Synthesis and characterization of PNA based molecules able to interfere with gene regulation
Avitabile, Concetta (2011) Synthesis and characterization of PNA based molecules able to interfere with gene regulation. [Tesi di dottorato] (Inedito)
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The control of gene expression is a fundamental process to bring the genome to life, and it pervades most of biology, from cell proliferation and differentiation to development. Mis-regulation of gene expression at any level can lead to disease.MicroRNAs, single-stranded RNAs (ssRNAs) of 19–25 nu-cleotides in length,function as guide molecules in post-transcriptional gene silencing by base pairing with target mRNAs, which leads to mRNA cleavage or translational repression. In this work PNA based molecules designed to interfere in the maturation of miRNA and gene expression were obtained.To achieve these goals, firstly a new, efficient and inexpensive protocol to obtain PNA by Fmoc solid-phase synthesis was developed. To interfere in the miRNA210 function PNA5 and PNA6 anti-premiR were obtained.To improve PNA delivery in cells, conjugation to cell-penetrating peptides, such as Tat peptide, and nuclear localization signal (NLS and biNLS) peptides was carried out. Furthermore, in order to demonstrate that the designed PNAs were able to bind to the pre-miRNA210 a Thiazole Orange (TO) modified PNA for fluorescence studies was obtained.A further aim of this project was the synthesis and the characterization of new modified PNA monomers bearing a sulphate group in gamma position of the backbone and of oligomers containing sulphate monomers were set up. The conformational preferences of the PNA monomers were investigated by NMR. Studies on the secondary structure of a polypirimidine oligomer were carried out by CD. The ability of the modified oligomer to interact with DNA, the specificity and affinity of binding were investigated by UV and CD. Finally, the ability of the sulphate PNA to interfere with the transcription of the ErbB2 gene on a human cell line overexpressing ErbB2 (SKBR3), by FACS and qPCR was explored.
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