les_9-10 - CHAPTER VIII INHIBITION OF GENE EXPRESSION...

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CHAPTER VIII. INHIBITION OF GENE EXPRESSION VIII.1. ANTI-SENSE DNA OLIGONUCLEOTIDES = short, synthetic, modified oligodeoxynucleotides (ODN) that hybridise with complementary mRNA sequences, and prevent protein expression from the target mRNA. Mechanisms of inhibition : - Inhibition of translation (interference with ribosome association at 5'-end) - Inhibition of splicing (binding at splice donor or acceptor site) - Rnase H mediated degradation of mRNA (specific for RNA/DNA hybrid, with specific cleavage of the RNA) oligonucleotide design : - length: 15-20 bp - modifications to increase the stability and efficiency (higher affinity for RNA): 2' sugar modifications: e.g. 2’-O-methyl, 2’-fluoro heterocyclic (base) modifications: e.g. 5-methylcytosine backbone modifications: e.g. phosphorothioate oligodeoxynucleotides (one of the O that are not involved in the phosphodiester binding is replaced by S), morpholinos, peptide nucleic acids (PNA)
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165 Applications : Functional genomics: analysis of the role of a specific gene Therapy: - Vitravene = ODN against the CMV immediate early RNA for the treatment of CMV infections of the eye. - Genasense = ODN against Bcl-2 for the treatment of various cancers (phase III)
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166 - Correction of splicing errors (due to genetic mutations: e.g.thalassemia, cystic fibrosis, muscular dystrophy, …). Normally DNA-RNA hybrids are recognized by RNaseH and RNA is degraded. This is not what you want here, you want correction of splicing error. Use modified ODN. VIII.2. RIBOZYMES e.g. Hammerhead, hairpin, group I intron, hepatitis delta virus, RNase P = RNA with endoribonuclease activity = catalytic RNA: catalyze their own cleavage and the cleavage of other RNAs; endogenous or synthetic VIII.2.1. Hammerhead ribozyme First identified in satellite RNA of plant viruses (self-cleavage of RNA is part of the rolling circle genome replication) ~30 nucleotides long = 3 helical stem structures, in which 9 conserved nucleotides form the catalytic domain Target = every sequence with UH (H= every nucleotide, except G) Specificity is determined by the hybridizing arms that hybridize with the complementary mRNA. Upon binding, the RNA is cleaved by the
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167 ribozyme. Upon dissociation, the ribozyme can bind and cleave other RNA molecules (= enzyme). Substitution of 2’-hydroxyl group of pyrimidines with 2’-fluoro, 2’-amino, or 2’-O- methyl analogues results in more stable RNAs. VIII.2.2. RNase P Normal function = removal of 5’leader sequence of e.g. pre-tRNA, E. coli 4.5S RNA (role in secretion) = ribonucleotide protein in all cells; catalytic activity in the RNA component. Specificity is dependent of the structure of the substrate ( hammerhead ribozyme): - dsRNA region (corresponding to aminoacyl or T-stem of pre-tRNA) - 3’ CCA unpaired sequence (not required for mammalian RnaseP) - 5’leader sequence as ssRNA, which is part of the cleaved strand - guanine and a pyrimidine base at the 3’ and 5’ place of the cleavage site, respectively, increases the catalytic activity.
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168 Use of ‘guiding sequence’ (GS) which hybridises with the target mRNA: - external (EGS)
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