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Riboswitches are regulatory RNA elements that undergo functionally important allosteric conformational switching upon binding of specific ligands. The here investigated guanidine-II riboswitch binds the small cation, guanidinium, and forms a kissing loop-loop interaction between its P1 and P2 hairpins. We investigated the structural changes to support previous studies regarding the binding mechanism. Using NMR spectroscopy, we confirmed the structure as observed in crystal structures and we characterized the kissing loop interaction upon addition of Mg2+ and ligand for the riboswitch aptamer from Escherichia coli. We further investigated closely related mutant constructs providing further insight into functional differences between the two (different) hairpins P1 and P2. Formation of intermolecular interactions were probed by small-angle X-ray scattering (SAXS) and NMR DOSY data. All data are consistent and show the formation of oligomeric states of the riboswitch induced by Mg2+ and ligand binding.
We report here the nuclear magnetic resonance 19F screening of 14 RNA targets with different secondary and tertiary structure to systematically assess the druggability of RNAs. Our RNA targets include representative bacterial riboswitches that naturally bind with nanomolar affinity and high specificity to cellular metabolites of low molecular weight. Based on counter-screens against five DNAs and five proteins, we can show that RNA can be specifically targeted. To demonstrate the quality of the initial fragment library that has been designed for easy follow-up chemistry, we further show how to increase binding affinity from an initial fragment hit by chemistry that links the identified fragment to the intercalator acridine. Thus, we achieve low-micromolar binding affinity without losing binding specificity between two different terminator structures.
In der vorgelegten kumulativen Arbeit wurden strukturelle und funktionale Untersuchungen an Nukleinsäuren durchgeführt, hauptsächlich, aber nicht ausschließlich unter Verwendung von NMR-Spektroskopie (Kernspin Resonanzspektroskopie) als Analysemethode. Die untersuchten Biomoleküle umfassten kleinere und größere biologisch relevante RNAs sowie einen artifiziellen DNA G-Quadruplex. Hierbei konnten Ergebnisse im Bereich der Bestimmung der molekularen Struktur, der Aufklärung der biologischen Funktion und der Wirkstoffentwicklung gewonnen werden, die in sechs verschiedenen Publikationen dargelegt sind, an deren Erstellung der Autor maßgeblich oder hauptverantwortlich beteiligt war. Des Weiteren wird in einem mehrgliedrigen Einleitungssegment auf den Stand der aktuellen Forschung in den jeweiligen Teilgebieten eingegangen.