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Auriane Guitton

Formation of covalent crosslinks between an aptamer and its target via specific oxidation of an 8-oxoguanine

Published on 25 October 2023
Thesis presented October 25, 2023

Abstract:
Covalent crosslink between proteins and DNA strands containing a guanine oxidation product, the 8-oxoguanine (OG), via the specific oxidation of this base has been demonstrated in various works. This project aims to investigate whether this concept can be applied to an OG-containing aptamer to generate an adduct with its target. Aptamers are short, single-stranded RNA or DNA oligonucleotides selected to recognize a target with high affinity and specificity via low-energy interactions. These recognition elements offer a number of advantages, such as the ability to undergo multiple denaturations, or the facility to introduce modifications in their sequence during chemical synthesis. The model system chosen is an aptamer recognizing L-argininamide. Three guanine bases of this aptamer, located in the recognition site, were replaced one at a time by an 8-oxoguanine. Fluorescence anisotropy measurements demonstrated that such replacement results to a decrease or increase in affinity for the target, depending on the position of the oxidized base. Analysis by capillary electrophoresis and HPLC-MS demonstrated the effective formation of adducts between the three OG-containing aptamers and the target following oxidation of this base by an iridium salt. Characterization of the products shows that their structures are spiro and guanidino, which is consistent with the literature. Finally, various strategies were implemented in an attempt to reduce the non-specific interactions existing between the polyanionic aptamer and the cationic target.

Keywords:
Aptamer, Covalent crosslink, Specific oxidation, Capillary electrophoresis, HPLC-MS