RNA Based Therapeutics Flashcards
What are the 4 different types of RNA-based therapeutics?
- Antisense oligonucleotides (ASO’s): inhibit mRNA translation.
- RNA interference (RNAi): RNA inhibitors of gene expression by degradation of
mRNA (destroys Mrna). Forms a RNA –induced silencing complex with a protein (Argonaute) - Ribozymes: catalytically active RNAs. Cleave covalent bonds in a target RNA.
- Aptamers: protein-binding RNAs. Length of RNA that binds to a protein (like a small molecule inhibitor)
Define Antisense Oligonucleotides (ASOs)?
Antisense oligonucleotide is a short strand oligonucleotide (ca. 20 bases pairs in length) that hybridizes with complementary mRNA in a sequence- specific manner via Watson-Crick base pairing.
What is the mechanism of action of Antisense oligonucleotides?
Entry of this RNA into the cell can lead to target protein knockdown in the cytoplasm or the nucleus:
Cytoplasm: binding of ASO to mRNA
- triggers RNase H activity OR
- inhibits ribosomal mRNA translation by steric hindrance. Both processes lead to target protein knockdown.
Nucleus: ASOs regulates mRNA maturation via three distinct mechanisms. - inhibition of 5’ cap being formed OR - inhibition of RNA splicing OR - recruits RNase H These lead to knock down of target genes and so target protein knockdown by preventing mature mRNA from forming.
Describe RNA interference.
- siRNA enters into a cell as a duplex (2 strands of RNA) and is incorporated into Argonaute (AGO), a protein within cells to form a part of the RNA-Induced Silencing Complex (RISC). Protein will get rid of one of the strands (the passenger strand in this case). Single strand stays in the AGO. (Described below aswell)
- When part of RISC, the siRNA is unwound (and the passenger strand is removed) with the guide strand (red one – identifies wich RNA gets taken by the ago) left behind to be used to find the desired target RNA in the cell.
- Target mRNA, bound in the AGO, is then cleaved (cut up) and digested or, if partially mismatched, silenced due to inhibited translation or once again degradation.
- If not perfectly matched (every RNA is not completely complementary i.e missing a base) – it doesn’t form a complex therefore cannot translate it.
What is the difference between ASO’s and RNAi?
ASO – only one strand, single strand that finds its targets. When it does, it either recruit’s other enzymes or blocks or stops it from being spliced. Does its task in the nucleus or the cytoplasm
siRNA – add double strand that finds a protein which gets rid of the passenger strand, the guide strand gets rid of the RNA, by cutting it up (cleaves) or gets in the way of translation.
What are ribozymes (other RNA-Based Therapeutics)?
Ribozymes = catalytic RNA enzymes used to inhibit gene expression.
These can cleave mRNA molecules, next to a recognition sequence (as they are made of RNA – PREVENTS RNA from being whole so can’t be translated into a protein), destroying the complete RNA sequence in the process.
Enzymatic mechanism used by ribozymes is very similar to that of protein ribonucleases.
What are Aptamers (other RNA-Based Therapeutics)?
Aptamer = RNA sequence that binds to a protein, preventing it from functioning (acts very similarly to a small molecule inhibitor).
What factors have an impact on the strength and stability of the interactions between ASOs and complementary mRNA target (Designing of Antisense Oligonucleotides)?
- Thermodynamic stability.
how tightly bound is the RNA to ASO?
how stable is the complex? - The secondary structure of the target mRNA transcript
what does the RNA look like in 3D?
which sites are accessible to the ASO? - The proximity of the hybridization site to the functional motifs on the designated transcript.
strategy can use proximity (how close) to translation start factor eg the methionine (AUG) initiation codon (blocks translation) or 5’cap.
alternatively, can block splice points in nucleus.
How can you increase the ‘hit rate’ of AGOs?
- Prediction of the secondary structure of the target RNA
> Various pieces of software are available for predicting secondary structures of
RNA -> use these to design ASOs. - Identification of preferable secondary local structures
> Due to the complex folding of mRNAs, effective ASOs should be designed to target accessible regions of the mRNA.
> Usually, these are located at terminal end, internal loops, joint sequences, hairpin and bulges of 10 or more consecutive nucleotides.
> Numerous softwares are available for identifying mRNA regions that can be targeted by ASOs.
What motifs are known to increase the mRNA knockdown effects of ASOs?
CCAC, TCCC, ACTC, GCCA and CTCT motifs
What motifs are known to weaken ASOs activity?
GGGG, ACTG, AAA and TAA motifs
What binding of two bases produces strong thermodynamic stability?
GC content is strongly correlated with thermodynamic stability of the complex and RNase H activity – WHY? – 3 hydrogen bonds so stronger – increases RNAse H activity
The presence of > 11 G or C residues gave strong ASO activity while < 9 gave minimal effect
What are the main disadvantages of RNA therapeutics?
- Natural RNAs are unstable in vivo due to the activity of ribonucleases – need to get it into the cells before its cut up (cleavage by DNase and RNase).
- Additionally, unmodified oligonucleotides have poor PK as they have weak binding to plasma proteins and they are filtered by the kidney and excreted into the urine.
- Typical antisense oligonucleotides, 20 bases in length, have a charges
backbone (formal charge of -19 – have to get this through membranes this won’t really happen) = very poor cellular uptake.
Give an example of generation one Modified Antisense Oligonucleotides?
In generation one modifications, changes to the backbone were made:
Most successful was PS-modified ASOs, the non-bridging oxygen atom of the
phosphodiester bond is replaced by a sulphur atom (phosphorothioate bond).
Phosphorothioate modifications:
- Increases nuclease‐resistance…
- and so increase bioavailability (less cleavage (breakdown) of ASO).
However, Reduces binding affinity. (Tm reduces by around 0.5 °C per nucleotide). Increases non-specific effects by interactions with cell surface and other cellular proteins.
Give an example of generation two Modified Antisense Oligonucleotides?
Generation two modifications: changes to the 2’ position of the sugar were made:
- 2’‐O‐methyl (2’-OMe) and 2’‐O‐methoxyethyl (2’-MOE) are widely used.
- Increase resistance to nucleases
- Tight binding between ASO and RNA
X Reduce RNase H activity (WHICH breaks down)
- To overcome this, ASOs with central 10‐PS‐modified 2’‐ deoxynucleotides flanked by five 2’‐OMe/O‐MOE nucleotides each side. (Can still recruit RNASE H)
5 of 2 OME on one end and 5 of 2 OME at the other end. In middle we have 10 x our PS modified RNA base pairs. High binding at both ends, tight binding, and more resistant at broken down and the 10 in the middle retains activity for the enzyme. It is mainly used with the sulfur modified version.
