Rivieccio, Elisa (2017) Synthesis, structural and biological characterization of modified aptamers and of azocompound derivatives having potential pharmacological activity. [Tesi di dottorato]


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Item Type: Tesi di dottorato
Lingua: English
Title: Synthesis, structural and biological characterization of modified aptamers and of azocompound derivatives having potential pharmacological activity
Rivieccio, Elisaelisa.rivieccio@unina.it
Date: 10 December 2017
Number of Pages: 160
Institution: Università degli Studi di Napoli Federico II
Department: dep05
Dottorato: phd071
Ciclo di dottorato: 30
Coordinatore del Corso di dottorato:
D'Auria, Maria Valeriamariavaleria.dauria@unina.it
Date: 10 December 2017
Number of Pages: 160
Uncontrolled Keywords: Antiproliferative and anti-HIV G-quadruplexes; aptamers; azo compounds; photoswitchers
Settori scientifico-disciplinari del MIUR: Area 03 - Scienze chimiche > CHIM/06 - Chimica organica
Date Deposited: 19 Dec 2017 12:52
Last Modified: 22 Mar 2019 11:13
URI: http://www.fedoa.unina.it/id/eprint/12145


Single-stranded RNA and DNA aptamers have been developed in order to selectively inhibit specific biological targets. The tridimensional structural arrangement of aptamers guarantees their selective and specific binding to the target. A large number of G-rich aptamers are able to form G-quadruplex (G-4) structures. The high polymorphism of G-4 structure provides for a range of stable structures having specific recognition sites on the corresponding target. Several aptamers based on the G-4 structures are attractive tools for the detection and treatment of vascular, cancer and viral diseases, many of which are currently being tested in preclinical and clinical trials. Often, in order to increase the in vivo nuclease aptamer resistance and/or to elucidate the aptamer-target structure-activity relationships, the initial aptamers, obtained by SELEX (Systematic Evolution of LIgands by EXponential enrichment), are subjected to chemical modifications. In this context, a piece of my research activity has been centred on the synthesis of modified G-quadruplex forming aptamers endowed with potential pharmacological activities. In order to explore the potential of the TBA G-quadruplex as antiproliferative agent, I searched for that TBA single residue modification capable both to turn off the antithrombin activity and to preserve the aptamer antiproliferative action. I reached this goal modifying the TBA with a particular biphenyl linker that was used instead of T residue at position 3, 4, 7, 9, 12 or 13. Each of six TBA variants was studied from structural point of view by CD, CD melting, 1 and 2D NMR techniques. The acquired data demonstrated that almost all the sequences were able to fold into monomolecular “chair-like” G-Quadruplex structure, strongly similar to that of TBA. On the other hand, the results of biological studies showed that two TBA variants, containing the modification at T4 or T13, lacked of the TBA antithrombin activity but preserved the TBA antiproliferative activity on He-La cells. Results concerning this research are discussed in the chapter 2, part I. Another G-quadruplex forming aptamer, named T30695 was been chemically modified at T-loop residues. T30695 has been selected as HIV-Integrase (HIV-In) inhibitor and its biological action is today still ambiguous. In fact, the T30695 virus inhibition is also connected with the binding to Gp120. The formation of T30695-Gp120 complex impairs the host-viral interaction and thus reduces the infectivity of HIV. Furthermore, in view of the results of recent studies on the G-quadruplex T30695 typology, such previously reported T30695-HIV-In structure-activity relationships require to be revised. A part of my studies has been performed to acquire new information about T30695 anti-HIV-In activity. In particular, I synthesised a new set of modified T30695 aptamers in which single T residue at position 4, 8 or 12 was modified as R or S glycerol T. Structural studies on modified aptamers, performed using CD, CD melting, EMSA, HRMS and NMR, highlighted that almost all modified sequences were able to form the same G-Quadruplex typology of that of T30695. LEDGF-p75 dependent and independent integration assays provided information about the biological behaviour of the modified aptamers. The results of these studies are described in the chapter 3, part I. During the third year of my PhD course I have focused my studies on the synthesis of azobenzene photowitchers useful both as antiproliferative agents and as modifying units of conjugated aptamers. Some newly synthesized chiral azo-heteroarenes showed promising photo-regulated antiproliferative activity on HCT116-p53-/- cancer cells. Results concerning the synthesis, the structural characterization and the biological behaviours of these new molecules are described in the chapter 2, part II. Finally, I carried out the synthesis of 1-(4-dimethyaminobenzyl)-2-(4'-(R-2,3-dihydroxypropoxy))diazobenzene as phosphoramidite building block useful to obtain 5'-conjugated oligonucleotides. I performed the conjugation of the synthesized building block at 5'-end of the TBA and of the T30695 aptamers. Preliminary spectroscopic studies concerning the behaviour of conjugated aptamers under photo-illumination are reported in chapter 3, part II.

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