Bipyridine modified DNA G-quadruplexes
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Date
2025
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From structure to function
Abstract
DNA G-quadruplexes are four-stranded secondary DNA structures formed through Hoogsten hydrogen bonds of four individual guanines, creating a G-tetrad whereby several of such tetrads can stack on top of each other to create the final construct. They are closely involved in the regulation of several biological processes such as transcription and translation, core mechanisms of life on earth. This makes them prime targets for several therapeutic approaches. Amongst them is the development of small molecule ligands capable of targeting and stabilising or destabilising G-quadruplex structures. Yet their polymorphic character and their complex interaction with other biomolecules, primarily proteins, remains a challenging aspect of understanding G-quadruplex chemical and biological features. Concomitantly this renders them an interesting playground to introduce modifications into these systems that potentially offer a novel understanding of their characteristics. In this thesis, a novel artificial bipyridine ligandoside was incorporated covalently into short DNA oligonucleotides capable of forming G-quadruplex structures. It was shown that these modifications had little to no impact on the parental structure, retaining the original topology and thermal stability. Although almost all sequences showed thermal stabilisation to some degree through complex formation of the bipyridine moieties and divalent metal cations such as Cu2+, Ni2+, Zn2+, Co2+, Cd2+ and Fe2+, only few G-quadruplexes experienced a structural reorganisation resulting in a novel distinct topology. Of these particular examples, kinetic experiments revealed how the refolding was mostly driven by hydrogen bond and π-stacking interactions of the DNA structure and merely kinetically enables through the initial fast formation of the metal complex. Furthermore, it was showcased that these artificial G-quadruplexes retained interaction capabilities with proteins and in particular helicases Pif1 and DHX36. As the topology of some modified G-quadruplexes was metal dependent, the resulting binding constants (Kd) where impacted as preferential interaction with either hybrid, antiparallel or parallel topologies was observed. For the best systems low nM Kd values could be obtained while taking advantage of the thermal stabilisation by the metal complex and thus greatly impeding the unwinding the G-quadruplex structure proven by luciferase assays. Lastly, these modified systems were transfected into human cancer cell lines HeLa and U2OS where their spontaneous localisation into the cell nucleus as well as the retention of their structure stabilised ideally by Ni2+ cations be proven by fluorescence based microscopy. Overall, these novel modified G-quadruplex systems bearing bipyridine modifications granted a more detailed insight into structural properties of these DNA secondary structures and also made for promising novel therapeutic approaches and an application as potential molecular decoy.
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Chemie, Biochemie, DNA
Subjects based on RSWK
DNS