L’acide désoxyribonucléique se structure chez les êtres vivants de différentes façons. La plus connue est sa forme double hélice mais de nombreuses autres structures secondaires existent et notamment les G-quadruplex. Il s’agit d’une structure basée sur le repliement d’un brin d’ADN possédant des répétitions de guanines. L’association de quatre guanines entre elles par liaisons hydrogène forme un plan appelé G-quartet. Ce réseau de liaisons hydrogène est appelé appariement de Hoogsteen. L’empilement d’au moins deux quartets autour d’un cation monovalent comme le potassium ou le sodium constitue la structure G-quadruplex. Ces structures ont été très étudiées lors des vingt dernières années et il a été montré qu’elles sont impliquées dans de nombreux mécanismes biologiques tels que la réplication, la transcription, la traduction et également le maintien des télomères. La présence des G-quadruplex peut provoquer une instabilité importante aussi bien génétique qu’épigénétique. C’est pourquoi de nombreuses méthodes ont été développées afin de localiser et comprendre le rôle de ces structures in vivo. Pour cela, un large panel d’outils moléculaires a été utilisé cependant il est encore difficile, à partir de ce panel, d’apporter une réponse à toutes les questions sur l’implication des G-quadruplex au niveau du génome. Lors de ce travail de thèse, nous avons alors développés de nouvelles molécules capables de cibler sélectivement les G-quadruplex au sein d’un milieu biologique complexe à partir de deux ligands PDC et PhenDC3 affins et sélectifs pour les structures G-quadruplex.Sur la base de molécules de référence que sont PhenDC3 et PDC, de nombreux ligands ont été mis au point. D’une part, des ligands fonctionnalisés avec une biotine et/ou un groupement photoactivable ont été synthétisés afin de capturer et d’extraire des structures G-quadruplex dans un milieu biologique. D’autre part, des dérivés capables d’être fonctionnalisés in cellulo par l’utilisation de chimie bioorthogonale ont également été obtenus. Ceci permet d’ajouter une fonction (fluorescente ou biotine…) après que le dérivé ait interagi avec sa cible cellulaire. L’ensemble des composés a été évalué par des techniques biophysiques, l’expérience de FRET-melting et l’expérience de FID, afin de mesurer leur affinité pour différentes structures G-quadruplex et leur sélectivité. Nous avons proposé une relation entre les deux expériences afin d’avoir un classement de ligands le plus approprié pour les G-quadruplex.Un des objectifs majeurs de ce travail était de localiser les ligands de G-quadruplex au sein de cellules cancéreuses humaines. Dans un premier temps, toute une étude au sein de cellules fixées a été réalisée en utilisant deux réactions de chimie « click », la réaction de cycloaddition d’un azoture et d’un alcyne catalysée par le cuivre (CuAAC) et la réaction de cycloaddition d’une cyclooctyne et d’un azoture (SPAAC). L’étude s’est, dans un second temps, poursuivie au sein de cellules vivantes en utilisant uniquement la réaction SPAAC à cause de la toxicité in cellulo du cuivre.Ces composés ont également été testés pour l’extraction de G-quadruplex à l’aide de billes magnétiques recouvertes d’une fonction cyclooctyne. Cependant, les résultats observés, lors de cette étude préliminaire, n’ont pas été concluants et demandent une mise au point pour optimiser le système. / Deoxyribonucleic acid has different structures in human beings. The most known is the double helix but a lot of secondary structures exist and particularly G-quadruplex. It consists of guanine-rich nucleic acid sequences. The association of four guanines through hydrogen bonds forms a plan called G-quartet. This set of hydrogen bonds is called Hoogsteen base pairs. The stacking of at least two quartets around a monovalent cation like potassium or sodium establishes the G-quadruplex. These structures have been much studied over the past twenty years. They are involved in numerous biological mechanisms like replication, transcription, translation and also telomere maintenance. G-quadruplex presence can cause an important genetic as well as epigenetic instability. That is why many methods have been developed in order to localize these structures and to understand their role in vivo. To this end, a broad panel of molecular tools has been used. However, it is still difficult to bring an answer to all the questions about the involvement of G-quadruplex at the genomic level with this panel. In this thesis work, we developed new molecular tools able to target selectively G-quadruplex in a complex biological medium from two benchmark ligands, PhenDC3 and PDC, which have very good affinity and selectivity for G-quadruplex.On the one hand, functionalized ligands have been synthetized with a biotin and/or a photoactivatable group in order to trap and pull-down G-quadruplex in various cellular contexts. On the other hand, derivative compounds which are able to be functionalized in cellulo by bioorthogonal reactions have been obtained. Once the compound interacts with its cellular target, a function (fluorophore or biotin) can be added through an orthogonal reaction. The new panel of compounds has been evaluated by biophysical techniques, FRET-melting experiment and FID assay, in order to determine their affinity to G-quadruplex and their selectivity. We proposed a relation between the two biophysical experiments in order to have a good ranking of ligands for G-quadruplex structures.One of the most important objectives of this work was to localize G-quadruplex ligands in human cancer cells. First, a complete study in fixed cells has been performed using two reactions of click chemistry: reaction of copper-catalyzed-alkyne-azide-cycloaddition (CuAAC) and reaction of strain-promoted alkyne-azide cycloaddition (SPAAC). Secondly, the study has been pursued in living cells using SPAAC reaction because of the toxicity of copper in cells.These compounds have also been used to extract G-quadruplex from biological systems with cyclooctyne-coated magnetic beads. However, results obtained in this preliminary study are not decisive so it could be interesting to optimize the system before concluding.

No description available.

Angiogenesis is known to be induced and maintained in tumors by the constant expression of the hypoxia inducible factor 1 alpha (HIF-1α) and human vascular endothelial growth factor (VEGF). In fact, tumor recurrence, aggressive metastatic legions and patient mortality rates are known to be positively correlated with overexpression of these two proteins. The HIF-1α and VEGF promoters contain a polypurine/polypyrimidine (pPu/pPy) tract, which are known to play critical roles in their transcriptional regulation, and are structurally dynamic where they can undergo a conformational transition between B-DNA, single stranded DNA and atypical secondary DNA structures such as G-quadruplexes and i-motifs. We hypothesize that the i-motif and G-quadruplex structures can form within the pPu/pPy tracts of the HIF-1α and VEGF proximal promoters, which play important roles in the transcriptional regulation of these genes by acting as scaffolds for alternative transcription factor binding sites. The purpose of this dissertation was to elucidate the transcriptional regulation of the HIF-1α and VEGF genes through the atypical DNA structures that form within the pPu/pPy tracts of their proximal promoters. We investigated the interaction of the C-rich and guanine-rich (G-rich) strands of both of these tracts with transcription factors heterogeneous nuclear ribonucleoprotein (hnRNP) K and nucleolin, respectively, both in vitro and in vivo and their potential role in the transcriptional control of HIF-1α and VEGF. In this dissertation, we demonstrate that both nucleolin and hnRNP K bind selectively to the G- and C-rich sequences, respectively, in the pPu/pPy tract of the HIF-1α and VEGF promoters. Specifically, the small interfering RNA-mediated silencing of either nucleolin or hnRNP K resulted in the down-regulation of basal VEGF gene, and the opposite effect was seen when the transcription factors were overexpressed, suggesting that they act as activators of VEGF transcription. Taken together, the identification of transcription factors that can recognize and bind to atypical DNA structures within pPu/pPy tracts will provide new insight into mechanisms of transcriptional regulation of the HIF-1α and VEGF gene.

In this dissertation, we explore the transcriptional regulatory roles of Gquadruplex- forming motifs and the involvement of specific transcriptional factors, which interact with the same elements, in the control of human c-myc and bcl-2 gene expression. The G-quadruplex structures within the NHE III1 region of the c-myc promoter and their ability to repress transcription has been well established. However, a longstanding unanswered question is how these stable DNA secondary structures are transformed to activate c-myc transcription. NDPK-B has been recognized as an activator of c-myc transcription via interactions with NHE III1 region of the c-myc gene promoter. Through the use of RNAi, we confirmed the transcriptional regulatory role of NDPK-B. We demonstrate that NDPK-B has DNA binding activity and the nuclease activity results from a contaminating protein. NDPK-B preferentially binds to the singlestranded guanine-rich strand of the c-myc NHE III₁. Potassium ions and G-quadruplexinteractive agents, which stabilize G-quadruplex structures, had an inhibitory effect on NDPK-B DNA binding activity. Based on our studies, we have proposed a stepwise trapping-out of the NHE III1 region in a single-stranded form, thus allowing singlestranded transcription factors to bind and activate c-myc transcription. This model provides a rationale for how the stabilization of G-quadruplexes within the c-myc gene promoter region can inhibit NDPK-B from activating c-myc transcription. Similarly, the human bcl-2 gene contains a GC-rich region within its promoter region, which is critical in the regulation of bcl-2 expression. We demonstrate that the guanine rich strand within this region can form three intramolecular G-quadruplex structures. Based on NMR studies, the central G-quadruplex forms a mixed parallel/antiparallel structure with three tetrads connected by loops of one, seven, and three bases. The Gquadruplex structures in the bcl-2 promoter extends beyond the ability to form any one of three separate G-quadruplexes to each having the capacity to form either three or six different loop isomers. This suggests that targeting these individual structures could lead to different biological outcomes. We also found that Telomestatin upregulates bcl-2 gene expression, which we propose is a result of inhibiting the binding of the WT1 repressor protein by the formation of a drug-stabilized G-quadruplex structure.

The activated product of the c-MYC proto-oncogene is one of the strongest known activators of carcinogenesis. It has been estimated that as many as one-seventh of all cancer deaths are associated with alterations in the c-MYC gene or its expression [1]. Therefore, understanding the regulation of c-MYC expression is a key factor in understanding carcinogenesis in many histologic classes of malignancy. The nuclease hypersensitive element (NHE) III₁ region of the c-MYC promoter has been shown to be particularly important in regulating c-MYC expression. Specifically, the formation of a G-quadruplex structure appears to promote repression of c-MYC transcription. In this dissertation, we investigate the role that nucleolin, a critical player in ribosome biogenesis and cell stress sensing, plays on the transcriptional regulation of the c-MYC promoter through its interaction with the c-MYC G-quadruplex structure. Our studies initiated with the design of a c-MYC G-quadruplex affinity column intended to trap potential c-MYC G-quadruplex-binding proteins that were then identified by LC-MS/MS. After careful examination of the literature of the list of potential c-MYC G-quadruplexbinding proteins, we realized that several of the proteins identified had been previously reported to interact directly with nucleolin. Consequently, we chose to focus our studies on nucleolin, as it could be a central regulator of the (NHE) III region. By performing chromatin immunoprecipitation in HeLa cells, we found that nucleolin indeed interacts with the c-MYC promoter region containing the NHE III₁ element. This binding activity was confirmed by both electromobility shift assay and polymerase stop assay. We provide evidence that nucleolin can induce the formation of the c-MYC G-quadruplex structure from single-stranded DNA, both in linear and circular DNA forms. We show that upon binding, nucleolin increases the stability of the c-MYC G-quadruplex structure leading to repression of c-MYC promoter activity. We also show that nucleolin binds with much higher affinity to G-quadruplex structures with topology similar to that of the parallel c-MYC G-quadruplex, such as those found in the VEGF and PDGF-A promoters; in comparison to G-quadruplexes found in telomeres or the c-MYB promoter, whose have significantly different topology. Interestingly, we also demonstrate that nucleolin binds with higher affinity to the c-MYC G-quadruplex than to its consensus RNA substrate, the nucleolin recognition element (NRE). Furthermore, we show that the C-terminal domain of nucleolin is critical for its interaction and stabilization of the c-MYC G-quadruplex structure. Lastly, we show that the binding of nucleolin to the (NHE) III region causes repression of c-MYC transcription. On the basis of these results, we propose that nucleolin may play an important role in the transcriptional regulation of c-MYC in vivo by inducing the formation of the c-MYC G-quadruplex structure.

Guanine-rich DNA and RNA sequences can fold, in the presence of alkali metal ions such as Na+ and K+, into G-quadruplex structures. These alkali metal ions are necessary for the stabilization of G-quadruplex structures. However, little is known about the ion dynamics in G-quadruplex structures. In this thesis, we used molecular dynamics (MD) simulations to study the energetics of ion transport in G-quadruplex DNA channels. In particular, we applied, for the first time, adaptive biasing force (ABF) and umbrella sampling (US) methods to obtain potential of mean force (PMF) profiles for Na+, K+, and NH4+ ion movement along [d(TG4T)]4 and [d(G3T4G4)]2 channels. We found that the ABF and US methods produce very similar PMF profiles, in qualitative agreement with the very limited experimental data in the literature. We found that, within a G-quadruplex channel, K+ and NH4+ ions experience significant energy barriers (13-17 kcal/mol) to cross a G-quartet, whereas the Na+ movement encounters minimal resistance (5-7 kcal/mol). All ions are nearly fully dehydrated inside the channel but quickly become hydrated after exiting the channel. Our simulations suggested that the free energy landscapes for ion movement between the channel exit points and bulk solution are quite flat (ca. 2-4 kcal/mol) regardless of the loop topology in the region. We discovered that the directional symmetry of the ion movement within any G-quadruplex channel depends critically on both the DNA sequence and the folding of the G-quadrupelx structure. While the ion movement inside the [d(TG4T)]4 channel shows the same free energy barrier in either direction, the [d(G3T4G4)]2 channel exhibits a free energy difference of 3-4 kcal/mol for NH4+ ions exiting from the two ends. We hypothesized that the mode of base-stacking is the determining factor for the G-quartet stiffness and this stiffness then contributes to the free energy barrier for any ion to across it. This hypothesis appears to be consistent with all currently available experimental observations. When a G-quadruplex channel contains multiple ions, we found that the ion-ion repulsion is an important factor that must be considered in order to have a complete understanding of the ion movement within G-quadruplex DNA channels. / Thesis (Ph.D, Chemistry) -- Queen's University, 2013-01-25 17:38:07.489

La déstabilisation des structures G-quadruplex au niveau des acides nucléiques a des répercussions physiologiques importantes. L’accentuation des connaissances concernant les processus cellulaires associés au métabolisme des structures des G4 est primordiale. Une panoplie d’hélicases à G4 est impliquée dans le métabolisme des structures G4, notamment l’hélicase humaine DHX36. Il a été déterminé au préalable par certains groupes de recherche que l’hélicase DHX36 se lie à son substrat l’ARN G4 et utilise des nucléosides triphosphates afin de catalyser le dépliement de la structure G-quadruplex. Toutefois, l’interaction avec l’ARN G4 a été sommairement caractérisée et la spécificité nucléotidique n’a toujours pas été évaluée. Ainsi, nous avons décidé d’approfondir les connaissances du mécanisme de dépliement de la structure du G4 d’ARN par l’hélicase DHX36. Notamment, en évaluant la thermodynamique de l’interaction entre l’hélicase et l’ARN G4 afin de révéler particulièrement l’efficacité de liaison mais également en évaluant la spécificité nucléotidique de l’hélicase DHX36 afin d’effectuer le dépliement de l’ARN G4. La combinaison des analogues de nucléotides et le modèle structural permettent de révéler les caractéristiques structurales et fonctionnelles de l’interaction entre l’hélicase humaine DHX36 et l’ATP. Nos analyses permettent de constater que l’enzyme DHX36 est en mesure d’utiliser autant l’ATP que GTP afin de dérouler les structures G4 d’ARN ayant, par contre, une spécificité accrue pour la molécule d’ATP.

The loss of expression of the fragile X mental retardation protein (FMRP) leads to fragile X syndrome. Fragile X syndrome is the most prevalent inheritable mental retardation. FMRP has two types of RNA binding domains, two K-homology domains and an arginine-glycine-glycine box domain, and is proposed to act as a translation regulator of specific mRNA. Despite extensive research, the mechanism by which FMRP loss leads to the fragile X syndrome remains unclear. Thus, there is high interest to produce sufficient quantities of pure recombinant FMRP for biochemical and biophysical studies of the protein function. However, the recombinant bacterial expression of FMRP has had limited success, and subsequent recombinant eukaryotic and in vitro systems may produce FMRP which is posttranslationally modified, as phosphorylation and arginine methylation have been shown to occur on FMRP. In this study, we have successfully isolated the conditions for recombinant expression, purification and dialysis of full-length FMRP using Escherichia coli, with a high yield. The expression of FMRP using E. coli renders the protein devoid of the posttranslational modifications of phosphorylation and arginine methylation, allowing for the further study of the direct effects of these modifications individually and simultaneously. Additionally, FMRP has been shown to undergo alternative splicing, with one of the splicing sites in close proximity to the FMRP domain shown to be involved in binding G quadruplex mRNA with high affinity and specificity. We have analyzed how naturally occurring truncations in the FMRP sequence affect its RNA binding affinity, by applying the expression, purification and dialysis process to the second and third longest FMRP isoforms, followed by subsequent analysis of the G quadruplex mRNA binding properties by fluorescence spectroscopy. Our results show that as FMRP gets truncated by alternative splicing, its mRNA binding affinity increases. To test a model we proposed for FMRP translation regulation activity, we developed a luciferase reporter gene construct that contains the G quadruplex structure in the mRNA 5���-untranslated region. Using luminescence spectroscopy to analyze luciferase translation, we showed that low levels of full-length FMRP reduces luciferase translation, and as the concentration of full-length FMRP increases the luciferase translation increases. / Bayer School of Natural and Environmental Sciences / Chemistry and Biochemistry; / PhD; / Dissertation;

Structure-specific recognition of nucleic acids is a promising method to reduce the size of the recognition unit required to achieve the necessary selectivity and binding affinity for small molecules. It has been demonstrated recently that G-quadruplex DNA structures can be targeted by organic cations in a structure-specific manner. Structural targets of quadruplexes include the planar end surfaces of the G-tetrad stacked columns as well as four grooves. The significant structural differences between quadruplex DNA and duplex DNA make quadruplex DNA a very attractive target for highly selective, structure-specific drug design. We have used a variety of biophysical techniques including circular dichroism, surface plasmon resonance, thermal melting and absorbance spectroscopy to investigate small molecules that can selectively bind to the ends of human telomeric DNA as well as the ends of the G-quadruplex structure formed by the purine-rich promoter region of the c-MYC oncogene. We have also screened a library of heterocyclic diamidines, and identified one that binds selectively in the grooves of human telomeric quadruplex DNA. This compound is an excellent starting point for the design of new anti-cancer and anti-parasitic compounds with high affinity and selectivity for human telomeric DNA.

The synthetic methodology of tri- and pentamethine carbocyanines and their interactions with biomolecules will be discussed in two chapters. The first chapter describes the preparation of halogenated carbocyanine dyes that display multiple charges; furthermore, these particular compounds were examined for their ability to bind G-quadruplex DNA with selectivity over duplex DNA and have potential for developing novel chemotherapeutic agents. The second section discusses the synthetic methods utilized to prepare trimethine cyanine fluorophores. This chapter will show how varying the N-indolenyl substituients’ hydrophobicity from ethyl to phenylpropyl influences the binding to Human Serum Albumin (HSA); additionally, alternating the terminal heterocyclic moieties of the cyanine dye has a direct quantitative effect on the biomolecular interaction. These identical compounds were recognized to be structurally analogous to agents that commonly interact with Protein Arginine Methyl Transferase (PRMT) and these compounds display low IC50 values toward inhibition of PRMT1 with unique NIR imaging properties.