<p>The process of in vitro selection has led to the isolation of many catalytic DNA molecules, called deoxyribozymes, which can catalyze a range of biologically-relevant reactions. Despite these advances, questions still remain as to why DNA, which seems more suited to information storage than catalysis can efficiently catalyze chemical reactions. In this thesis, a group of deoxyribozymes that can catalyze their own phosphorylation using NTP substrates are used a model system to study how DNA is able to fold into complex structures necessary for catalysis. Using a variety of structural probing techniques, these studies elucidated a common secondary structure shared by three deoxyribozymes, which do not appear to share a common ancestor sequence. This suggests that this motif may be most efficient motif to catalyze self-phosphorylation by DNA. It also more generally demonstrates that DNA can undergo convergent evolution to reach the same complex folding arrangement. A fourth deoxyribozyme was found to fold into a complex tertiary structure containing a novel quadruplex-helix pseudoknot motif. The finding of this pseudoknot and comparison with other quadruplexes found in other functional nucleic acids led us to investigate whether these stable motifs could be incorporated into nucleic acid libraries to improve the process of in vitro selection and give researchers a better chance of isolating functional nucleic acids. Design and characterization of structured libraries revealed that DNA libraries could be made in which the majority of sequences are folded into quadruplex arrangements. The incorporation of this quadruplex scaffold into DNA sequence libraries may ease the isolation of functional nucleic acids that contain this useful structural motif. In the final part of this thesis, a self-phosphorylating deoxyribozyme was converted from a tool for study of DNA structure to a sensor for GTP and Mn²⁺, demonstrating that deoxyribozyme substrates can be converted into targets for biosensors.</p> / Doctor of Philosophy (PhD)

G-quadruplexes (GQs) are highly stable noncanonical nucleic acid structures that form in the DNA of human cells and play fundamental roles in maintaining genomic stability and regulating gene expression. These unique structures exert broad influence over biologically important processes and can modulate cell survival and human health. In fact, mutations, hyper-stability, and dissociation of GQs are implicated in neurodegenerative disease, mental retardation, premature-aging conditions, and various cancers. As such, GQs are novel drug targets. GQ-targeting therapeutics are developed to influence the folding and genetic interactions of GQs that are implicated in diseased states. To do so requires a greater understanding of GQ structure and dynamics and molecular dynamics (MD) simulations are well suited to provide these fundamental insights. Previous MD simulations of GQs have provided limited information due to inaccuracies in their models, namely the nonpolarizable nature of their force fields (FFs). The cutting-edge Drude polarizable FF models electronic degrees of freedom, allowing charge distribution to change in response to its environment. This is an important component for modeling ion-ion and ion-DNA interactions and can influence the overall stability of GQ structures. The work herein employs the Drude polarizable FF to 1) describe the role of electronic structure on the dynamics and folded stability of GQs, 2) determine the impact of ion interaction on GQ stability, and 3) characterize the role of G-hairpin motifs in GQ intermediates. Such fundamental investigations will help clarify GQs role in healthy and diseased states and transform our understanding of noncanonical DNA, improving human health, therapeutic design, and fundamental science. / Doctor of Philosophy / Human health and disease are influenced by unique nucleic acid structures called G-quadruplexes (GQs). GQs form when DNA or RNA fold into a square-shaped structure that is stabilized by ion interactions and special hydrogen bonding patterns. These GQ structures exert broad influence over normal biological processes, but also play a role in neurodegeneration, intellectual disabilities, premature-aging conditions, and various cancers, many of which are chemotherapeutic resistant. As such, modulating GQ structures, or their interactions with proteins, is a promising therapeutic approach. However, a greater understanding of GQ folding, folded structure, and interactions with other biomolecules is needed to do so. Computational techniques such as molecular dynamics (MD) simulations use experimental data and fundamental biophysics to gain new insights on these properties and inform novel drug design. In this project, we explored the dynamics of several distinct GQ structures and folding intermediates with state-of-the-art MD simulation methods. In doing so, we provided new insight on their structural features as well as their interactions with extended DNA sequences and different ion types, which serve as fundamental information for future structural or computer-aided drug design studies.

Gene expression regulation encompasses a wide range of mechanisms that govern cellular processes. Among these, post-transcriptional regulation, including translation control, plays a pivotal role in ensuring precise protein synthesis, timing, and quantity. Perturbations of mechanisms such as RNA modifications, and interactions between RNA-binding proteins (RBPs) and specific RNA motifs, can lead to dysregulation of essential cellular processes. These alterations contribute to the development of various disorders, including cancer, neurodegenerative diseases, and metabolic disorders. Many publicly available datasets and studies offer opportunities to investigate the link between alterations in these mechanisms and disease manifestations. However, the limited availability of datasets for certain conditions or notable inconsistencies among reported associations prevent complete understanding of the underlying processes. Therefore, extending the investigations to encompass a diverse range of genes and/or diseases will enhance our comprehension of these intricate regulatory and disease mechanisms, aiding in the identification of potential therapeutic targets and innovative interventions to mitigate pathological conditions. In particular, we focused on three separate aspects involved in gene expression regulation: RNA modifications, RBPs interactions with RNA secondary structures, and the Kozak consensus sequence as a translational modulator. Each part uncovers essential mechanisms that govern post-transcriptional control of gene expression, shedding light on their roles in cellular processes and disease contexts. At first, we performed a comprehensive exploration of 15 RBPs involved in the regulation of the N6-methyladenosine (m6A) methylation. Leveraging data from The Cancer Genome Atlas (TCGA), we conducted a pan-cancer analysis across 31 tumor types to uncover the distribution of alterations of these factors, and we developed a user-friendly web application to enable users to conduct similar analyses. Additionally, we performed a parallel analysis focused on neuroblastoma, using data from publicly available and in-house datasets. These investigations unveil the potential impact of a subset of m6A factors on cancer development and progression. While in the first case, VIRMA and YTHDF reader proteins, emerged as the most frequently altered genes with significant pan-cancer prognostic implications, in the context of neuroblastoma, the writer METTL14 and the reader ALKBH5, showed the most prominent roles. Subsequently, our focus shifted to a distinct subset of RBPs capable of interacting with RNA secondary structures, particularly with RNA G-quadruplexes (RG4s). We established a comprehensive database cataloging RBPs with potential RG4-binding capabilities. This resource represents a valuable tool for researchers aiming to explore the intricate interplays between RBPs and RG4s, and their putative implications in diverse biological processes and diseases. Finally, attention was directed to the Kozak sequence, a pivotal determinant of the regulation of translation initiation. Exploiting the power of base editors, we developed a method to optimize translation initiation by modifying the Kozak sequence. This strategy offers promise in addressing haploinsufficiency-related disorders, where enhancing the functional protein is essential. Overall, these findings present opportunities for the identification of potential therapeutic targets and precision medicine strategies to alleviate a spectrum of pathological conditions, thus fostering advancements in the field of molecular biology and disease management.

G-quadruplexes, characterized by stacked G-tetrad rings held together by Hoogsteen hydrogen bonds, have been visualized in human cells and implicated in transcriptional and translational control, telomere maintenance and disease. RHA Helicase associated with AU-rich element (RHAU), a DEAH-box helicase, is a major G-quadruplex resolvase in human cell lysates. It binds G-quadruplexes through the RHAU specific motif in its N-terminus. In order to investigate the recognition of G-quadruplexes by helicases, the binding between the N-terminal construct of RHAU, RHAU53-105, and the DNA analog of the quadruplex formed by the 5’ terminus of human telomerase RNA component, hTR1-20, was investigated in a comprehensive biophysical approach followed by crystallization screening. RHAU53-105, hTR1-20 DNA and their complexes were analysed by gel electrophoresis, UV-visible spectroscopy, spectropolarimetry, dynamic light scattering and small angle X-ray scattering (SAXS). The findings reveal that hTR1-20 DNA, separated in two conformations by size exclusion chromatography in the presence of potassium cations, assumes a disk-like parallel G-quadruplex secondary structure in solution. Far-UV circular dichroism spectra and SAXS demonstrate that RHAU53-105 assumes an extended (Dmax = 7.8 nm , rG = 2.1 (±0.2) nm) and ordered conformation in solution. The analysis confirms the binding between RHAU53-105 and each conformation of the hTR1-20 DNA quadruplex. Circular dichroism spectra indicate the retention of quadruplex secondary structure in both RHAU53-105•hTR1-20 DNAc1 and RHAU53-105•hTR1-20 DNAc2 complexes. This analysis provides some insight into the interaction between G-quadruplexes and the N-terminal domain of RHAU and identifies 0.2 M sodium formate, 20 % (w/v) polyethylene glycol 3350 and 1.5 M sodium chloride, 10 % (v/v) ethanol as preliminary conditions for crystallization of the complex of RHAU53-105 and hTR1-20 DNAc2. / October 2014

Les triarylamines sont de petites molécules largement utilisées comme porteurs de charges dans le domaine de l'électronique organique, car elles présentent des mobilités de transport de trous élevées. En 2010, notre groupe a démontré pour la première fois que les molécules de triarylamine décorées avec des groupements amide subissent une polymérisation supramoléculaire. D'autre part, les propriétés de reconnaissance des résidus de nucléobases ont été largement utilisées au cours des 25 dernières années pour déclencher des processus d'auto-assemblage de polymères ou de petites molécules en polymères supramoléculaires bien définis. Dans cette thèse, une série de molécules triarylamine décorées avec des amides sur leurs chaînes latérales avec différentes nucléobases comme la guanine, la thymine et la cytosine ont été synthétisées. Nous avons démontré que les monomères de triarylamine conservent leurs propriétés d'auto-assemblage dans les solvants chlorés lors de l'irradiation lumineuse, à condition que le résidu de la nucléobase n'affecte pas les interactions non covalentes nécessaires pour l'auto-assemblage du cœur de triarylamine. En outre, nous avons démontré que la présence d'amines primaires sur le résidu de la nucléobase interdit la formation de structures auto-assemblées dès qu'elles ne sont pas incorporées dans des réseaux de liaisons hydrogène. Dans un deuxième chapitre, nous avons ensuite étudié les auto-assemblages de nos molécules triarylamine-nucléobase dans des solvants organiques en utilisant des ions ou de petites molécules comme matrice. Tout d'abord, la polymérisation supramoléculaire de la triarylamine-monothymine à l'aide de mélamine dans divers solvants a été étudiée. Par ailleurs, l'influence sélective des ions de mercure sur les propriétés sensibles légères de triarylamine-monothymine a été soigneusement analysé. Enfin, les polymères supramoléculaires hybrides de triarylamine-monoguanine conjugué en absence et en présence d'ions potassium ont été obtenus. En particulier, nous avons décrit le premier exemple de polymères supramoléculaires construits à partir de mélamine dans des solvants organiques. Dans l'ensemble, l'impact de ce travail est triple: a) il conduit à une meilleure compréhension du comportement d'auto-assemblage des conjugués de triarylamine, b) il influence la conception des structures de triarylamine auto-assemblées et c) il offre de nouvelles approches pour l'auto-assemblage des molécules de triarylamine. / Triarylamines are small molecules widely used as charge carriers in the field of organic electronics as they display high hole-transport mobilities. In 2010, our group demonstrated for the first time that chemically-tailored triarylamine amide molecules undergo supramolecular polymerization. On the other hand, the recognition properties of nucleobase residues have been widely used in the last 25 years to trigger self-assembling processes of polymers or small molecules into well-defined supramolecular polymers.In this thesis, a series of triarylamine amide molecules decorated on their side chains with various nucleobases such as guanine, thymine and cytosine have been synthesized. We have demonstrated that the triarylamine monomers retain their self-assembling properties in chlorinated solvent upon light irradiation, provided that the nucleobase residue does not affect the non-covalent interactions necessary for the self-assembly of the triarylamine core. In addition, we have demonstrated that the presence of primary amines on the nucleobase residue prohibit the formation of self-assembled structures, as soon as they are not embedded in hydrogen bonding arrays. In a second chapter, the templated self-assemblies of our triarylamine-nucleobase molecules in organic solvents were studied. Firstly, templated supramolecular polymerization of triarylamine-monothymine using melamine in various solvents was investigated. Besides, selective influence of mercury ion on the light responsive properties of triarylamine-monothymine was analyzed carefully. At last, hybrid supramolecular polymers of triarylamine-monoguanine conjugate in absence and in presence of potassium ion were obtained. In particular, we have described the first example of supramolecular polymers build from melamine in organic solvents. Overall, the impact of this work is three-fold: a) it leads to a better understanding of the self-assembly behavior of triarylamine conjugates, b) it influences the design of self-assembling triarylamine structures and c) it offers new approaches for the self-assembly of triarylamine molecules.

Methods for the rapid detection and differentiation of the Burkholderia pseudomallei complex comprising B. pseudomallei, B. mallei, and B. thailandensis, have been the topic of recent research due to the high degree of phenotypic and genotypic similarities of these species. B. pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively. B. pseudomallei and B. mallei are recognized by the CDC as tier 1 select agents. Although B. thailandensis is generally avirulent in mammals, this species displays very similar phenotypic characteristics to that of B. pseudomallei. Optimal identification of these species remains problematic, due to the difficulty in developing a sensitive, selective, and accurate assay. To date, no real-time, multiplex PCR assay has been developed that can detect and differentiate between B. pseudomallei, B. mallei, and B. thailandensis in a single tube format. Here, we describe the development of such an assay that detects and differentiates the species of the B. pseudomallei complex. A real-time quadruplex qPCR assay, Bcom, was designed to target unique genomic regions of B. pseudomallei, B. mallei, B. thailandensis, and the B. pseudomallei complex that detects and differentiates the three species. A total of 299 isolates within the B. pseudomallei complex was evaluated in this study, as well as 15 near-neighbors and other bacterial species. The results showed that this quadruplex assay was capable of detecting the respective species in a given sample at a sensitivity between 288 fg and 277 pg of genomic DNA. The B. pseudomallei- and B. pseudomallei complex-specific assays tested negative on two presumed B. pseudomallei isolates. In addition, a third presumed B. pseudomallei isolate tested negative by the B. pseudomallei-specific test, but was detected by the B. thailandensis and B. pseudomallei complex-specific assays. After cultural and biochemical characterization, 16s rRNA sequencing, and multiple loci sequencing, it is proposed that B. pseudomallei 34 is B. thailandensis 82172 (Accession No. DQ388536), B. pseudomallei Darwin 175 is Elizabethkingia meningoseptica, and B. pseudomallei 135 is a new strain of B. ubonensis 135.

Les télomères sont des structures nucléoprotéiques nécessaires à la protection des extrémités des chromosomes contre les dégradations ou fusions induites par les processus de réparation de lADN. Ils sont constitués de complexes protéiques associés à des répétitions en tandem dun motif 5-(TTAGGG)-3 sous forme double brin de plusieurs kilobases, et finalisés par une extrémité 3simple brin de la même séquence de quelques centaines de bases. On observe in vitro un raccourcissement des télomères à chaque division cellulaire, ce même fait est corrélé in vivo avec le vieillissement. Lorsque les télomères se raccourcissent et atteignent une taille critique, les cellules entrent en sénescence réplicative qui se définit par un arrêt de croissance définitif et viable des cellules. La télomérase est une ADN polymerase ARN-dépendante qui allonge le télomère en lui ajoutant des séquences répétitives TTAGGG. Elle comprend une composante ARN (hTR) qui sert de matrice et une composante catalytique à activité transcriptase inverse (hTERT). Lexpression seule dhTERT suffit à immortaliser différents types cellulaires. La télomérase est fortement exprimée dans la majorité des cellules tumorales alors que son activité est difficilement détectable dans la plupart des cellules somatiques. Ces observations font de la télomérase une cible dintérêt pour des nouvelles thérapies anticancéreuses. Une de ces nouvelles stratégies consiste en lutilisation de molécules capables de stabiliser les structures en G-quadruplexe de lADN. La stabilisation des G-quadruplexes télomériques rend les télomères inaccessibles pour la télomérase et inhibe son activité par la séquestration de son substrat. Lobjectif de cette thèse est dévaluer la réponse cellulaire induite par le traitement cellulaire de deux ligands des G-quadruplexes au niveau du protéome des cellules WI38 transfectées pour exprimer hTERT. Les deux ligands, TMPyP4 et la télomestatine, inhibent la télomérase mais ont une spécificité différente pour les diverses structures G-quadruplexes. En premier lieu, nous avons étudié leffet de la transfection dhTERT sur des cellules fibroblastiques humaines (WI38). Cette première étude a été conduite afin de caractériser ladaptation cellulaire résultante de limmortalisation des cellules WI38. Par la suite, celle-ci permettra de comparer ces résultats avec ceux obtenus lors de létude protéomique de leffet des ligands des G-quadruplexes. Nous avons montré que hTERT induit une augmentation de la capacité fonctionnelle du réticulum endoplasmique ainsi quune modulation des signaux cellulaire Ca2+-dépendant. Nous proposons que cette adaptation cellulaire est responsable dune résistance accrue vis-à-vis de différents stress environnementaux. Dautres protéines impliquées dans des mécanismes doncogenèse ont été identifiées et sont différentiellement exprimées entre les cellules parentales et les cellules transfectées. Lanalyse protéomique des traitements cellulaires indique que TMPyP4 induit une altération du protéome beaucoup plus prononcée que celle induite par la télomestatine. Ceci est probablement dû au manque de spécificité de TMPyP4 pour les G-quadruplexes télomériques. TMPyP4 induit, entre autres, une sous-expression massive des hnRNPs, une modulation de la voie protéasomale, une diminution probable de la traduction et une surexpression de plusieurs chaperonnes moléculaires. La télomestatine induit notamment une surexpression de la protéine BCL2A1 qui est impliquée dans les processus de résistance aux agents anticancéreux et une probable augmentation de la traduction. Les deux ligands ont des effets communs sur la variation dexpression des chaperons CCT (sou-expression), de lHSP90 alpha (surexpression) et de lhnRNP D (sous-expression). LHSP90 est également surexprimée dans les cellules hTERT-WI38 par rapport aux cellules parentales. Cette protéine fait actuellement lobjet de nombreuses recherches visant à inhiber son activité du fait de son implication en oncogenèse ainsi que dans la modulation de lactivité de la télomérase. Enfin, nous avons montré lintérêt de ce type détude protéomique dans lévaluation dagents à vocation thérapeutique préalablement aux études cliniques.

There is a high demand for quadruplex-specific compounds that not only bind preferentially to quadruplex DNA over duplex DNA, but also bind to one quadruplex motif over other motifs. Quadruplex structures are recognized as common occurrences in cancer cells, and if a compound could stabilize this structure, it may serve as an effective anti-cancer treatment with minimal side effects. In this study, cyanine dyes’ interactions with DNA were analyzed with fluorescence titrations, UV-Vis thermal studies, circular dichroism titrations, and surface plasmon resonance (SPR) analysis. With these techniques, binding affinity, DNA stabilization, and conformational shifts were analyzed to determine if cyanine dyes could act as quadruplex-specific binding compounds for possible cancer treatments.

Orientador: Mônica Alonso Cotta / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin / Made available in DSpace on 2018-08-19T11:07:57Z (GMT). No. of bitstreams: 1 MurilloMunar_DuberMarcel_M.pdf: 2543124 bytes, checksum: 3cf9b797cba5e450d6c025e609885768 (MD5) Previous issue date: 2011 / Resumo: Este trabalho tem como finalidade mostrar a importância dos diferentes modos da Microscopia de Varredura por Ponta de Prova (SPM) numa abordagem complementar para o estudo de dois diferentes sistemas biológicos. O processo de formação de biofilmes da bactéria fitopatogênica Xylella fastidiosa (Xf) foi o primeiro sistema abordado neste trabalho. Neste caso nosso objetivo é levantar informações que possam complementar o modelo mais aceito atualmente e corroborar os resultados obtidos anteriormente em nosso grupo. As amostras foram preparadas sobre substratos de silício recoberto com ouro e cultivadas durante tempos de crescimento de 7, 14 e 21 dias. O principal modo utilizado foi a Microscopia de Força Atômica por Kelvin Probe por modulação de amplitude (AM-KPFM) que fornece o potencial de superfície com resolução nanométrica. Imagens por KPFM foram adquiridas simultaneamente com as de topografia e fase obtidas por Microscopia de Força Atômica no modo não-contato (NC-AFM). Os resultados obtidos revelaram um processo de recobrimento gradual das bactérias por um filme de substância polimérica extracelular (EPS), concordando com os modelos propostos na literatura, porém ainda não comprovados. Imagens adquiridas por microscopia óptica (MO) mostram um desenvolvimento mais lento dos biofilmes (BF) em comparação aos resultados de G. S. Lorite para BF sobre substratos de silício obtidos anteriormente em nosso grupo. Isto está de acordo com a preferência das bactérias por superfícies com potenciais mais altos. Um resultado original está na observação de protuberâncias encontradas nas extremidades das bactérias, que mostram sinal elétrico diferenciado do resto da célula. Acreditamos que estas estruturas estão relacionadas ao processo de reprodução, pois aparecem tanto em bactérias isoladas como nas que estão no BF para todos os tempos de crescimento. O segundo sistema estudado foi a formação de estrutura quadrúplex-G de DNA. As amostras de DNA foram preparadas sobre mica e medidas no modo NC-AFM. Embora o DNA seja um sistema muito estudado em AFM, o protocolo de preparação das amostras muda segundo o tipo de estrutura que se queira visualizar. Assim, a maior parte do trabalho neste caso consistiu em desenvolver este protocolo de preparação que permitisse a visualização por AFM das estruturas de interesse. Usando concentrações altas de DNA (5ng/µL) as imagens apresentaram estruturas auto-organizadas que impedem a visualizaçã da estrutura quadrúlex·G. Para concentraçõs menores que 0,5ng/µL conseguimos visualizar moléulas isoladas, mas ainda assim as moléulas nã ficaram num estado relaxado. Um resultado interessante foi encontrado nas imagens de fase de moléulas isoladas (com concentraçã de DNA de 0,1ng/µL) onde se observam diferençs estruturais no interior das moléulas, possivelmente devido àformaçã da estrutura quadrúlex-G. Estas diferençs de fase representam um resultado original e mostram a importâcia da complementaridade dos modos AFM na observaçã de fenôenos biolóicos / Abstract: The aim of this work is to show the importance of different modes of Scanning Probe Microscopy (SPM) in a complementary approach to the study of two different biological systems. The process of biofilm formation of the phytopathogenic bacterium Xylella fastidiosa (Xf) was the first system discussed in this work. In this case our goal is to gather information that complements the currently most accepted model and corroborates the results obtained previously in our group. The samples were prepared on silicon substrates coated with gold and cultivated during 7, 14 and 21 days. The main results were obtained using Kelvin Probe Atomic Force Microscopy with amplitude modulation (AM-KPFM) which provides the surface potential with a nanometric resolution. Images were acquired by KPFM simultaneously with topography and phase images obtained by Atomic Force Microscopy in non-contact mode (NC-AFM). The results revealed a process of gradual coating of the bacteria by a film of extracellular polymeric substance (EPS), in agreement with the models proposed in the literature, but not yet proven. Images acquired by optical microscopy (OM) show a slower development of the biofilms (BF) compared to the results of G. S. Lorite for BF on silicon substrates obtained previously in our group. This result agrees with the preference of the bacteria for surfaces with large potentials. An original result is the observation of lumps found at the extremities of the bacteria, which show electrical signal different from the rest of the cell. We believe that these structures are related to the reproduction process as they appear both in isolated bacteria and those on BF for all growth times. The second system studied was the formation of G-quadruplex structure of DNA. DNA samples were prepared on mica and measured in the NC-AFM mode. Although DNA is a well studied system in AFM, the sample preparation protocol changes depending on the type of structure to be viewed. Thus, most of the work described here was to develop the preparation protocol that allows the visualization of the structures of interest by AFM. Using high concentrations of DNA (5ng/µL) the images showed self-organized structures that prevent visualization of G-quadruplex structure. For concentrations less than 0.5 ng/mL we got isolated molecules, but still the molecules were not in a relaxed state. An interesting result was found in the phase images of isolated molecules (with DNA concentration of 0.1 ng/mL) where structural differences are observed within the molecules, possibly due to the formation of G-quadruplex structure. These phase differences represent an original result and show the importance of the complementarity of AFM modes in the observation of biological phenomena / Mestrado / Mestre em Física

Un G-quadruplex (G4) est une structure non-canonique d’acides nucléiques formée par des séquences riches en guanines. Certains G4s sont polymorphiques, une même séquence peut former desG4s de différentes topologies. Les G4s sont proposés comme régulateurs de processus biologiques car ils sont trouvés dans des régions génomiques clés telles que dans des promoteurs de gènes et au niveau des télomères. Stabiliser ces G4s par rapport à la forme duplexe est une stratégie proposée pour combattre le cancer. Pour ce faire, des ligands spécifiques et affins sont utilisés. Le design de ces ligands implique habituellement de larges plans aromatiques, optimisés pour se lier par des interactions π-π sur les Gquartets extérieurs. Cependant, si ce type d’interaction était le seul mode de liaison, tous les ligands auraient des affinités similaires pour tous les G4s.Afin de caractériser les structures ciblées et de quelle manière les ligands vont interagir avec celles-ci, nous avons utilisé la spectrométrie de masse de type native (MS). D’abord, nous avons développé une méthode de préparation d’échantillons en conditions KCl pour former les G4s dans des conditions biologiquement pertinentes. Ensuite, nous avons caractérisé les équilibres de liaison du K+ aux G4s et caractérisé leur mécanisme de repliement. Ce mécanisme implique la présence d’une impasse constituée de G4s antiparallèles à 1- et 2-K+ qui sont formés rapidement. Enfin, nos études de liaison de ligands ont montré que certains des ligands les plus affins pouvaient influencer la structure des G4s comme observé par le nombre d’ions potassium liés. Les ligands Phen-DC3, 360A et PDS sont capables de déplacer les équilibres vers la forme à 1-K+ antiparallèle. La structure antiparallèle à 2-K+ est favorisée par la liaison coopérative de deux ligands Cu-ttpy. Ces résultats démontrent l’importance de la caractérisation des stoechiométries de complexes ternaires (G4:ligand:K+), obtenue par la spectrométrie de masse native. / A G-quadruplex (G4) is a non-canonical nucleic acids structure formed by guanine-rich sequences. Some G4s are polymorphic, a given sequence can form G4s of different topologies. G4s are proposed to be biological regulators because they are found in key regions of the genome, for example, ingene promoters or at the telomeres. Stabilizing G4s formed in those regions as compared to the duplex form is a strategy to fight cancer. To do so, specific and affine ligands are used. Ligand design usually implies the optimization of large aromatic planes to π-π stack on external G-quartets. However, if this was the only binding mode, all ligands would bind with similar affinities to all G4s.To characterize which structures should be targeted and how the ligands interact with these structures, we used native mass spectrometry (MS).First, we developed a MS-compatible sample preparation method in KCl conditions in which G4s are folded with similar topologies as compared to those obtained in biologically relevant conditions. Then, we characterized the K+ binding equilibria and G4s folding pathways. This folding pathway involves the presence of a dead-end constituted by antiparallel G4s with either 1- or 2-K+ cations that are folded first. Finally, our ligand binding studies showed that some of the most affine ligands can influence G4’sstructures, as probed by the number of K+ ions bound. Ligands Phen-DC3, 360A and PDS are able to shift the equilibria towards the 1-K+ antiparallel G4s. The formation of antiparallel with 2-K+ complexes is induced by the cooperative binding of two Cu-ttpy ligands. Our results demonstrate the importance to characterize ternary complex stoichiometries (G4:ligand:K+) as obtained from native mass spectrometry.