The 3′ Splice Site of Influenza A Segment 7 mRNA Can Exist in Two Conformations: A Pseudoknot and a Hairpin

The 3′ splice site of influenza A segment 7 is used to produce mRNA for the M2 ion-channel protein, which is critical to the formation of viable influenza virions. Native gel analysis, enzymatic/chemical structure probing, and oligonucleotide binding studies of a 63 nt fragment, containing the 3′ splice site, key residues of an SF2/ASF splicing factor binding site, and a polypyrimidine tract, provide evidence for an equilibrium between pseudoknot and hairpin structures. This equilibrium is sensitive to multivalent cations, and can be forced towards the pseudoknot by addition of 5 mM cobalt hexammine. In the two conformations, the splice site and other functional elements exist in very different structural environments. In particular, the splice site is sequestered in the middle of a double helix in the pseudoknot conformation, while in the hairpin it resides in a two-by-two nucleotide internal loop. The results suggest that segment 7 mRNA splicing can be controlled by a conformational switch that exposes or hides the splice site

BIOPHYSICAL INVESTIGATIONS OF THE THREE-WAY JUNCTION IN PHI29-LIKE BACTERIOPHAGE PROHEAD RNA

The knowledge gained from biophysical studies of RNA can contribute to basic research and inform new directions for translational research. In particular, information that develops the relationships among RNA energetics, physiologic stability, and structure can contribute to better predictions of RNA structure from sequence and guide the rational design of functional supramolecular structures such as targeted therapeutics. A conserved three-way junction (3WJ) in noncoding viral prohead or packaging RNA (pRNA) uniquely captures many aspects of RNA structure and function and represents a model system for studying these relationships. This dissertation presents thermodynamics for pRNA 3WJs. Phylogenetically related 3WJs have a wide range of thermodynamic stabilities, and some sequences are more stable than a phi29 3WJ sequence that is used commonly in RNA nanotechnology. The range of thermodynamic stabilities observed is not explained fully by differences in sequence at the 3WJ core. The prototype phi29 pRNA sequence is used commonly in nanotechnology, for example as a nanoparticle delivery vector for therapeutic drugs. Related sequences are underexplored. The successful translation of phi29-alternative 3WJs into clinical practice will depend on energetic stability and the ability to overcome several key physiological barriers. This dissertation also presents 3WJ stabilities in human blood serum and in cells. Energetic and physiologic stabilities are not always correlated, but some 3WJs are both more thermodynamically stable and more stable in serum than the phi29 3WJ. The results of cell uptake studies support the idea that phi29-alternative 3WJs may be useful in nanotechnology, for example, in stabilizing RNAs for drug delivery. The coaxial stacking of helices across RNA 3WJs can be thermodynamically favorable and may account for some of the observed differences in 3WJ stabilities. Transmission electron microscopy (TEM) data for a helix-extended construct indicate that helices are distributed equally around the phi29 3WJ. The determination of the relative orientation of helices around the pRNA 3WJ can help shed light on how sequence and secondary structure at the 3WJ core influence global structure and may provide insight into why some 3WJs are more energetically and physiologically stable than others. The work presented here contributes to parameters for better RNA structure prediction from sequence, expands the number of useful building blocks for RNA nanotechnology, and provides a method for further investigating structural mechanisms underlying pRNA stability and self-assembly

Complexity in the binding of minor groove agents: netropsin has two thermodynamically different DNA binding modes at a single site

Structural results with minor groove binding agents, such as netropsin, have provided detailed, atomic level views of DNA molecular recognition. Solution studies, however, indicate that there is complexity in the binding of minor groove agents to a single site. Netropsin, for example, has two DNA binding enthalpies in isothermal titration calorimetry (ITC) experiments that indicate the compound simultaneously forms two thermodynamically different complexes at a single AATT site. Two proposals for the origin of this unusual observation have been developed: (i) two different bound species of netropsin at single binding sites and (ii) a netropsin induced DNA hairpin to duplex transition. To develop a better understanding of DNA recognition complexity, the two proposals have been tested with several DNAs and the methods of mass spectrometry (MS), polyacrylamide gel electrophoresis (PAGE) and nuclear magnetic resonance spectroscopy in addition to ITC. All of the methods with all of the DNAs investigated clearly shows that netropsin forms two different complexes at AATT sites, and that the proposal for an induced hairpin to duplex transition in this system is incorrect

Stability and Kinetics of DNA Pseudoknots: Formation of T∗A•T Base-Triplets and Their Targeting Reactions

Pseudoknots have been found to play important roles in the biology of RNA. These stem-loop motifs are considered to be very compact and the targeting of their loops with complementary strands is accompanied with lower favorable free energy terms. We used a combination of spectroscopic (UV, CD and fluorescence), calorimetric (DSC, PPC and ITC) and kinetic (SPR) techniques to investigate: 1) Local base-triplet formation in pseudoknots; 2) energetic contributions for the association of pseudoknots with their complementary strands; and 3) the kinetic rates as a function of targeting strand length. We investigated a set of DNA pseudoknots with sequence: d(TCTCTTnAAAAAAAAGAGAT5TTTTTTT), where “Tn” is a thymine loop with n = 5, 7, 9, and 11. The favorable folding of each pseudoknot resulted in favorable enthalpy-entropy compensation, correlated to favorable base-pair stacking contributions and unfavorable uptakes of ions and water molecules. The increase in the length of the loop yielded higher TMs, 53°C to 59°C and folding enthalpies ranging from -60 to -110 kcal/mol, resulting in a significant stabilization, ΔG°(5) = -8.5 to -16.6 kcal/mol, which is consistent with the formation of 1-2 TAT/TAT base-triplet stacks. The PPC results yielded folding volume changes, ΔVs, ranging from 18 to 23 ml/mol, indicating the higher volume of the folded pseudoknots is due to the uptake of both water (ΔnW of -11 to -24 mol H2O/mol) and ions (Δnion of -2.5 to -4.1 mol Na+/mol). We use ITC and DSC to determine thermodynamic profiles for the reaction of pseudoknots with partially complementary strands. We obtained favorable reaction free energies terms. However, the targeting of compact pseudoknots containing local base-triplets is less favorable due to their larger folding free energy term. The SPR data indicated that the rate of association, kon, decreases while the rate of dissociation, koff, increases as the length of the targeting strand increases, which yielded increasing KD, app.. This indicates the affinity of the target strand to the pseudoknot decreases as the length of the target strand increases. A similar trend was obtained when dissociation constants, KD, DSC, were measured from DSC Hess cycles. However, the KD, DSC were much smaller. This apparent discrepancy between these techniques is that SPR is measuring both the initial association and initial dissociation rates of steady state equilibrium states, while DSC measures true equilibrium states of the entire molecules

Whole-genome mapping of APOBEC mutagenesis in metastatic urothelial carcinoma identifies driver hotspot mutations and a novel mutational signature

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) enzymes mutate specific DNA sequences and hairpin-loop structures, challenging the distinction between passenger and driver hotspot mutations. Here, we characterized 115 whole genomes of metastatic urothelial carcinoma (mUC) to identify APOBEC mutagenic hotspot drivers. APOBEC-associated mutations were detected in 92% of mUCs and were equally distributed across the genome, while APOBEC hotspot mutations (ApoHMs) were enriched in open chromatin. Hairpin loops were frequent targets of didymi (twins in Greek), two hotspot mutations characterized by the APOBEC SBS2 signature, in conjunction with an uncharacterized mutational context (Ap[C&gt;T]). Next, we developed a statistical framework that identified ApoHMs as drivers in coding and non-coding genomic regions of mUCs. Our results and statistical framework were validated in independent cohorts of 23 non-metastatic UCs and 3,744 samples of 17 metastatic cancers, identifying cancer-type-specific drivers. Our study highlights the role of APOBEC in cancer development and may contribute to developing novel targeted therapy options for APOBEC-driven cancers.</p

Whole-genome mapping of APOBEC mutagenesis in metastatic urothelial carcinoma identifies driver hotspot mutations and a novel mutational signature

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) enzymes mutate specific DNA sequences and hairpin-loop structures, challenging the distinction between passenger and driver hotspot mutations. Here, we characterized 115 whole genomes of metastatic urothelial carcinoma (mUC) to identify APOBEC mutagenic hotspot drivers. APOBEC-associated mutations were detected in 92% of mUCs and were equally distributed across the genome, while APOBEC hotspot mutations (ApoHMs) were enriched in open chromatin. Hairpin loops were frequent targets of didymi (twins in Greek), two hotspot mutations characterized by the APOBEC SBS2 signature, in conjunction with an uncharacterized mutational context (Ap[C&gt;T]). Next, we developed a statistical framework that identified ApoHMs as drivers in coding and non-coding genomic regions of mUCs. Our results and statistical framework were validated in independent cohorts of 23 non-metastatic UCs and 3,744 samples of 17 metastatic cancers, identifying cancer-type-specific drivers. Our study highlights the role of APOBEC in cancer development and may contribute to developing novel targeted therapy options for APOBEC-driven cancers.</p

In silico modelling of RNA-RNA dimer and its application for rational siRNA design and ncRNA target search

Non-protein coding region, which constitutes 98.5% of the human genome, were long depreciated as evolutive relict. It is only recently that the biological relevance of\ud the non-coding RNAs associated with these non-coding regions was recognized. The development of experimental and bioinformatical methods aimed at detecting these non-coding RNAs (ncRNAs) lead to the discovery of more than 29,000,000 sequences, grouped into more than 1300 families. More often than not these ncRNAs function by binding to other RNAs, either pro- tein coding or non-protein coding. Compared to the number of tools to detect and classify ncRNAs, the number of tools to search for putative RNA binding partners is negligible. This leads to the actual situation where the function of the majority of the annotated ncRNAs genes is completely unknown. The aim of this work is to assess the function of different families of ncRNAs by developing new algorithms and methods to study RNA-RNA interactions. These new methods are extensions of RNA-folding algorithms applied to the problem of RNA- RNA interactions. Depending on the class of ncRNA studied, different methods were developed and tested. This work shows that the development of RNA-folding algorithms to study RNA- RNA interactions is a promising way to functionally annotate ncRNAs. Still other factors like RNA-proteins interaction, RNA-concentration or RNA-expression, play an important role in the process of RNA hybridization and will have to be taken into account in future works in order to achieve reliable prediction of RNA binding partners.Non-protein coding region, which constitutes 98.5% of the human genome, were long depreciated as evolutive relict. It is only recently that the biological relevance of the non-coding RNAs associated with these non-coding regions was recognized. The development of experimental and bioinformatical methods aimed at detecting these non-coding RNAs (ncRNAs) lead to the discovery of more than 29,000,000 sequences, grouped into more than 1300 families. More often than not these ncRNAs function by binding to other RNAs, either pro- tein coding or non-protein coding. Compared to the number of tools to detect and classify ncRNAs, the number of tools to search for putative RNA binding partners is negligible. This leads to the actual situation where the function of the majority of the annotated ncRNAs genes is completely unknown. The aim of this work is to assess the function of different families of ncRNAs by developing new algorithms and methods to study RNA-RNA interactions. These new methods are extensions of RNA-folding algorithms applied to the problem of RNA- RNA interactions. Depending on the class of ncRNA studied, different methods were developed and tested. This work shows that the development of RNA-folding algorithms to study RNA- RNA interactions is a promising way to functionally annotate ncRNAs. Still other factors like RNA-proteins interaction, RNA-concentration or RNA-expression, play an important role in the process of RNA hybridization and will have to be taken into account in future works in order to achieve reliable prediction of RNA binding partners

Approaches for studying RNA aptamers with molecular dynamics simulation

The objective of this dissertation is to study RNA aptamers with molecular dynamics simulation. It addresses fundamental challenges associated with RNA aptamers that can be investigated via molecular dynamics simulation, including the unavailability of 3D structures for the apo state, the challenge of ensuring good sampling for a flexible molecule, and the uncertainties that accompany molecular properties. The results presented in this dissertation focus on the application of multiple independent simulations to address these issues. I present results from multiple independent molecular dynamics simulations that are started from selected de novo predicted structures, according to experimentally determined base stacking, as a workflow to characterize the flexible apo state of an aptamer. I systematically investigate the sampling of multiple independent simulations by studying the nonlinear dynamic behavior, including principal component analysis and multivariate recurrent quantification analysis. I further propose a simulation assessment approach based on the root mean square deviation (RMSD) matrix eigenvalue and estimate molecular properties of interest with rigorous statistical analysis. I first develop a workflow that combines computational modeling and fluorescence experiments to study the structure and dynamics of the aptamer apo state. The selected predicted structures pass rounds of clustering and satisfy the stacking condition of critical bases in apo state determined from experiments. Multiple independent simulations from these selected structures effectively achieve better sampling than using the available NMR complex structure with ligand removed. It is also noticed that when the backbone is well aligned, a different base at the same position might also be potential binding site. This provides insight to the ligand binding mechanism, specifically, whether the flexible terminal loop adjust its whole structure or a critical base flips to fit the ligand. With the evidence that multiple molecular dynamics simulations can be used to investigate the conformation of aptamer for situations where a 3D structure is not available, I next investigate how well multiple independent simulations from different initial conformations sample the conformational space. The sampling of simulations started from different predicted structures is compared both qualitatively and quantitatively. The projection of sampled structures on selected principal components axes shows overlap among different groups of simulations as well as regions visited only by a specific group. The sampling of different groups of simulations is then further compared via recurrence quantification analysis using the top 10 principal components. The minimum length required for each independent simulation is determined. The number of independent simulations for sufficient sampling of the system is recommended based on the standard error of the mean for the molecular property of interest. Once the number of independent simulations and the minimum length of each simulation are known, it is necessary to systematically perform rigorous statistical analysis on any property of interest. Examination of the simulation quality can be done by looking at the progress of the largest eigenvalue from the RMSD matrix. Simulations or sections of simulations can be grouped as repeated measurements or enrichment, which further determines the uncertainty calculation. I recommend such a procedure because the sampling achieved with molecular dynamics simulations performed with limited timescales might display dependence on the initial conditions. This would lead to an outcome where different simulations could exhibit different error. I urge that care be taken in analyzing simulation outcomes and emphasize that taking the average is not sufficient

On the performance characterization and evaluation of RNA structure prediction algorithms for high performance systems

Ph.DDOCTOR OF PHILOSOPH

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