Gas-phase dissociaton of sugar-modified nucleic acids

Hari, Yvonne Ilona (2017). Gas-phase dissociaton of sugar-modified nucleic acids. (Dissertation, Department of Chemistry and Biochemistry, Facultiy of Sciences)

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The application of modified oligonucleotides in medicine or biochemical engineering necessitates reliable sequencing tools and analytical procedures to study the interaction of synthetic oligonucleotides with biological targets. Mass spectrometry ideally meets the double challenge of characterising single- as well as double-stranded nucleic acids.

The first focus of the present thesis lies in the sequencing of the antisense drug-candidate tricyclo-DNA (tcDNA) by collision-induced dissociation and the characterisation of tcDNA duplexes. No correlation was observed between the collision energy required to fragment modified and unmodified duplexes and their in-solution stability. Due to the conformationally restricted backbone in tcDNA, nucleobase loss is preferred over the entropy-driven strand separation in modified duplexes. The limited flexibility of tcDNA oligonucleotides in the gas-phase was further evidenced by ion mobility spectrometry. The experimental collision cross sections of sequence-identical DNA, RNA, and tcDNA 15mers suggest that the modified sugar-moiety prevents tight folding of the backbone. Moreover, ion mobility data give evidence for asymmetric contraction of the two strands in tcDNA:RNA heteroduplexes after desolvation.

Radical-based activation techniques can provide valuable structural information complementary to collision-induced dissociation, as exemplified by the DNA analogue homo-DNA. Collisional activation of homo-DNA only gave rise to unspecific nucleobase loss, but full sequence information was obtained after electron transfer dissociation. Furthermore, homo-DNA oligonucleotides exhibited high dissociation yields, whereas DNA cations formed stable reduced species. Pi-pi-stacking is thought to impede backbone cleavage in DNA but not in homo-DNA. This hypothesis was sustained by experiments on oligonucleotides harbouring abasic sites.

In conclusion, the presented work highlights the impact of sugar-modified nucleotides on the activation energy of dissociation pathways and the nucleic acid conformation, thereby contributing to a better understanding of fragmentation mechanisms and the accessible reaction pathways in the gas-phase.

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