Date of Award

Fall 11-16-2017

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Professor Mauro F.A. Adamo

Second Supervisor

Dr Maria Moccia

Funder/Sponsor

Higher Education Authority

Keywords

Neuroblastoma, PNA, miR-34a, mimics, PNA building block

Abstract

Chapters 1 and 2

Neuroblastoma is the most common extra-cranial paediatric solid tumour, responsible for 15% of childhood cancer deaths. Overexpression of the MYCN proto-oncogene remains the most significant biomarker of the disease, and it is found amplified in ~25% of neuroblastoma tumours. Because of its correlation with aggressive tumour progression, and poor outcome, MYCN is an ideal therapeutic target in neuroblastoma. It has recently been demonstrated that miR-34a directly targets the MYCN 3’UTR, resulting in inhibition of the MYCN protein. By silencing MYCN expression, it exerts a massive inhibition on cell proliferation and is considered a strong tumour suppressor. Peptide Nucleic Acids (PNA) have been identified as suitable analogues to develop miRNA-34a mimics, with enhanced properties such as stability and resistance to endonucleases and proteases. Considering the higher affinity and binding stability of PNAs towards target mRNAs, the aim of this project was the identification of the minimum portion of miR-34a sequence that can be synthesized as a PNA and used to successfully inhibit MYCN. PNA constructs of different lengths have been designed, including (a) a seed sequence and/or 3’-supplementary binding site, recognising 3’UTR region of MYCN mRNA, (b) two peptide carriers, able to improve the internalization of the construct, (c) a linker, and (d) a fluorescent tag. The PNA-based miRNAs, described herein, were prepared by microwave assisted solid phase synthesis; their cellular uptake and biological activity were tested in vitro on Neuroblastoma cell lines. The best peptide carrier identified includes a tri-lysine fragment, on the C-term, and a lysine-cysteine fragment, on the N-term. The longest sequence, including 22 nucleobases, was internalized, with a greater efficiency into the cell, and it exerted the highest suppression of neuroblastoma cell growth.

Chapter 3

Peptide Nucleic Acids (PNA) are synthetic nucleic acid analogues, with wide biomedical application. However, despite their ability to recognise and hybridise DNA/RNA targets, these oligomers suffer from: 1) low water solubility, 2) poor cell permeability, and 3) ambiguity in nucleic acids recognition (parallel/antiparallel). Hence, the design of novel PNA building blocks, for the preparation of second-generation polymers, with enhanced properties, is still a challenge. The introduction of one chiral monomer into a PNA chain has been shown to induce right- or left-handed helicity to the strand, hence improving the binding affinity towards the target, as well as selective parallel/antiparallel binding. A plethora of chiral monomers have been reported over the last two decades, bearing different functionalities on the backbone, in an -α, -β or -γ position to the carboxyl group. In this work, we propose a novel chiral PNA monomer, in which the central nitrogen is replaced by a chiral carbon. The presence of inherent chirality is expected to impact on the ability, if the new polymers selectively recognise their RNA or DNA targets. It should be noted that the handedness induced by the R or S enantiomer to the PNA helix is not accurately predictable. For this reason, we have planned a synthetic layout and developed a route, to afford both R and S enantiomers for PNA synthesis. The synthesis started from commercially available cyclopentenone and malonate and provides, in 13 steps, the required carbon framework.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.

File Size

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Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2017

Available for download on Monday, November 30, 2020

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