Antisense

Question 1

What is Antisense RNA?

Answer 1

Strong strands of nucleic acids that bind mRNA and change the behaviour (degrade/supress translation etc) and affect protein production

Question 2

What are the main types of antisense?

Answer 2

Oligodioxynucelotides (AO)
Ribozymes
RNAi

Question 3

What steps can we regulate?

Answer 3

Processing (splicing, polyadenylation, capping)
Editing
Nuclear Export
Localization
Protein degradation

Question 4

What is the key stage of translation that we can regulate?

Answer 4

Initiation

Question 5

How does initiation work?

Answer 5

Binding of initiation complex (eIF4 a, b, g, e) with preinitiation complex to the 5’ cap (modified guanine)

Scans down until it reaches AUG (start codon) - recruits 60S ribosome

Question 6

What is mRNA circularisation?

Answer 6

eIF4E/G bind PABPI on 3’ polyA tail - circularisation of mRNA - recycling ribosomes - ensures only intact mRNA are transcribed (with polyA tail)

Question 7

What are some examples of natural AO?

Answer 7

MSX-1 - Murine tooth development

Frq - circadian rhythm

Question 8

What are some examples of therapeutic AO?

Answer 8

Thallasaemia - Mutation in intron 2 - insertion of a cryptic splice site - insertion of intron 2 in final mRNA - leads to truncated B-globin protein. AO can bind to intron 2 and prevent insertion of cryptic splice site - prevents insertion of intron 2 - normal protein

DMD - Mutation in exon 23 - nonsense mutation (C-T = STOP codon) - leads to truncated Dystrophin - AO binds to exon 23 and stops it being included in final mRNA - short but functional dystrophin protein.

SMA - Mutation in exon7 - prevention of SF2/ASF binding to ESE - truncated unstable protein - AO binds ESE and recruits SF2/ASF - normal protein

Question 9

What are the problems with antisense?

Answer 9

RNA unstable
Difficult to get into cell
Cell natural mechanisms to target antisense for degradation

Question 10

How can we modify AO?

Answer 10

Modified Ribose - add methyl/sulphur groups onto phosphate - prevent recognition by cell

Modified Backbone - change sugar/phosphate backbone to peptide backbone - prevent recognition and reduce charge so easier to get into cell

Question 11

What are some additional examples of AO as therapeutics?

Answer 11

Genasense - BCL-2 - CLL/Breast cancer

Gem92 - HIV Gag (proteins in assembly and vision function) - AIDS

Question 12

What is a ribozyme?

Answer 12

RNA enzyme

Question 13

What are some types of ribozyme?

Answer 13

RNAse P
Group 1 and 2 Introns
Hammerhead (plant virus)

Question 14

What are aptamers?

Answer 14

Short RNA sequences that can bind specific substrate (e.g. peptide) and block its activity (e.g. catalytic function)

Question 15

How do we produce aptamers?

Answer 15

SELEX

Question 16

What is an example of an aptamer for therapeutic use?

Answer 16

TAR decoy aptamer - binds TAT - inhibits gene expression - HIV

Question 17

What are some uses of aptamers?

Answer 17

Diagnostics
Target validation
High Throughput screening
Therapeutics

Question 18

What are the two classes of RNAi?

Answer 18

siRNA

miRNA

Question 19

What are siRNAs?

Answer 19

21-23 nucleotides
Perfect complimentary - degradation
Viral defence mechanisms

Question 20

What are miRNAs?

Answer 20

21-23 nucleotides
Imperfect complimentary - suppression of translation
Gene regulation

Question 21

How are miRNAs produced?

Answer 21

miRNA genes transcribed in nucleus by RNA Pol II to produce pre-miRNA

Pre-miRNA cleaved by Drosha and exported from nucleus via exportin 5

Cleaved by Dicer enzyme to produce miRNA

Combined with Argonaute complex to produce RISC

Targets mRNA with imperfect complimentary binding - translational supression

Question 22

How are siRNAs produced?

Answer 22

dsRNA in cell is degraded by Dicer enzyme

siRNA combine with Argonaute to produce RISC

Ago2 cleaves passenger strand

siRISC binds with perfect complimentarity to mRNA and causes degradation

Question 23

What is the origin of dsRNA within the cell?

Answer 23

Immune defence against virus and transposons

Question 24

What are the domains of the DICER enzyme?

Answer 24

PAZ domain - recognises 3’ of dsRNA and positions for 21-23 nucleotide cleavage

RNAse domain - cleavage of dsRNA - contains two catalytic sites for both strands - Mn divalent cation containing

Question 25

What is the RISC complex?

Answer 25

RNA induced silencing complex - contains Argonaute complex as catalytic component

Question 26

What are the domains of the Argo complex?

Answer 26

PAZ - recognises 3’ RNA and positions miRNA

PIWI domain - cleaves mRNA (siRNA) or suppresses (miRNA )

Question 27

How does mRNA suppress translation?

Answer 27

Two step - Translation block + Turnover

miRISC complex binds 3’ UTR of mRNA and recruits GW182 which blocks 60S ribosome binding and recruits deadeylation complex

Deadenylation beings degradation process - recruits decapping proteins - lose 5’ cap - lose initiation complex

mRNA degraded from 5’ using XRN1

Question 28

What is an example of a multiple approach method of antisense?

Answer 28

HIV

Aptamer - u6 TAR decoy - target TAT and stop HIV gene expression

Ribozyme - target CCR5 mRNA for cleavage - slows down disease

siRNA - targets REV - inhibits HIV

Question 29

What is the next phase of the HIV therapy?

Answer 29

Use of an aptamer that targets CCR5 and contains a siRNA which targets REV - single therapy