Characterization of therapeutical oligonucleotides by ion mobility coupled to mass spectrometry

Abstract

The subject of this thesis is based on the study of short nucleic acids, known as oligonucleotides, which have been increasingly investigated and implemented for their various interesting properties, including in the pharmaceutical field. Within the scope of this work, different models of oligonucleotides and their higher-order structures are examined in the gas phase, relying on ion mobility coupled with mass spectrometry and various collisional activation methods. Through this coupling, a mass characterization (m/z ratios) and ion mobility of the species could be achieved, and, in combination with activation methods, relative stabilities, fragmentation pathways, as well as the presence of potential structural changes could be investigated. All these experiments were conducted on three types of different higher-order structures of oligonucleotides. The initial models consist of DNA duplexes with an increasing content of G and C bases. These were studied as a continuation of a prior laboratory investigation into the effect of this compositional modification on duplex properties. In this study, ion mobility dimension was added to determine if differences in terms of structural changes were observed between the models. In this context, in addition to confirming the previously observed trend, slight changes in travel times were observed before dissociation. Subsequently, chemically modified (conjugated) RNA duplexes were studied. For these, the variable under investigation was the presence or absence of different conjugates on the models to determine their potential impact on the various properties of RNA duplexes. It was shown that, for these models, their behaviors are generally similar despite the presence of these conjugates. Finally, specific DNA structures, G-quadruplexes, were investigated. Three different G-quadruplex models were compared to determine the influence of their structures and sequences on the properties under investigation. It was demonstrated that the presence of a loop in the quadruplex makes its structure more sensitive to activation. Their fragmentation pathways were also compared, and similarities were found among structurally similar models.