mattossovich, rosanna (2021) DEVELOPMENT OF A NEW ENZYME BASED BIOSENSOR FOR DETECTION OF DAMAGED DNA, BY USING THE THERMOSNAP-DISPLAY TECHNOLOGY. [Tesi di dottorato]

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Cellular DNA is constantly subjected to covalent modifications by intracellular chemical compounds and coming from the external environment. The most dangerous types of molecular changes include the formation of covalent bonds between the strands of the DNA , or their breakage, and the loss or the bases' modification . Among these, alkylating agents are very reactive molecules that bind chemical groups in nucleic acids, causing alterations in their functions. The presence of alkylating agents in industrial waste, as well as in combustion and food products, is one of the main problems for the environment. The determination and the measure of damaged DNA is really important for: the identification and/or the optimization of DNA protective molecules against the action of alkylating agents ,in cosmetic and pharmaceutical productions ;the assessment of environmental pollution; the analysis of the quality of production, packaging and storage of food. Among the DNA modifications by alkylating agents, the most mutagenic adduct introduced into DNA by methylating agents is the O6-MG. This modification alters during DNA replication with thymine leading to the GC → AT transition, which could be fixed in the genome. The strategy used by nature to repair alkylation damage is the direct transfer of these alkyl groups to the active site of the O6- alkylguanine-DNA alkyl-transferase(AGTs).AGTs are highly interesting not only for investigating critical biological processes, such as DNA repair, but also for the development of new and simple, intuitive and inexpensive assays to optimize inhibitory molecules of therapeutic and environmental interest. The aim of this project is the development of innovative analytical methods for the determination of the O6-alkyl-guanine nucleobases (O6-AGs) content in DNA samples from different sources, locations and cell treatments (mDNA). In general, this project proposes an innovative, rapid and reproducible method to identify particular DNA damages. The goal was pursued through two different approaches, the first based on the employing of particular enzymes that, unlike the complex repair mechanisms, are able to repair DNA with a single irreversible; and the second on the use of suitably modified oligonucleotides. The bottleneck of this assay is the need to perform the reaction at moderately high temperatures (ca. 60 °C), which avoids any endogenous AGTs' activities still present in DNA samples.

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