No description available.

It is now widely known that some nucleic acid molecules, either DNA or RNA, are capable of forming intricate three-dimensional structures and carrying out functions of molecular recognition and catalysis. Most of known functional nucleic acids are isolated from DNA or RNA pools with random sequences using the technique of in vitro selection. With intensive research for the past three decades, a variety of functional nucleic acids have been discovered and examined for potential applications. The general objective of this thesis is to expand the repertoire of functional nucleic acids via new in vitro selection experiments and pursue their biosensing applications. I started by asking the question of whether it is possible to develop a new kind of functional nucleic acids: chimeric RNA/DNA substrates that have high activity for ribonuclease H2 from the important bacterial pathogen Clostridium difficile but much reduced activity towards the same enzymes from other bacterial species. The key rationale behind pursuing these special functional nucleic acids is my hypothesis that these molecules can eventually be developed into useful biosensors for diagnosing Clostridium difficile infection. For this reason, in my first project, I applied the in vitro selection technique to a random-sequence DNA pool, obtained several highly selective chimeric RNA/DNA substrates, and carried out in-depth analysis of their reactivities and their structural properties. During this study, I accidentally discovered a family of highly guanine-rich DNA molecules that are able to form an unusual guanine-quadruplex structure in 7 molar urea, a strong denaturing condition for nucleic acid structures. This discovery constitutes a novel observation and therefore, in my second project, I fully characterized the sequence and structural properties of these special DNA molecules and established the conditions that allow these molecules to create stable structures in 7 molar urea. I then got interested in devising a unique application to take advantage of the urea-resistant property exhibited by these molecules. Towards this end, in my third project, I used one such DNA molecule to set up a DNA detection method capable of detecting single nucleotide polymorphism in very long DNA sequences, a desired application that has never been demonstrated before. The findings made in these projects contribute to the ever-growing appreciation of functional capability and practical utility of nucleic acids. / Thesis / Doctor of Philosophy (PhD)

A G-quadruplex (GQ) is a type of noncanonical nucleic acid structure that can form in regions of nucleic acids rich in guanine nucleotides. The guanine bases form a square planar conformation via Hoogsteen hydrogen bonding. These stacked tetrads have inward-facing carbonyl oxygens, facilitating the coordination of ions. Improper GQ conformations can lead to improper regulation of gene expression, potentially resulting in genetic diseases or cancer. Here, we performed molecular dynamics simulations using the Drude polarizable force field (FF) to gain insight into factors contributing to the stability of two GQs. One is the bcl-2 promoter region GQ, which is implicated in several types of cancer including B-cell lymphoma, and the second is the sc1 RNA GQ, which binds to the Fragile-X Mental Retardation Protein (FMRP) and is implicated in the development of Fragile X Syndrome (FXS). Aberrant bcl-2 GQ conformations result in increased production of the BCL2 protein, which is an apoptosis inhibitor. As such, we aim to characterize the factors stabilizing the GQ for future small-molecule development to prevent apoptosis inhibition and therefore cancer. The FMRP protein functions as a regulator of sc1 conformation to control the translation of proteins required for frontal lobe development. FXS arises from a nonsense mutation that causes the deletion of the C-terminal region of FMRP, rendering it non-function. Therefore, we aim to simulate sc1 when FMRP is bound as well as unbound to provide insight into the types of interactions that must be maintained and therefore mimicked by a small molecule drug. / Master of Science in Life Sciences / DNA is commonly represented as a double helix and RNA is thought of as a simple single stranded, disordered molecule, but DNA and RNA can both adopt more complicated structures. An example of this is the G-quadruplex (GQ), a structure that can form in regions of DNA and RNA that are rich in guanine. These guanine bases form a stable core structure that can act as an "on-off" switch for different processes in the cell. Alterations to GQ structure can lead to dysfunction and different types of disease. Here, we perform atomistic computer simulations to further understand factors that contribute to GQ stability, focusing on two different GQs, one of plays a role in several types of cancer, and the other whose regulation is in Fragile X Syndrome (FXS). Furthermore, we study the Fragile X Mental Retardation Protein, which is what brain cells normally use to regulate expression of proteins needed for frontal lobe development by modulating specific GQ structure. The information from these simulations can be used to potentially develop drugs for these conditions.

Les acides nucléiques simple-Brins contenant des répétitions de guanines peuvent former des structures secondaires non canoniques dites G-Quadruplexes, composées de plusieurs couches de quartets de guanine. Malgré de nombreuses études in vivo, les preuves de présence de structures quadruplexes in vivo restent indirectes. L’objectif de ce travail était la recherche de sondes fluorescentes capables de signaler la présence d'ADN quadruplex et détecter sa structure (topologie).Deux séries de sondes fluorescentes ont été envisagées et préparées : les colorants styryles (majoritairement distyryles) et les dérivés PDC-Coumarines. La conception de ces deux séries est basée sur l’échafaudage bisquinolinium pyrido¬dicarboxamide (PDC-360A), un ligand sélectif ayant une bonne affinité vis-À-Vis des structures d’ADN quadruplexes, mais qui est non-Fluorescent. En s’inspirant de cette molécule et du motif styryle, connu pour ses propriétés spectroscopiques, nous avons préparé une librairie de colorants distyryles. Une deuxième série, les dérivés PDC-Coumarine, est synthétisée afin d’introduire la propriété fluorescente de la coumarine dans le PDC par une liaison covalente.Les propriétés de colorant de ces deux librairies (65 composés) ont été étudiées en présence de nombreuses structures d’ADN (quadruplex et duplex) en utilisant un criblage par fluorescence sur microplaques et des méthodes de titration. Nos résultats montrent que certains colorants synthétisés possèdent une haute réponse fluorimétrique (facteur d’augmentation de fluorescence de 200 à 600) vis-À-Vis de différentes structures d’ADN et d’ARN quadruplex, ayant une très faible réponse fluorimétrique vis-À-Vis de l’ADN duplex. Cela permet de marquer sélectivement l’ADN quadruplex dans la solution ou sur les gels d’électrophorèse. Ces résultats représentent une première étape vers l’utilisation de ces sondes dans un contexte biologique, par exemple dans l’imagerie de fluorescence. / Single-Stranded nucleic acids containing guanine repeats can form non-Canonical secondary structures called G-Quadruplexes. These structures are composed of several guanine quartets, maintained by hydrogen bonds and metal cations (K+ or Na+) coordinated between G-Quartets. In spite of being well-Studied in vitro, the evidence for the presence of quadruplex DNA structures in vivo remains mainly indirect. The objective of this work was research of fluorescent probes that can signal the presence of quadruplex DNA and detect its structure (topology).Two series of fluorescent probes were considered and prepared: styryls dyes (mostly distyryls) and PDC-Coumarin derivatives. The design of these two series is based on the molecular scaffold of bisquinolinium pyridodicarboxamide (PDC-360A), a selective ligand with good affinity for quadruplex DNA structures but which is not fluorescent. Inspired by this molecule and the styryl motif, which is known for its spectroscopic properties, we considered a library of distyryles dyes. A second series, the PDC-Coumarin derivatives, was developed to introduce the fluorescence property of coumarin in the PDC by a covalent link. The properties of dyes of these two libraries (65 compounds) were studied in the presence of a number of DNA structures (quadruplex and duplex) by a fluorescent screening using microplate and titration methods. Our results show that some of synthesized dyes display high fluorescence response (i.e. fluorescence increase factor from 200 to 600) for different quadruplex DNA and RNA structures, while having a very low fluorimetric response for duplex DNA. This allows a selective visualization of quadruplex DNA in solution or in electrophoresis gel. These results represent the first steps towards the use of these probes in a biological context, for example in fluorescence imaging

Le développement des nanoparticules fluorescentes, appelées quantum dots (QDs) est devenu l'un des domaines les plus prometteurs de la science des matériaux. Dans cette étude une procédure de synthèse de QDs a été mise au point, comprenant la synthèse de noyaux ultra-minces de CdSe, la purification de noyau haute performance, le revêtement central avec une coquille épitaxiale en ZnS. Cette approche a permis d’obtenir des QDs d’une taille de 3,7 nm possédant un rendement quantique supérieur à 70%. Les QDs développés ont été utilisés pour concevoir des conjugués de QDs compacts avec les nouveaux dérivés d'acridine, ayant une affinité élevée pour le G-quadruplex des télomères, ainsi que leur effet inhibiteur sur la télomérase, une cible importante du traitement du cancer. Les résultats de cette étude ouvrent la voie à l'ingénierie de nanosondes multifonctionnelles possédant une meilleure pénétration intracellulaire, une plus forte brillance et une stabilité colloïdale plus importante. / Development of the fluorescent nanoparticles referred to as quantum dots (QDs) has become one of the most promising areas of materials sciences. In this study, a procedure of synthesis of QDs, which includes the synthesis of ultrasmall CdSe cores, high-performance purification, core coating with an epitaxial ZnS shell has been developed. This approach has allowed obtaining 3.7-nm QDs with a quantum yield exceeding 70%. The QDs have been used: to engineer compact conjugates of QDs with the novel acridine derivatives, which have a high affinity for the telomere G-quadruplex; to demonstrate their inhibitory effect on telomerase, an important target of anticancer therapy; and to accelerate transmembrane penetration of ultrasmall QDs into cancer cells while retaining a high brightness and colloidal stability. The results of this study pave the way to the engineering of multifunctional nanoprobes with improved intracellular penetration, brightness, and colloidal stability.

G-quadruplex structures are found in important regions of the eukaryotic genome, such as telomeres and regulatory sequences of genes, and are likely to play important roles in regulation of biological events. The significant structural differences with duplex DNA make quadruplex DNA a very attractive target for anticancer drug design. The purpose of this study is to explore conformational space in a series of heterocyclic cations to discover novel structural motifs that can selectively bind and stabilize specific G-quadruplex arrangements. A variety of biophysical techniques such as thermal melting experiments, biosensor surface plasmon resonance, circular dichroism, fluorescence displacement assay and mass spectrometry were employed to evaluate the affinity of the compounds and their recognition properties. The screening of the molecules allowed the identification of not only selective G-quadruplex ligands but also potential quadruplex groove binders. These results can be useful for the development of new efficient telomerase inhibitors which are endowed with pharmacological activity.

G-quadruplex forming human telomere sequence (HTS) DNA, has been widely studied due to the telomere's implied role in biological processes, including cellular ageing and cancer physiology. The goal of these previous efforts has been to characterize the physiologically relevant structures and their stability and dynamics in order to develop therapeutic applications. Unfortunately, understanding the biologically relevant form of the human telomere DNA is complicated by the fact that HTS-derived sequences are highly polymorphic. To further complicate the issue, recent investigations have demonstrated the ability of "cell-like" co-solvents to alter the preferred G-quadruplex fold of HTS DNA. However, the origins of G-quadruplex structure selection, the relative contributions of crowding versus dehydration, and the possible effects of co-solvents on kinetically determined folding pathways remain unresolved. Towards answering these questions, I investigated HTS DNA G-quadruplex in extreme anhydrous and high viscosity conditions utilizing a deep eutectic solvent (DES) consisting of choline chloride and urea. Herein I report that the water-free DES supports an extremely stable parallel stranded structure, consistent with observations that diminished water activity is the main cause of structural transitions to the "parallel-propeller" form. Furthermore, my research shows that the highly viscous nature of the solvent enables significant diffusion based control over HTS g-quadruplex folding rates and topology, fully consistent with Kramers rate theory. To the best of my knowledge, this is the first example of the kinetic exploration of G-quadruplex folding utilizing high friction solvent; the results of which display a decreased intramolecular folding rate of HTS DNA to a never before encountered time scale on the order of days at physiological temperature. Moreover, I have demonstrated that the folding pathway of a G-quadruplex can be altered with increased solvent friction. These discoveries are important because they highlight the need to consider the viscosity when exploring the dynamics of human telomeres specifically drug binding and folding of G-quadruplexes in vivo where cellular viscosity has been reported to be as high as 140cP. Lastly, it appears that tuning solvent viscosity could prove useful to the continued study of G-quadruplex dynamics.

Les structures G-quadruplexes (G4) sont considérées comme des obstacles qui s’opposent à la progression du réplisome. Les séquences capables de former des G4 dans le génome humain se trouvent dans les régions d’ADN double brin au niveau des oncogènes et des proto-oncongènes et sur l’extrémité simple brin des télomères. La plupart des études biochimiques et biophysiques ont caractérisé les propriétés thermodynamiques des G4 en utilisant par exemple la température de fusion Tm pour déduire la thermodynamique de la formation/résolution du G4. Cependant, les expériences en solution donnent seulement une information indirecte concernant la dynamique du G4. Dans ce travail de thèse en molécule unique utilisant la technique des pinces magnétiques, nous avons pu caractériser la cinétique de la formation et résolution des G4s ainsi que la stabilité d’une structure G4 insérée dans une région d’ADN double brin : une situation qui ressemble aux G4 dans les promoteurs de gènes, où la séquence complémentaire est en compétition avec la formation de la structure de G4. Nous avons trouvé que le G4 télomérique a une très courte durée de vie (~20 s) et donc ce G4 se résout sans qu’une hélicase soit nécessaire. Au contraire, ce n’est pas le cas pour le G4 du c-MYC qui est très stable (~2h). Nous avons observé en temps réel la collision entre les hélicases et les polymérases et le G4 du c-MYC. Nous avons trouvé que l’hélicase Pif1 ouvre l’ADN puis résout le G4 après avoir effectué une pause et reprend l’ouverture de l’ADN, alors que l’hélicase RecQ et l’hélicase réplicative du bactériophage T4 ne peuvent pas le résoudre, mais peuvent le sauter. Nous avons aussi trouvé que la RPA ne peut pas résoudre le G4 du c-MYC. D’autre part, nous avons observé que la polyémrase du virus T4, la gp43, ainsi que la polymérase de T7, et la polymérase ε de la levure peuvent répliquer le G4 du c-MYC qui de façon étonnante ne constitue pas une barrière infranchissable. / G-quadruplex (G4) structures are considered as the major impediments for the replisome progression. The putative G4 forming sequences in the human genome are mostly located in the double-stranded DNA regions of oncogenes and proto-oncogenes and on the single-stranded overhangs of telomeres. Most of the biochemical and biophysical studies have characterized the G4 thermodynamics properties using melting temperature Tm as a proxy to infer thermodynamics of G4 folding/unfolding energetic. However, these thermodynamics properties give only indirect information about G4 dynamics. In this work, using single molecule magnetic tweezers technique, we first characterize the kinetics of folding and unfolding and thus the stability of a single G4 inserted in a dsDNA: a situation that mimics the G4s in promoters, where the complementary sequence competes with the G-rich structure. We find that the lifetime of telomeric G4 is short (~20 s) and thus that this G4 unfolds without the need of a helicase. This is not the case for the very stable c-MYC G4 (~2 hr). We observe in real time how helicases or polymerases behave as they collide with the c-MYC G4 on their track. We find that the Pif1 helicase unwinds dsDNA, resolves this G4 after pausing and resume unwinding, while RecQ helicase and the bacteriophage T4 replicative helicase do not resolve the G4 but may jump it. We also find that RPA does not unfold the c-MYC G4. Besides, we find that T4 bacteriophage gp43 polymerase, T7 polymerase and Yeast Pol ε can replicate the G4 which surprisingly does not appear as a major roadblock for them.

No description available.

Les oligopyridine-dicarboxamides et les oligoquinoline-carboxamides sont des oligomères synthétiques capables d’adopter des conformations hélicoïdales stables et bien définies. Les premiers sont comparables à des ressorts moléculaires qui peuvent s’étirer puis s’autoassembler pour former des doubles hélices artificielles. L’étude structurale d’oligopyridines de différentes tailles par diffraction des rayons X et RMN a permis d’éclaircir les principes de l’hybridation en double hélice. Par exemple, nous constatons que la stabilité du duplex est d’autant plus grande que l’oligomère est long mais la cinétique de l’hybridation décroit avec la taille des hélices. Ces propriétés sont modulables en fonction de divers paramètres tels que le solvant ou les substituants des pyridines. Les seconds forment de simples hélices moléculaires stables dans les solvants organiques mais aussi dans l’eau. Nous avons développé leur synthèse sur support solide afin de disposer de séquences variées, à l’image des alpha-peptides. Des études par RMN suggèrent que l’introduction d’unités aminométhylpyridines au sein d’un oligoquinoline hydrosoluble apporte de la flexibilité sans perturber sa structure hélicoïdale. Cela témoigne de la stabilité de ces structures secondaires dans les solvants protiques. Par ailleurs, certains de ces peptidomimes s’avèrent capables de reconnaitre et stabiliser des motifs structuraux particuliers de l’ADN : les G-quadruplex. Etant donné que ces architectures se forment à des endroits clés du génome impliqués dans des cancers, ces hélices moléculaires font figure de potentiels agents antitumoraux d’un nouveau genre. / Oligopyridine-dicarboxamides and oligoquinoline-carboxamides are synthetic oligomers able to fold into stable and well defined helical conformations. The first ones are comparable to molecular springs which can extend then associate to form artificial double helices. A structural study of oligopyridines of various sizes by X-ray diffraction and NMR provided a better understanding of the hybridization process. For example, we noticed that the stability of the duplex is all the higher as the oligomer is long but the kinetics of hybridization decrease with length. These properties depend on diverse parameters such as the solvent or the substituants of pyridine rings. The second family forms stable single helices in organic solvents and also in water. We adapted their synthesis on solid support to promote accessibility to a variety of sequences, just like for alphas-peptides. NMR studies suggested that the introduction of aminomethylpyridine units within a hydrophilic oligoquinoline strand brings some flexibility without disrupting its helical structure, showing the high stability of these secondary structures in protic solvents. Besides, some of these peptidomimetics turn out to be capable of recognizing and stabilizing a particular DNA motif: G-quadruplex structure. Given that these architectures form in critical places of the genome involved in cancers, these molecular helices may represent a new class of potential antitumoral agents.