Antisense Flashcards
What is Antisense RNA?
Strong strands of nucleic acids that bind mRNA and change the behaviour (degrade/supress translation etc) and affect protein production
What are the main types of antisense?
Oligodioxynucelotides (AO)
Ribozymes
RNAi
What steps can we regulate?
Processing (splicing, polyadenylation, capping) Editing Nuclear Export Localization Protein degradation
What is the key stage of translation that we can regulate?
Initiation
How does initiation work?
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
What is mRNA circularisation?
eIF4E/G bind PABPI on 3’ polyA tail - circularisation of mRNA - recycling ribosomes - ensures only intact mRNA are transcribed (with polyA tail)
What are some examples of natural AO?
MSX-1 - Murine tooth development
Frq - circadian rhythm
What are some examples of therapeutic AO?
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
What are the problems with antisense?
RNA unstable
Difficult to get into cell
Cell natural mechanisms to target antisense for degradation
How can we modify AO?
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
What are some additional examples of AO as therapeutics?
Genasense - BCL-2 - CLL/Breast cancer
Gem92 - HIV Gag (proteins in assembly and vision function) - AIDS
What is a ribozyme?
RNA enzyme
What are some types of ribozyme?
RNAse P
Group 1 and 2 Introns
Hammerhead (plant virus)
What are aptamers?
Short RNA sequences that can bind specific substrate (e.g. peptide) and block its activity (e.g. catalytic function)
How do we produce aptamers?
SELEX
What is an example of an aptamer for therapeutic use?
TAR decoy aptamer - binds TAT - inhibits gene expression - HIV
What are some uses of aptamers?
Diagnostics
Target validation
High Throughput screening
Therapeutics
What are the two classes of RNAi?
siRNA
miRNA
What are siRNAs?
21-23 nucleotides
Perfect complimentary - degradation
Viral defence mechanisms
What are miRNAs?
21-23 nucleotides
Imperfect complimentary - suppression of translation
Gene regulation
How are miRNAs produced?
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
How are siRNAs produced?
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
What is the origin of dsRNA within the cell?
Immune defence against virus and transposons
What are the domains of the DICER enzyme?
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
What is the RISC complex?
RNA induced silencing complex - contains Argonaute complex as catalytic component
What are the domains of the Argo complex?
PAZ - recognises 3’ RNA and positions miRNA
PIWI domain - cleaves mRNA (siRNA) or suppresses (miRNA )
How does mRNA suppress translation?
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
What is an example of a multiple approach method of antisense?
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
What is the next phase of the HIV therapy?
Use of an aptamer that targets CCR5 and contains a siRNA which targets REV - single therapy
