Spektroskopische Untersuchungen zur Bestimmung von RNA-Ligand-Wechselwirkungen und RNA-Dynamiken

This thesis describes the structural characterization of interactions between biological relevant ribonucleic acid biomacromolecules (RNAs) and selected ligands to optimize the methodologies for the design of pharmacological lead compounds. To achieve this aim, not only the structures of the RNA, the ligand and their complexes need to be known, but also information about the inherent dynamics, especially of the target RNA, are necessary. To determine the structure and dynamics of these molecules and their complexes, liquid state nuclear magnetic resonance spectroscopy (NMR) is a suitable and powerful method. The necessity for these investigations arises from the lack of knowledge in RNA-ligand interactions, e.g. for the development of new medicinal drugs targeting crucial RNA sequences. In the first chapters of this thesis (Chapters II to IV), an introduction into RNA research is given with a focus on RNA structural features (Chapter II), into the interacting molecules, the biology of the specific RNA targets and the further development of their ligands (Chapter III) and into the NMR theory and methodologies used within this thesis (Chapter IV). Chapter II begins with a description of RNA characteristics and functions, placing the focus on the increasing attention that these biomacromolecules have attracted in recent years due to their diverse biological functionalities. This is followed by a detailed description of general structural features of RNA molecules. The biological functions of the RNAs investigated in this thesis (Human immunodeficiency virus PSI- and TAR-RNA and Coxsackievirus B3 Stemloop D in the 5’-cloverleaf element), together with their known structural characteristics are introduced in Chapter III. Furthermore, a description of the investigated ligands is given, focusing on the methods how their affinity and specificity were determined. The introduction is completed in Chapter IV, where the relevant NMR theory and methodologies are explained. First, kinetics and thermodynamics of ligand binding are summarized from an NMR point of view. Subsequently, a detailed description of the resonance assignment procedures for RNAs and peptidic ligands is given. This procedure mainly concentrates on the assignment of the proton resonances, which are essential for the later structure calculation from NMR restraints. The procedure for NMR structure calculation of RNA and its complexes follows with a short introduction into the programs ARIA and HADDOCK. The final part of this chapter explains the relaxation theory and the methodology to extract dynamic information from autocorrelated relaxation rates via the model-free formalism. In the Chapters V to VII of this thesis, the original publications are included and grouped into three topics. Chapter V comprehends the publications on the investigations of HIV PSI-RNA and its hexapeptidic ligand. These three publications[1-3] focus on the characterization of the ligand and its binding properties, its structure and the optimization of its composition aiming to improve its usage for further spectroscopic investigations.Die vorliegende Doktorarbeit behandelt die strukturelle Aufklärung von Wechselwirkungen zwischen biologisch relevanten Ribonukleinsäuren (RNA) und ausgewählten Liganden, sowie die Bestimmung der inhärenten Dynamik der RNA, um zur Methodenentwicklung für den Entwurf neuer Pharmaka beizutragen. Zur Bestimmung sowohl der Strukturen, als auch der Dynamiken stellt die Flüssig-Kernspinresonanz-Spektroskopie (NMR) eine ideale biophysikalische Methode dar. Die ersten Hälfte dieser Doktorarbeit gibt zum einen eine Einleitung in die RNA-Forschung mit besonderem Fokus auf den allgemeinen strukturellen und dynamischen Eigenschaften von Ribonukleinsäuren, stellt zweitens die ausgewählten RNA-Zielstrukturen und deren mit verschiedenen Methoden bestimmten Liganden vor, und erklärt drittens die zugrundeliegende NMR-Theorie und die verwendeten Methoden zur Untersuchung der Bindungs¬charakteristika, zur Strukturbestimmung der RNA und der Liganden und zur Ableitung dynamischer Parameter aus experimentellen Daten. Die zweite Hälfte dieser Arbeit ist der kumulative Teil und enthält die Originalpublikationen, die in drei Themenbereiche eingeteilt sind. Zuerst sind die drei Publikationen gruppiert, in denen die Bestimmung und Charakterisierung peptidischer Liganden der HIV Psi-RNA und deren Wechselwirkungen miteinander behandelt werden. Durch einen Phage-Display Assay wurde zunächst eine Konsensus¬sequenz eines peptidischen Liganden identifiziert (HWWPWW). Zur Verbesserung der Bindungseigenschaften wurde das Hexapeptid mittels einer Sequenzvariierung auf einer Membranoberfläche (SPOT-Assay) weiter optimiert (HKWPWW). Die weiteren strukturellen Untersuchungen der RNA-Ligand-Wechselwirkungen wurden per Fluoreszenz- und NMR-Spektroskopie durchgeführt, wobei die NMR-Spektroskopie aufzeigen konnte, dass das Peptid HKWPWW in zwei Konformationen der zentralen Prolinpeptidbindung zu beinahe gleichen Anteilen vorliegt. Die nächsten zwei Publikationen beschreiben die Ligandselektion gegen die Zielstruktur HIV TAR und die Strukturaufklärung des Komplexes mittels NMR-Spektroskopie. Als Liganden wurden Tripeptide synthetisiert, in denen zwei Arginine eine synthetische Aminosäure mit aromatischen oder hetero¬aromatischen Gruppierungen in ihrer Seitenkette flankieren. Mittels Fluoreszenz-Resonanz¬energietransfersichtung (FRET-Assay) wurde eine Vorauswahl der Liganden vorgenommen und die Interaktionen der ausgewählten Liganden mit der RNA per NMR-Spektroskopie konkretisiert. Eine intensive strukturelle Untersuchung des Liganden mit einer Pyrimidinylgruppe in der Seitenkette der zentralen Aminosäure in Komplex mit der TAR RNA ergab eine 2:1 Bindungsstöchiometrie des Liganden. Die erste stärkere Bindungsstelle im Bulge der RNA war bereits weitgehend bekannt als Ziel von Arginin-tragenden Liganden. Die strukturellen Untersuchungen konnten jedoch auch die zweite Bindungsstelle des Tripeptids unterhalb des Bulges lokalisieren. Zuletzt sind die zwei Publikationen zur Untersuchung der RNA-Dynamik zusammengefasst. Aus autokorrelierten Relaxationsraten der Kerne C1’ und C8 (für Purine) bzw. C6 (für Pyrimidine) in Nukleotiden der RNA Tetraloopsequenzen UUCG und CACG wurden mittels des Model-Free Formalismus Parameter abgeleitet, die über Dynamiken auf der Zeitskala von Pico- bis Nanosekunden der C-H Vektoren berichten. Die Verwendung optimierter und neuer Werte der C-H Bindungslänge und der Anisotropie der 13C-chemischen Verschiebung (13C-CSA) ermöglichte eine genauere Ableitung der inhärenten Dynamiken dieser RNA Moleküle. Diese Informationen konnten in die strukturellen Untersuchungen der glykosidischen Bindung durch kreuzkorrelierte Relaxationsraten eingebaut werden. Des Weiteren konnten die dynamischen Parameter bei verschiedenen Temperaturen mit Parametern abgeglichen werden, die aus Molekular-Dynamischen (MD) Trajektorien abgeleitet wurden. Dies ermöglichte die Visualisierung der internen Bewegungen zweier strukturell ähnlicher Tetraloops aus der YNMG-Familie, die sich aber in ihrer Stabilität unterscheiden. Bei Temperaturen nahe dem Schmelzpunkt des weniger stabilen CACG-Tetraloops offenbarten sich die Änderungen in der Dynamik, die zum Aufschmelzen des Loops führen

Structural and functional analysis of the archaeal endonuclease Nob1

Eukaryotic ribosome biogenesis requires the concerted action of numerous ribosome assembly factors, for most of which structural and functional information is currently lacking. Nob1, which can be identified in eukaryotes and archaea, is required for the final maturation of the small subunit ribosomal RNA in yeast by catalyzing cleavage at site D after export of the preribosomal subunit into the cytoplasm. Here, we show that this also holds true for Nob1 from the archaeon Pyrococcus horikoshii, which efficiently cleaves RNA-substrates containing the D-site of the preribosomal RNA in a manganese-dependent manner. The structure of PhNob1 solved by nuclear magnetic resonance spectroscopy revealed a PIN domain common with many nucleases and a zinc ribbon domain, which are structurally connected by a flexible linker. We show that amino acid residues required for substrate binding reside in the PIN domain whereas the zinc ribbon domain alone is sufficient to bind helix 40 of the small subunit rRNA. This suggests that the zinc ribbon domain acts as an anchor point for the protein on the nascent subunit positioning it in the proximity of the cleavage site

Multi‐Site Conformational Exchange in the Synthetic Neomycin‐Sensing Riboswitch Studied by 19 F NMR

The synthetic neomycin-sensing riboswitch interacts with its cognate ligand neomycin as well as with the related antibiotics ribostamycin and paromomycin. Binding of these aminoglycosides induces a very similar ground state structure in the RNA, however, only neomycin can efficiently repress translation initiation. The molecular origin of these differences has been traced back to differences in the dynamics of the ligand:riboswitch complexes. Here, we combine five complementary fluorine based NMR methods to accurately quantify seconds to microseconds dynamics in the three riboswitch complexes. Our data reveal complex exchange processes with up to four structurally different states. We interpret our findings in a model that shows an interplay between different chemical groups in the antibiotics and specific bases in the riboswitch. More generally, our data underscore the potential of 19F NMR methods to characterize complex exchange processes with multiple excited states

¹⁹F NMR Untersuchung des Konformationsaustauschs mehrerer Zustände im synthetischen Neomycin‐bindenden Riboschalter

Der synthetische Neomycin-bindende Riboschalter interagiert mit seinem Liganden Neomycin sowie mit den verwandten Antibiotika Ribostamycin und Paromomycin. Die Bindung dieser Aminoglykoside induziert sehr ähnliche Grundzustandsstrukturen in der RNA, allerdings kann nur Neomycin die Initiierung der Translation effizient unterdrücken. Der molekulare Ursprung dieser Unterschiede wurde auf Unterschiede in der Dynamik der Ligand-Riboschalter-Komplexe zurückgeführt. In diesem Artikel kombinieren wir fünf komplementäre fluorbasierte NMR-Methoden, um die Dynamik der drei Riboschalter-Komplexe im Sekunden- bis Mikrosekundenbereich genau zu quantifizieren. Unsere Daten offenbaren komplexe Austauschprozesse mit bis zu vier strukturell unterschiedlichen Zuständen. Wir interpretieren unsere Ergebnisse in einem Modell, das ein Zusammenspiel zwischen verschiedenen chemischen Gruppen in den Antibiotika und spezifischen Basen im Riboschalter zeigt. Allgemeiner unterstreichen unsere Daten das Potenzial von 19F NMR-Methoden, komplexe Austauschprozesse mit mehreren angeregten Zuständen zu charakterisieren

NMR and MD studies of the temperature-dependent dynamics of RNA YNMG-tetraloops

In a combined NMR/MD study, the temperature-dependent changes in the conformation of two members of the RNA YNMG-tetraloop motif (cUUCGg and uCACGg) have been investigated at temperatures of 298, 317 and 325 K. The two members have considerable different thermal stability and biological functions. In order to address these differences, the combined NMR/MD study was performed. The large temperature range represents a challenge for both, NMR relaxation analysis (consistent choice of effective bond length and CSA parameter) and all-atom MD simulation with explicit solvent (necessity to rescale the temperature). A convincing agreement of experiment and theory is found. Employing a principle component analysis of the MD trajectories, the conformational distribution of both hairpins at various temperatures is investigated. The ground state conformation and dynamics of the two tetraloops are indeed found to be very similar. Furthermore, both systems are initially destabilized by a loss of the stacking interactions between the first and the third nucleobase in the loop region. While the global fold is still preserved, this initiation of unfolding is already observed at 317 K for the uCACGg hairpin but at a significantly higher temperature for the cUUCGg hairpin

Revised diagnostic criteria for neurofibromatosis type 1 and Legius syndrome: an international consensus recommendation

Purpose By incorporating major developments in genetics, ophthalmology, dermatology, and neuroimaging, to revise the diagnostic criteria for neurofibromatosis type 1 (NF1) and to establish diagnostic criteria for Legius syndrome (LGSS). Methods We used a multistep process, beginning with a Delphi method involving global experts and subsequently involving non-NF experts, patients, and foundations/patient advocacy groups. Results We reached consensus on the minimal clinical and genetic criteria for diagnosing and differentiating NF1 and LGSS, which have phenotypic overlap in young patients with pigmentary findings. Criteria for the mosaic forms of these conditions are also recommended. Conclusion The revised criteria for NF1 incorporate new clinical features and genetic testing, whereas the criteria for LGSS were created to differentiate the two conditions. It is likely that continued refinement of these new criteria will be necessary as investigators (1) study the diagnostic properties of the revised criteria, (2) reconsider criteria not included in this process, and (3) identify new clinical and other features of these conditions. For this reason, we propose an initiative to update periodically the diagnostic criteria for NF1 and LGSS.</p

Revised diagnostic criteria for neurofibromatosis type 1 and Legius syndrome: an international consensus recommendation.

Funder: Children’s Tumor Foundation; doi: https://doi.org/10.13039/http://dx.doi.org/10.13039/100001545PURPOSE: By incorporating major developments in genetics, ophthalmology, dermatology, and neuroimaging, to revise the diagnostic criteria for neurofibromatosis type 1 (NF1) and to establish diagnostic criteria for Legius syndrome (LGSS). METHODS: We used a multistep process, beginning with a Delphi method involving global experts and subsequently involving non-NF experts, patients, and foundations/patient advocacy groups. RESULTS: We reached consensus on the minimal clinical and genetic criteria for diagnosing and differentiating NF1 and LGSS, which have phenotypic overlap in young patients with pigmentary findings. Criteria for the mosaic forms of these conditions are also recommended. CONCLUSION: The revised criteria for NF1 incorporate new clinical features and genetic testing, whereas the criteria for LGSS were created to differentiate the two conditions. It is likely that continued refinement of these new criteria will be necessary as investigators (1) study the diagnostic properties of the revised criteria, (2) reconsider criteria not included in this process, and (3) identify new clinical and other features of these conditions. For this reason, we propose an initiative to update periodically the diagnostic criteria for NF1 and LGSS

Guidelines for the Li–Fraumeni and heritable TP53 -related cancer syndromes

Abstract: Fifty years after the recognition of the Li–Fraumeni syndrome (LFS), our perception of cancers related to germline alterations of TP53 has drastically changed: (i) germline TP53 alterations are often identified among children with cancers, in particular soft-tissue sarcomas, adrenocortical carcinomas, central nervous system tumours, or among adult females with early breast cancers, without familial history. This justifies the expansion of the LFS concept to a wider cancer predisposition syndrome designated heritable TP53-related cancer (hTP53rc) syndrome; (ii) the interpretation of germline TP53 variants remains challenging and should integrate epidemiological, phenotypical, bioinformatics prediction, and functional data; (iii) the penetrance of germline disease-causing TP53 variants is variable, depending both on the type of variant (dominant-negative variants being associated with a higher cancer risk) and on modifying factors; (iv) whole-body MRI (WBMRI) allows early detection of tumours in variant carriers and (v) in cancer patients with germline disease-causing TP53 variants, radiotherapy, and conventional genotoxic chemotherapy contribute to the development of subsequent primary tumours. It is critical to perform TP53 testing before the initiation of treatment in order to avoid in carriers, if possible, radiotherapy and genotoxic chemotherapies. In children, the recommendations are to perform clinical examination and abdominal ultrasound every 6 months, annual WBMRI and brain MRI from the first year of life, if the TP53 variant is known to be associated with childhood cancers. In adults, the surveillance should include every year clinical examination, WBMRI, breast MRI in females from 20 until 65 years and brain MRI until 50 years

Guidelines for the Li–Fraumeni and heritable TP53 -related cancer syndromes

Abstract: Fifty years after the recognition of the Li–Fraumeni syndrome (LFS), our perception of cancers related to germline alterations of TP53 has drastically changed: (i) germline TP53 alterations are often identified among children with cancers, in particular soft-tissue sarcomas, adrenocortical carcinomas, central nervous system tumours, or among adult females with early breast cancers, without familial history. This justifies the expansion of the LFS concept to a wider cancer predisposition syndrome designated heritable TP53-related cancer (hTP53rc) syndrome; (ii) the interpretation of germline TP53 variants remains challenging and should integrate epidemiological, phenotypical, bioinformatics prediction, and functional data; (iii) the penetrance of germline disease-causing TP53 variants is variable, depending both on the type of variant (dominant-negative variants being associated with a higher cancer risk) and on modifying factors; (iv) whole-body MRI (WBMRI) allows early detection of tumours in variant carriers and (v) in cancer patients with germline disease-causing TP53 variants, radiotherapy, and conventional genotoxic chemotherapy contribute to the development of subsequent primary tumours. It is critical to perform TP53 testing before the initiation of treatment in order to avoid in carriers, if possible, radiotherapy and genotoxic chemotherapies. In children, the recommendations are to perform clinical examination and abdominal ultrasound every 6 months, annual WBMRI and brain MRI from the first year of life, if the TP53 variant is known to be associated with childhood cancers. In adults, the surveillance should include every year clinical examination, WBMRI, breast MRI in females from 20 until 65 years and brain MRI until 50 years