery build up on?

Answer 30

That the cloning of the desired gene takes place between two T7 promoters on a RNA plasmid called L4440

Question 31

How does bacterial RNAi delivery work in theory?

Answer 31

• 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

Question 32

How can we bypass the negative charge of the siRNA?

Answer 32

By binding it to the PTB-DRBD domain

Question 33

Why do we want to bypass the negative siRNA charge?

Answer 33

So they can be uptake by PTD mediated cells

Question 34

Why do we want our siRNA to be uptake by PTD mediated cells?

Answer 34

Because it doubles the dsRNA efficiency