Macromolecules Flashcards
what are the types of RNA therapeutics?
Antisense oligonucleotides (ASO’s): inhibit mRNA translation.
RNA interference (RNAi): RNA inhibitors of gene expression by degradation of 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)
RNA vaccines: introduces mRNA sequence coded for a disease specific antigen into cells to build up an immune response
what are Antisense Oligonucleotides (ASOs)?
short strand
Entry of this RNA into the cell can lead to target protein knockdown in the cytoplasm or the nucleus:
what happens when ASOs bind in the cytoplasm?
bind to mRNA
– triggers ribonuclease (RNase H) activity OR
– inhibits ribosomal mRNA translation by steric hindrance.
what happens when ASOs bind in the nucleus?
regulates mRNA maturation
how do ASOs regulate mRNA maturation?
– inhibition of 5’ cap being formed OR
– inhibition of RNA splicing OR
– recruits ribonuclease (RNase H)
what are some examples of RNA interference?
siRNA
RNAi
whats the difference between ASO’s and RNAi?
what are RNA interferences?
how do ribozymes work?
cleave mRNA molecules next to a recognition sequence = destroying RNA sequences
similar to protein nucleases
how do aptamers work?
aptamer sequence = functional aptamer = target binding = preventing functionality of bound molecule
half life of RNA
short half life = fast degradation
when may RNA vaccines not be safe to administer?
for patients susceptible to autoimmune response
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what is the disadvantage of RNA therapeutics?
- enzyme degradation - activity of ribonuclease
- fast excretion - unmodified oligonucleotides have a poor PK due to weak plasma binding = filtration thru the kidney
- poor absorption/bioavailability= poor cellular uptake
what can chemical modifications do to oligonucleotides?
stabilise ASOs and resistant to ribonucleases = increase half life
retain/improve RNAse H efficacy
reduce immunogenicity
increase affinity and potency
decrease non-sequence specific toxicity
how do you prevent ribonuclease attacking RNA therapeutics?
chemically modify the antisense oligonucleotide
what are generation one modified antisense oligonucleotides?
phosphorothioate (PS) modifications to the backbone
what are generation two modified antisense oligonucleotides?
changes to the 2’ position of the sugar
2’-MOE, 2’-O-methyl (2’OMe) , 2’-fluoro
what are generation three modified antisense oligonucleotides?
ASOs with modifications in the sugar region with a non-charged backbone
Peptide Nucleic Acid (PNA)
Locked nucleic acid (LNA)
what are the advantages/disadvantages of PS-modifications?
adv
- increase nuclease-resistance
- increase bioavailability
disadv
- reduces binding affinity
- non-specific interactions with cell surface and other proteins
what are the advantages/disadvantages of 2’-MOE, 2’-O-methyl (2’OMe) , 2’-fluoro -modifications?
adv
- increase nuclease-resistance
- tight binding between ASO and RNA
disadv
reduced RNase H activity
how do you overcome the disadvantages of using generation 2
modified antisense oligonucleotides?
reduced RNase H activity - disadvantage
overcome by using in combination with generation 1 modified antisense oligonucleotides
what are the advantages/disadvantages of Peptide Nucleic Acid (PNA)?
adv
- high binding affinity to RNA/DNA than RNA-RNA and DNA-DNA
- stable towards nucleases and peptidases
- bind double-stranded DNA = transcriptional arrest
disadv
- effects produced by steric inhibition of mRNA translation
what are the advantages/disadvantages of Locked Nucleic Acid (LNA)?
adv
- increased binding affinity towards target mRNA
- resistance to nuclease degradation
disadv
- not processed by RNase H
what are the PK of antisense oligonucleotides?
- uncharged oligonucleotides such as PNA and morpholino = low plasma protein binding = rapidly cleared
- single stranded, PS-modified ASOs = high affinity for plasma protein = decrease excretion
- ASO bind to plasma protein = decrease excretion
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