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Sélection d’aptamères anti-adénine ADN modifiés en présence de solvant organique. Application au développement de biocapteurs / Selection of DNA modified aptamer-recognizing adenine in organic media : application to biosensor developmentChaou, Thinhinane 16 December 2015 (has links)
Le développement d’outils de détection in situ (IDT) est indispensable pour rapporter entemps réel la présence d’une signature moléculaire spécifique. L’efficacité d’un IDT estliée à son affinité, à sa spécificité, mais aussi à son potentiel à opérer dans des conditionsimposées par le contexte d’application. Parmi les contraintes imposées, la variation detempérature, le pH et la présence de solvant d’extraction. Le but du projet est dedévelopper des aptamères capables d’opérer en présence de solvant organique ; à ceteffet, nous avons opté pour une sélection en présence de méthanol. La limitation decette stratégie est principalement liée à la nature chimique des acides nucléiques. Parconséquent, nous avons choisi d’utiliser une banque ADN incorporant le (5-(octa1,7-diynyl)-2’-deoxyuridine) dDOTP, au lieu du dTTP. Notre stratégie expérimentale aabouti à la sélection d’un aptamère qui lie spécifiquement l’adénine en présence de 25 %de méthanol. Nous avons montré que les dDOTP sont essentiels à l’interaction del’aptamère avec l’adénine ; l’aptamère sélectionné comporte un motif riche en G partagéavec l’aptamère anti-adénosine, cependant, l’aptamère sélectionné dans le méthanolcomporte un motif structural indispensable à l’interaction avec la cible en présence deméthanol. Par ailleurs, nous avons montré que l’aptamère sélectionné pourraitfonctionner comme un module de reconnaissance spécifique d’un biocapteur. / Development of in situ detection tools (IDT) is required for specific real time monitoringof chemical species. Efficient IDT is not only related to its affinity and ability todiscriminate between molecular variants, but moreover it must be adapted for operatingunder conditions imposed by the context of application. Among others, hightemperature, pH variation and presence of organic solvents may be mentioned. The aimof our project is to develop an aptamer operating in the presence of organic solvents. Forthis purpose, we opted for selection in presence of methanol. Limitations of this strategyare adaptability of SELEX technology and chemical diversity of nucleic acids. For thispurpose, we used library incorporating (5-(octa1,7-diynyl)-2’-deoxyuridine) dDOTPinstead of conventional thymine nucleotide. Our experimental strategy led to theselection of an aptamer that specifically recognises adenine in the presence of 25 % ofmethanol. dDOTP nucleotides are essential for adenine recognition; the selectedaptamer shares a purine rich motif with a previously described adenosine aptamer, butdisplays a specific structure motif, essential for operating in the presence of methanol.Furthermore, the ability of truncated variants of the selected aptamer to form arecognition module of biosensor was assessed in this study.
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Investigation of RNA kissing complexes by native electrospray mass spectrometry : magnesium binding and ion mobility / Etudes de « kissing complexes » d’ARN par spectrométrie de masse native : liaison du magnésium et spectrométrie de mobilité ioniqueRabin, Clemence 19 December 2017 (has links)
En plus d’être l’intermédiaire entre l’ADN et les protéines, l’ARN est impliqué dans plusieurs processus biologiques : régulation et expression des gènes (riboswitches, ARNm et ARNt) ou encore catalyse (ribozymes). La fonction de chaque ARN est liée à sa structure et à sa dynamique de repliement. Des cations tel que le magnésium se lient à l’ARN et peuvent être essentiels au bon repliement et à la stabilité de ces structures. L’obtention de détails structuraux et thermodynamiques sur l’interaction avec le magnésium a donc une grande importance dans la compréhension de la relation structure-fonction. La première partie de ce travail a consisté en la caractérisation des équilibres de liaison entre le magnésium et des motifs d’ARN modèles, appelés « kissing complexes », par spectrométrie de masse native (SM). Grâce à la SM, il est possible de distinguer les stoechiométries de liaison du magnésium. Le travail présenté ici a permis l’élaboration d’une méthode pour quantifier chaque espèce en prenant en compte la distribution d’adduits non-spécifiques. Afin d’aller plus loin dans la localisation du magnésium, nous avons utilisé la spectrométrie de masse en tandem (SM/SM). Nous avons également étudié le comportement des complexes d’ARN en phase gazeuse en utilisant la spectrométrie de mobilité ionique (SMI), avec pour but de détecter des changements de conformation dus à la liaison de cations ou ligands. Contrairement à ce qui était anticipé, nous avons démontré que les duplexes d’ADN et ARN ainsi que les « kissing complexes » subissaient une compaction significative en phase gazeuse aux états de charge initialement obtenus par SM native, ce qui pourrait cacher l’effet des cations. Notre travail a montré comment la spectrométrie de masse peut apporter de nouvelles indications sur les stoechiométries et affinités entre ARN et cations, et discute de certaines limitations quant à l’utilisation de techniques en phase gazeuse pour explorer les structures en solution. / Besides being the molecular intermediate between DNA and proteins, RNA can have many other functions such as gene regulation (riboswitches), gene expression (mRNA and tRNA) or catalysis (ribozymes). RNA function is linked to its structure and its folding dynamics. Cations such as magnesium bind to RNA and are in some instances essential for proper folding and for stability. The need of structural and thermodynamic details about Mg2+ interactions is then of upmost importance in the study of the structurefunction relationships. The first part of our work consists in characterizing the binding equilibria between magnesium and RNA model motifs, called kissing complexes, using native mass spectrometry (MS). MS makes it possible to distinguish individual binding stoichiometries, and the present work consisted in developing a method to quantify each species, taking into account the contribution of nonspecific adducts. We also explored how tandem mass spectrometry (MS/MS) could further help localizing magnesium ions. Further, we explored the structures of RNA complexes in the gas phase using ion mobility mass spectrometry (IMMS), with the aim to detect shape changes upon cation or ligand binding. But in contrast with anticipations, we found that DNA and RNA duplexes as well as RNA kissing complexes undergo a significant compaction at charge states naturally produced by native ESI-MS, which may hide the effect of cations. Our work showcases how mass spectrometry can bring novel information on RNA-cation binding stoichiometries and affinities, but also discusses some limitations of a gas-phase method to probe solution structures.
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Exploring Protein-Nucleic Acid Interactions Using Graph And Network ApproachesSathyapriya, R 03 1900 (has links)
The flow of genetic information from genes to proteins is mediated through proteins which interact with the nucleic acids at several stages to successfully transmit the information from the nucleus to the cell cytoplasm. Unlike in the case of protein-protein interactions, the principles behind protein-nucleic acid interactions are still not very (Pabo and Nekludova, 2000) and efforts are still underway to arrive at the basic principles behind the specific recognition of nucleic acids by proteins (Prabakaran et al., 2006). This is mainly due to the innate complexity involved in recognition of nucleotides by proteins, where, even within a given family of DNA binding proteins, different modes of binding and recognition strategies are employed to suit their function (Luscomb et al., 2000). Such difficulties have also not made possible, a thorough classification of DNA/RNA binding proteins based on the mode of interaction as well as the specificity of recognition of the nucleotides.
The availability of a large number of structures of protein-nucleic acids complexes (albeit lesser than the number of protein structures present in the PDB) in the past few decades has provided the knowledge-base for understanding the details behind their molecular mechanisms (Berman et al., 1992). Previously, studies have been carried out to characterize these interactions by analyzing specific non-covalent interactions such as hydrogen bonds, van der Walls, and hydrophobic interactions between a given amino acid and the nucleic acid (DNA, RNA) in a pair-wise manner, or through the analysis of interface areas of the protein-nucleic acid complexes (Nadassy et al., 1998; Jones et al., 1999). Though the studies have deciphered the common pairing preferences of a particular amino acid with a given nucleotide of DNA or RNA, there is little room for understanding these specificities in the context of spatial interactions at a global level from the protein-nucleic acid complexes. The representation of the amino acids and the nucleotides as components of graphs, and trying to explore the nature of the interactions at a level higher than exploring the individual pair-wise interactions, could provide greater details about the nature of these interactions and their specificity. This thesis reports the study of protein-nucleic interactions using graph and network based approaches. The evaluation of the parameters for characterizing protein-nucleic acid graphs have been carried out for the first time and these parameters have been successfully employed to capture biologically important non-covalent interactions as clusters of interacting amino acids and nucleotides from different protein-DNA and protein-RNA complexes.
Graph and network based approaches are well established in the field of protein structure analysis for analyzing protein structure, stability and function (Kannan and Vishveshwara, 1999; Brinda and Vishveshwara, 2005). However, the use of graph and network principles for analyzing structures of protein-nucleic acid complexes is so far not accomplished and is being reported the first time in this thesis. The matter embodied in the thesis is presented as ten chapters. Chapter 1 lays the foundation for the study, surveying relevant literature from the field. Chapter 2 describes in detail the methods used in constructing graphs and networks from protein-nucleic acid complexes. Initially, only protein structure graphs and networks are constructed from proteins known to interact with specific DNA or RNA, and inferences with regard to nucleic acid binding and recognition were indirectly obtained . Subsequently, parameters were evaluated for representing both the interacting amino acids and the nucleotides as components of graphs and a direct evaluation of protein-DNA and Protein-RNA interactions as graphs has been carried out.
Chapter 3 and 4 discuss the graph and network approaches applied to proteins from a dataset of DNA binding proteins complexed with DNA. In chapter 3, the protein structure graphs were constructed on the basis of the non-covalent interactions existing between the side chains of amino acids. Clusters of interacting side chains from the graphs were obtained using the graph spectral method. The clusters from the protein-DNA interface were analyzed in detail for the interaction geometry and biological importance (Sathyapriya and Vishveshwara, 2004). Chapter 4 also uses the same dataset of DNA binding proteins, but a network-based approach is presented. From the analysis of the protein structure networks from these DNA binding proteins, interesting observations relating the presence of highly connected nodes(or hubs) of the network to functionally important amino acids in the structure, emerged. Also, the comparison between the hubs identified from the protein-protein and the protein-DNA interfaces in terms of their amino acid composition and their connectivity are also presented (Sathyapriya and Vishveshwara, 2006)
Chapter 5 and 6 deal with the graph and network applications to a specific system of protein-RNA complex (aminoacyl-tRNA synthetases) to gain insights into their interface biology based on amino acid connectivity. Chapter 5 deals with a dataset of aminoacyl-tRNA synthetase (aaRS) complexes obtained with various ligands like ATP, tRNA and L-amino acids. A graph based identification of side chain clusters from these ligand-bound aaRS structures has highlighted important features of ligand-binding at the catalytic sites of the two structurally different classes of aaRS (Class I and Class II). Side chain clusters from other regions of aaRS such as the anticodon binding region and the ligand-activation sites are discussed.
A network approach is used in a specific system of aaRS(E.coli Glutaminyl-tRNA synthetase (GlnRS) complexed with its ligands, to specifically understand the effects of different ligand binding., in chapter 6. The structure networks of E.coli GlnRS in the ligand-free and different ligand-bound states are constructed. The ligand-free and the ligand-bound complexes are compared by analyzing their network properties and the presence of hubs to understand the effect of ligand-binding. These properties have elegantly captured the effects of ligand-binding to the GlnRS structure and have also provided an alternate method for comparing three dimensional structures of proteins in different ligand-bound states (Sathyapriya and Vishveshwara, 2007).
In contrast to protein structure graphs (PSG), both the interacting amino acids and nucleotides (DNA/RNA) form the components of the protein-nucleic acid graphs (PNG) from protein-nucleic acid complexes. These graphs are constructed based on the non-covalent interactions existing between the side chains of the amino acids and nucleotides.
After representing the interacting nucleotides and amino acids as graphs, clusters of the interacting components are identified. These clusters are the strongly interacting amino acids and nucleotides from the protein-nucleic acid complexes. These clusters can be generated at different strengths of interaction between the amino acid side chain and the nucleotide (measured in terms of its atomic connectivity) and can be used for detecting clusters of non-specific as well as specific interactions of amino acids and nucleotides. Though the methodology of graph construction and cluster identification are given in chapter 2, the details of the parameters evaluated for constructing PNG are given in chapter 7. Unlike in the previous chapters, the succeeding chapters deal exclusively with results that are obtained from the analyses of PNG. Two examples of obtaining clusters from a PNG are given, one each for a protein-DNA and a protein-RNA complex. In the first example, a nucleosome core particle is subjected to the graph based analysis and different clusters of amino acids with different regions of the DNA chain such as phosphate, deoxyribose sugar and the base are identified. Another example of aminoacyl-tRNA synthetase complexed with its cognate tRNA is used to illustrate the method with a protein-RNA complex. Further, the method of constructing and analyzing protein-nucleic acid graphs has been applied to the macromolecular machinery of the pre-translocation complex of the T. thermophilus 70S ribosome. Chapter 8 deals exclusively with the results identified from the analysis of this magnificent macromolecular ensemble. The availability of the method that can handle interactions between both amino acids and the nucleotides of the protein-nucleic acid complexes has given us the basis fro evaluating these interactions in a level higher than that of analyzing pair-wise interactions.
A study on the evaluation of short hydrogen bonds(SHB) in proteins, which does not fall under the realm of the main objective of the thesis, is discussed in the Chapter 9. The short hydrogen bonds, defined by the geometrical distance and angle parameters, are identified from a non-redundant dataset of proteins. The insights into their occurrence, amino acid composition and secondary structural preferences are discussed. The SHB are present in distinct regions of protein three-dimensional structures, such that they mediate specific geometrical constraints that are necessary for stability of the structure (Sathyapriya and Vishveshwara, 2005).
The significant conclusions of various studies carried out are summarized in the last chapter (Chapter 10). In conclusion, this thesis reports the analyses performed with protein-nucleic acid complexes using graph and network based methods. The parameters necessary for representing both amino acids and the nucleotides as components of a graph, are evaluated for the first time and can be used subsequently for other analyses. More importantly, the use of graph-based methods has resulted in considering the interaction between the amino acids and the nucleotides at a global level with respect to their topology of the protein-nucleic acid complexes. Such studies performed on a wide variety of protein-nucleic acid complexes could provide more insights into the details of protein-nucleic acid recognition mechanisms. The results of these studies can be used for rational design of experimental mutations that ascertain the structure-function relationships in proteins and protein-nucleic acid complexes.
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Method development for biomolecular solid-state NMR spectroscopyAsami, Sam 17 October 2014 (has links)
Im Rahmen der vorliegenden Arbeit, wird ein neuartiges Markierungsschema für die Festkörper-NMR-Spektroskopie vorgestellt, das sogenannte Reduced Adjoining Protonation (RAP) Schema, welches die Protonendetektion sämtlicher Aliphaten erlaubt. Hochaufgelöste, 1H-detektierte 1H,13C Korrelationsspektren wurden erhalten. Des Weiteren wurde der Vorteil von hohen MAS-Frequenzen untersucht. 1H- und 13C-detektierte 3D Zuordnungsexperimente wurden implementiert, welche uns die Zuordnung von 90% aller aliphatischen Resonanzen von alpha-Spektrin SH3 erlaubten. Da die chemische Verschiebung abhängt vom Strukturmotiv, kann sie verwendet werden, um Sekundärstruktur-Informationen abzuleiten. Darüber hinaus wurde ein 1H-detektiertes H(H)CH 3D Experiment entwickelt, um weitreichende 1H,1H Kontakte zu ermitteln, welche für die Bestimmung der Tertiärstruktur genutzt werden können. Um artefaktfreie Relaxationsdaten zu erhalten, wurde das RAP-Markierungsschema modifiziert, um 1H- und 13C-verdünnte Proben zu erhalten, in denen Spindiffusion unterdrückt ist. Für die Untersuchung von Sub-Mikrosekunden-Dynamik werden Experimente vorgestellt zur Bestimmung von 13C T1 Relaxationszeiten und 1H,13C dipolaren Kopplungstensoren für Rückgrat- und Seitenketten-Resonanzen. Des weiteren zeigen wir, dass das RAP-Markierungsschema auf nicht-kristalline Systeme, wie Amyloidfibrillen des Abeta1-40 Peptids der Alzheimer-Krankheit, angewendet werden kann. Unter Verwendung von 1H-Detektion, erhielten wir hochaufgelöste 1H,13C Korrelationsspektren. Schließlich wurde der Perdeuterierungsansatz auf den L7Ae-box C/D Protein-RNA Komplex aus P. furiosus angewendet. Wir erhielten hochaufgelöste, 1H-detektierte 1H,15N, sowie 13C,13C Korrelationsspektren des Protein-RNA Komplexes. Weiterhin haben wir eine Methode zur Bestimmung genauer Abstands- und Winkelinformationen für die Protein-RNA Schnittstelle etabliert und schlagen Ansätze vor, für die Zuordnung der chemischen Verschiebungen von RNA-Resonanzen. / In this thesis, a novel labeling scheme for solid-state NMR spectroscopy, the Reduced Adjoining Protonation (RAP) scheme, is introduced, which allows proton detection of all aliphatic sites, as shown for the microcrystalline SH3 domain of alpha-spectrin. These samples yield high-resolution, 1H-detected 1H,13C correlation spectra. In addition, the benefit of high MAS frequencies was investigated. 1H- and 13C-detected 3D assignment experiments are implemented, which allowed us to assign 90% of all aliphatic resonances of alpha-spectrin SH3. As the chemical shift is dependent on the structural motif, it can be employed to derive secondary structure information. Furthermore, a 1H-detected H(H)CH 3D experiment is introduced, to obtain long-range 1H,1H contacts, which can be used for the determination of the tertiary structure. To obtain artifact-free relaxation data, the RAP labeling scheme was modified to obtain sparsely proton labeled, 13C dilute samples, in which spin diffusion is suppressed. To probe sub-microsecond dynamics, we report experiments to determine 13C T1 relaxation times and 1H,13C dipolar coupling tensors for backbone and side chain resonances, respectively. Furthermore, we show, that the RAP labeling scheme can be applied to non-crystalline systems, such as amyloid fibrils of the Alzheimer’s disease peptide Abeta1-40. Using 1H-detection, we obtained high-resolution 1H,13C correlation spectra. Finally, we applied the perdeuteration approach to the L7Ae-box C/D protein-RNA complex from P. furiosus. We obtained high-resolution, 1H-detected 1H,15N, as well as 13C,13C correlation spectra of the protein-RNA complex. In addition, we established a methodology to determine accurate distance and angular restraints for the protein-RNA interface and propose approaches for the chemical shift assignment of RNA resonances.
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