Academic literature on the topic 'ARN G-Quadruplexes'

Abstract Over 100 modifications to RNA are known to occur in human cells, where they influence many aspects of RNA biology, including protein and nucleic acid interactions. Our survey of human RNA-modifying enzymes with selectively essential phenotypes in specific cancers, suggests that these cancer-essential enzymes fall into four mechanistic categories: group transfer enzymes; base editors; nucleases; and helicases. Our team has discovered nanomolar or picomolar inhibitors of cancer-essential enzymes within each of these four categories. DHX9, a novel cancer target, is a multifunctional DEAH-box RNA helicase which can unwind regions of double-stranded DNA and RNA helices but has a greater propensity for secondary structures such as DNA/RNA hybrids (R-loops), circular RNA and DNA/RNA G-quadruplexes. DHX9 interacts with and regulate a large variety of proteins, including key proteins in DNA damage repair pathways such as BRCA1, ATR, Ku86, and WRN. We previously demonstrated that DHX9 inhibition is selectively efficacious in microsatellite high (MSI-H)/defective mismatch repair (dMMR) tumor models, in vitro and in vivo. Further profiling of DHX9 inhibitors across a broad panel of cancer cell lines reveals that tumor cells with Loss-of-Function (LOF) mutations in the DNA damage repair genes BRCA1 and/or BRCA2 (as defined by somatic mutations including single-nucleotide variants and/or copy number loss), are also selectively responsive to DHX9 inhibitor treatment. Selective dependence on DHX9 was observed in both ovarian and breast cancer cell lines that exhibit BRCA1 and/or BRCA2 LOF. DHX9 inhibition leads to increased RNA/DNA secondary structures such as R-loops and G-quadruplexes, resulting in subsequent DNA damage and increased replication stress. Cell lines that exhibit BRCA1 and/or BRCA2 LOF appear unable to resolve this replication stress and showed S-G2 phase cell cycle arrest prior to onset of apoptosis. An orally bioavailable DHX9 inhibitor was dosed in vivo to assess DHX9 dependency within multiple human xenografts representing triple negative breast cancer and high-grade serous ovarian cancer with BRCA1 and/or BRCA2 LOF. In all models, DHX9 inhibition was well tolerated for a period of up to 28 days, with robust and significant tumor growth inhibition - including tumor regression - observed in multiple BRCA1 and/or BRCA2 LOF models; in contrast, minimal tumor growth inhibition was observed in BRCA1 and BRCA2 wild type models. These results portend DHX9 inhibition as a novel treatment modality for patients with BRCA1 and/or BRCA2 LOF across multiple tumor types, including breast and ovarian cancer. Citation Format: Robert A. Copeland, Jennifer Castro, Matthew H. Daniels, Deepali Gotur, Young-Tae Lee, Shihua Yao, David Brennan, Brian T. Johnston, Monique Laidlaw, Sunaina Pai, Jie Wu, Rishabh Bansal, Anugraha Raman, Shane M. Buker, Julie Liu, E. Allen Sickmier, Kevin Knockenhauer, Chuang Lu, Stephen J. Blakemore, Serena J. Silver, P. Ann Boriack-Sjodin, Kenneth W. Duncan, Jason A. Sager. Small molecule inhibitors of RNA modifying enzymes as precision cancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY11-02.

Abstract DHX9 is a multifunctional DEAH-box RNA helicase which can unwind regions of double-stranded DNA and RNA helices but has a greater propensity for secondary structures such as DNA/RNA hybrids (R-loops), circular RNA and DNA/RNA G-quadruplexes. Given the delicate balance of R-loop formation and resolution in maintaining efficient transcription and replication, the ability of DHX9 to unwind R-loops is important in helping to maintain genomic stability. In addition, DHX9 can interact and regulate a large variety of proteins, including key proteins in DNA damage repair pathways such as BRCA1, ATR, Ku86, and WRN. Previously we demonstrated that DHX9 inhibition was efficacious in microsatellite high (MSI-H) CRC xenograft tumor models. Here we report results of in vitro and in vivo preclinical studies in ovarian and breast cancer that indicate patients with Loss-of-Function (LOF) mutations in the DNA damage repair genes BRCA1 and/or BRCA2, may also benefit from DHX9 inhibitor treatment. DHX9 small molecule inhibitors were tested for anti-proliferative activity in a panel of ovarian (N = 15) and breast cancer (N = 41) cell lines, and bioinformatic analyses performed to identify molecular variants that co-associate with sensitivity or resistance cell proliferation outcomes. Notably, selective dependency on DHX9 was observed in both ovarian and breast cancer cell lines that exhibit BRCA1 and/or BRCA2 LOF, as defined by somatic mutations including single-nucleotide variants and/or copy number loss. DHX9 inhibition leads to increased RNA/DNA secondary structures such as R-loops and G-quadruplexes, resulting in subsequent DNA damage and increased replication stress. Cell lines that exhibit BRCA1 and/or BRCA2 LOF appear unable to resolve this replication stress and show S-G2 phase cell cycle arrest prior to onset of apoptosis. Furthermore, a potent and selective DHX9 inhibitor ATX666 was dosed orally in vivo to assess DHX9 dependency within multiple human xenografts representing triple negative breast cancer and high-grade serous ovarian cancer with BRCA1 and/or BRCA2 LOF. In all models, ATX666 was well tolerated for a period of up to 28 days, with robust and significant tumor growth inhibition and regression observed in multiple BRCA1 and/or BRCA2 LOF models with minimal tumor growth inhibition observed in BRCA1 and BRCA2 wild type xenograft models. These results extend the opportunity for DHX9 inhibition to provide therapeutic benefit in solid tumor patients beyond what was previously reported for MSI-H CRC. Together, this preclinical data package validates DHX9 as a tractable new target with potential utility as a novel treatment for patients with BRCA1 and/or BRCA2 LOF across multiple tumor types including breast and ovarian cancer. Citation Format: Jennifer Castro, Matthew H. Daniels, Sunaina Pai, Monique Laidlaw, Jie Wu, David Brennan, Brian T. Johnston, Anugraha Raman, Chuang Lu, Stephen J. Blakemore, Serena J. Silver, P. Ann Boriack-Sjodin, Kenneth W. Duncan, Jason A. Sager, Robert A. Copeland. DHX9 inhibition as a novel therapeutic for ovarian and breast cancer with loss-of-function mutations in the DNA damage repair genes BRCA1 or BRCA2 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr PR003.

Abstract Over 100 modifications to RNA are known to occur in human cells, where they play critical roles in many aspects of normal cellular physiology, such as cell fate decisions and terminal differentiation, through effects on RNA biology such as protein and nucleic acid interactions. Our survey of human RNA-modifying enzymes suggests many are cancer essential enzymes with striking synthetic lethal profiles, including the XRN1 nuclease and DHX9 helicase. XRN1 degrades single stranded mRNA from the 5′→3′ direction and plays a key role in endogenous cellular mRNA turnover. XRN1 can also degrade double-stranded RNA (dsRNA), and as such plays a role in innate immunity. We identified XRN1 as a selective vulnerability in tumor cells with intrinsic elevation of a Type I Interferon Stimulated Gene (TISG) signature through analysis of publicly available CRISPR data across 483 tumor cell lines. XRN1 KO via CRISPR leads to robust anti-proliferative effects in TISG high cells, but not in TISG low cells, as well as upregulation of Interferon-β mRNA and induction of phosphorylated PKR. Exogenous treatment with Interferon-β to induce elevated Type I Interferon expression sensitizes TISG low cells to XRN1 KO, confirming this synthetic lethal relationship. Enzymatic and biophysical assays for XRN1 were developed and used to identify and optimize XRN1 inhibitors. We identified Compound 1, a substrate-competitive inhibitor with single-digit micromolar affinity for XRN1 which does not bind or inhibit XRN2. High-resolution crystal structures indicate that Compound 1 binds in an allosteric binding site, confirming the mechanism of inhibition and selectivity profile determined by in vitro assays. This data suggests that XRN1, through its regulation of dsRNA burden, is a compelling and druggable oncology target for TISG high tumors. DHX9 is a multifunctional DExH-box RNA helicase which can unwind regions of double-stranded DNA and RNA helices but has a greater propensity for secondary structures such as DNA/RNA hybrids (R-loops) and DNA/RNA G-quadruplexes. DHX9 interacts with and regulates many proteins, including key members of DNA damage repair pathways. We have found that DHX9 knockdown is synthetic lethal in microsatellite high (MSI-H) or defective mismatch repair (dMMR) tumor models. A suite of assays was developed to identify and optimize potent and selective inhibitors of the DHX9 helicase, which recapitulate our findings with genetic tools. Profiling of DHX9 inhibitors across a broad panel of cancer cell lines reveals that tumor cells with mutations in the DNA damage repair genes BRCA1 and/or BRCA2 are also responsive to DHX9 inhibitor treatment in vitro and in vivo. DHX9 inhibition leads to increased RNA/DNA secondary structures such as R-loops and G-quadruplexes, resulting in subsequent DNA damage and increased replication stress, leading to cell cycle arrest and apoptosis. These results suggest that DHX9 inhibitors are a novel treatment modality for patients with defective DNA damage repair pathways such as dMMR and/or BRCA mutations. Citation Format: Serena J Silver, Maureen M. Lynes, Brian A. Sparling, Jennifer Castro, Matthew H. Daniels, Sunaina Pai, Sophie A. Shen, David Brennan, Hyelee Lee, Gordon J. Lockbaum, Kevin Knockenhauer, Deepali Gotur, Simina Grigoriu, Shihua Yao, Shane Buker, Monique Laidlaw, Jie Wu, Stephen J. Blakemore, P. Ann Boriack-Sjodin, Kenneth W. Duncan, Jason A. Sager, Robert A. Copeland. RNA-modifying enzyme inhibitors as synthetic lethal cancer therapeutics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA024.

Abstract DHX9 is a DEAH-box RNA helicase which can unwind regions of double-stranded DNA and RNA but has a greater propensity for secondary structures such as DNA/RNA hybrids (R-loops), circular RNA and DNA/RNA G-quadruplexes. Given the delicate balance of R-loop formation and resolution in maintaining efficient transcription and replication, the ability of DHX9 to unwind R-loops is important in helping to maintain genomic stability. In addition, DHX9 can interact and regulate a large variety of proteins, including key proteins in DNA damage repair pathways such as BRCA1, ATR, Ku86, and WRN. Previously we demonstrated that DHX9 inhibition was efficacious in microsatellite high (MSI-H) CRC xenograft tumor models. Efficacy attributed to DHX9 loss in MSI-H correlates with defective DNA repair pathways such as mismatch repair (MMR). Here we report results of preclinical studies in ovarian and breast cancer models that indicate patients with Loss-of-Function (LOF) mutations in the DNA damage repair genes BRCA1 and/or BRCA2, may also benefit from DHX9 inhibitor treatment. DHX9 small molecule inhibitor ATX968 was tested for anti-proliferative activity in a large cell panel of 300 cell lines, and bioinformatic analyses was performed to identify molecular variants that co-associate with sensitivity or resistance cell proliferation outcomes. Notably, selective dependency on DHX9 was observed in both ovarian and breast cancer cell lines that exhibit BRCA LOF, as defined by somatic mutations including single-nucleotide variants and/or copy number loss in BRCA1 and/or BRCA2. DHX9 inhibition leads to increased RNA/DNA secondary structures such as R-loops and G-quadruplexes, resulting in subsequent DNA damage and increased replication stress. Cell lines that exhibit BRCA LOF appear unable to resolve this replication stress and show S-G2 phase cell cycle arrest prior to onset of apoptosis. Furthermore, a potent and selective DHX9 inhibitor tool compound was dosed orally in vivo to assess DHX9 dependency within multiple human xenografts representing triple negative breast cancer and high-grade serous ovarian cancer with BRCA LOF. In all models, the tool DHX9 inhibitor was dosed orally at 100 mg/kg BID and it was well tolerated for a period of up to 28 days. Robust and significant tumor growth inhibition and regression was observed in multiple BRCA LOF models with minimal tumor growth inhibition observed in BRCA1 and BRCA2 wild type xenograft models. These results extend the opportunity for DHX9 inhibition to provide therapeutic benefit for patients with solid tumors beyond what was previously reported for MSI-H CRC. Together, this preclinical data package validates DHX9 as a tractable new target with potential utility as a novel treatment for patients with defective DNA repair, such as MMR and BRCA1 and/or BRCA2 LOF, across multiple tumor types including colorectal, breast and ovarian cancer. Citation Format: Jennifer B. Castro, Matthew H. Daniels, Sunaina Nayak, Monique Laidlaw, David Brennan, Brian T. Johnston, Jie Wu, Anugraha Raman, Chuang Lu, Stephen J. Blakemore, Serena J. Silver, P. Ann Boriack-Sjodin, Kenneth W. Duncan, Jason A. Sager, Robert A. Copeland. DHX9 inhibition as a novel therapeutic for cancer with loss-of-function mutations in DNA damage repair genes BRCA1 and BRCA2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3908.

Abstract Background: DHX9 is a DEAH-box RNA helicase which has been reported to play important roles in replication, transcription, translation and RNA splicing/processing, all of which contribute to DHX9’s role in the maintenance of genomic stability. Functionally, DHX9 unwinds and/or resolves regions of double-stranded DNA and RNA helices but has a greater propensity for secondary structures such as DNA/RNA hybrids (R-loops), circular RNA (circRNA) and DNA/RNA G-quadraplexes. Overexpression of DHX9 is evident in multiple cancer types, including colorectal cancer (CRC) and lung cancer. In particular, microsatellite instable (MSI) tumors exhibiting defective mismatch repair (dMMR) show a strong dependence on DHX9, making this helicase an attractive target for oncology drug discovery. Here, we will demonstrate validation of DHX9 as a novel target for CRC-MSI, and the identification of potent and selective in vitro and in vivo small molecule inhibitors of DHX9. Materials and Methods: DHX9 targeted siRNA or DHX9 small molecule inhibitors were used to assess anti-proliferative activity in multiple different CRC-MSI and CRC-MSS cell lines through either CellTiter-Glo proliferation or colony formation assays. Downstream biological consequences of DHX9 knockdown or inhibition in vitro were determined using immuno-fluorescent imaging, western blot, flow cytometry to measure RNA/DNA secondary structure, cell cycle changes, apoptosis, and qPCR to measure circBRIP1 induction. CRC-MSI and CRC-MSS tumor xenografts were treated with DHX9 inhibitor ATX968 BID orally for 21-28 days. Tumor and plasma samples were collected for pharmacokinetics (PK) and pharmacodynamic (PD) (circBRIP1) measurement. Results: We demonstrate that DHX9 inhibition in CRC-MSI leads to an increase in RNA/DNA secondary structures such as R-loops, G-quadruplexes and Alu mediated circRNA such as circBRIP1, leads to subsequent DNA damage and increased replication stress. Cell lines that exhibit defective DNA repair pathways such as dMMR are unable to resolve this replication stress and demonstrate S-G2 phase cell cycle arrest prior to onset of apoptosis. We confirmed this selective dependency in a panel of cancer cell lines, where anti-proliferative effects mediated by DHX9 inhibition were associated with dMMR status. Furthermore, DHX9 tool compound ATX968 was well tolerated in vivo across a 28-day treatment period with robust and durable tumor regression observed in the MSI CRC tumor xenograft model. Following cessation of treatment, minimal tumor regrowth was observed in a 28-day post treatment window. Tumor and plasma concentrations of ATX968 and changes in PD markers of DHX9 inhibition were measured and resulting PK, PD and efficacy data were highly correlated. Conclusions: Together, these preclinical data validate DHX9 as a tractable new target with potential utility as a novel treatment for patients with CRC-MSI. Conflict of Interests: All authors are current or former employees and shareholders of Accent Therapeutics, Inc. Citation Format: Jennifer Castro, Matthew H Daniels, David Brennan, Brian T Johnston, Rishabh Bansal, Monique Laidlaw, Chuang Lu, Deepali Gotur, Young-Tae Lee, Kevin Knockenhauer, April Case, Jie Wu, Anugraha Raman, Jae Eun Cheong, Julie Liu, Shane M Buker, E. Allen Sickmier, Stephen J Blakemore, P. Ann Boriack-Sjodin, Kenneth W Duncan, Serena J Silver, Scott Ribich, Robert A Copeland. DHX9 inhibition as a novel therapeutic modality in microsatellite instable colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C087.

Les acides nucléiques riches en guanines ou en cytosines peuvent se replier sur eux-mêmes et former des systèmes tétramériques tels que les G-quadruplexes (G4) ou les i-motifs. Ces motifs, abondamment représentés dans certaines régions du génome humain semblent contribuer à la régulation cellulaire et suscitent depuis plusieurs années un intérêt grandissant. Ils sont notamment présents dans la région télomérique, mais aussi dans les promoteurs d’oncogènes ou au sein des génomes viraux et sont impliqués dans certaines pathologies humaines. Ils représentent ainsi des cibles thérapeutiques et diagnostiques potentielles. Cependant, les G4 adoptent in-vitro des topologies variées qui compliquent le développement de ligands spécifiques et affins. Dans ce contexte, le laboratoire a développé le concept du TASQ pour ‘‘Template Assembled Synthetic G-Quadruplex’’ dans le but d'accéder à des G4 se structurant en une topologie définie.Le premier chapitre décrit l’assemblage de mimes de motifs G4 contraints en une topologie unique. En utilisant un gabarit cyclodécapeptide rigide et différentes méthodes de conjugaison, nous avons assemblé des motifs G4 ARN parallèle et hybride ADN/ARN dérivant de la séquence télomérique ainsi qu’un motif G4 d’ADN présent dans la séquence promotrice du VIH-1. L’utilisation du concept TASQ nous a également permis de préparer un motif G-triplexe (G3), intermédiaire à la formation des motifs G4. Nous avons montré une forte stabilisation de tous les édifices G4 contraints ainsi préparés.Le second chapitre concerne les études de caractérisation et de sélection de ligands vis-à-vis des motifs G4 et G3 contraints. La caractérisation repose sur l’évaluation de l’affinité et de la sélectivité de différentes familles de ligands pour ces édifices, par résonance plasmonique de surface ou par interférométrie bio-couche. La sélection de ligands a été réalisée par la méthode SELEX dans le but d’obtenir des aptamères affins et spécifiques d’un motif G4 contraint
Guanines or cytosines rich nucleic acids can fold into tetrameric G-quadruplexes (G4) or i-motifs structures. G4 motifs are found within the human genome and should contribute to cellular regulation. In particular G4 are found at telomeric region and also in promoters of oncogenes or within viral genomes. They are suspected of participating in the regulation of human pathologies and have therefore been envisioned as potential therapeutic and diagnostic targets. However, the intrinsic conformational polymorphism of G4 motifs complicates the development of specific and affine ligands. In this context, the laboratory has developed the TASQ concept for "Template Assembled Synthetic G-Quadruplex" with the aim to obtain a defined G4 topology.The first chapter reports on the assembly on the peptide template of RNA and DNA:RNA hybrid G4 structures that derive from the human telomeric sequence as well as of DNA G4 structure found within the HIV virus promoter. G-triplex (G3) motif which is supposed to be an intermediate during the formation of the G4 motifs has also been prepared. By using appropriate ligations of the oligonucleotide strands on the peptide template we were able to control the folding of G-quadruplex motifs and stabilize them.The second chapter reports the studies for the characterization and the selection of ligands against G4 and G3 motifs. The evaluation of the affinity and selectivity of different families of ligands for these constrain motifs was performed by using surface plasmon resonance or by bio-layer interferometry. The selection of ligands was carried out by the SELEX method in order to obtain affine and specific aptamers of a constrained G4 motif

Le développement du cancer et la réponse aux traitements sont associés à des variations de la régulation post-transcriptionnelle qui entraîne une modification du protéome qualitativement et/ou quantitativement. La régulation post-transcriptionnelle implique des protéines de liaison à l’ARN (RBP), interagissant avec des éléments cis comme les séquences, les modifications ou les structures de l’ARN. Parmi ces éléments cis, les structures non-canoniques nommées ARN G-Quadruplexes (RG4), et les modifications N6-Méthyladénosines (m6A), jouent un rôle critique dans le modelage post-transcriptionnel guidant l’expression des gènes du cancer, et leur ciblage est actuellement envisagé par des essais pré-cliniques. L’un des défis majeurs de ce champ de recherches repose sur la compréhension des mécanismes contrôlants la sélectivité des sites modifiés en m6A, leur reconnaissance et leur suppression, ainsi que sur l’identification des régulateurs de la structuration des RG4. Pour répondre à ces défis, la colocalisation entre les RG4 et les m6A et leur régulation mutuelle doit encore être étudiée de manière claire. Un autre objectif clé consiste à relier les interactions entre les protéines liant les RG4 dans les transcrits et leurs fonctions biologiques liées au cancer en tirant parti des prédictions de la structuration des RG4 et des données expérimentales sur les RG4 et les RBP. Mon projet de thèse aborde ces deux défis centrés sur la régulation cis- et trans- des RG4, en utilisant des approches multidisciplinaires incluant la bioinformatique, la biologie moléculaire et cellulaire. Pour cartographier et caractériser globalement les régulateurs agissant en trans sur les RG4, nous avons développé QUADRatlas (https://rg4db.cibio.unitn.it), une base de données de RG4 identifiés expérimentalement et prédits par ordinateur dans le transcriptome humain, liés à leurs fonctions biologiques et à leurs associations aux pathologies (Bourdon et al, NAR, 2023). Ce travail offre un accès à un large catalogue construit manuellement de protéines de liaison aux RG4 connues, complété par un vaste ensemble de données de sites de liaison de RBP pour découvrir de nouvelles interactions potentielles RG4-RBP. Notre étude sur l'interaction entre les RG4 et les m6A a révélé leur colocalisation dans le transcriptome codant humain. Nous avons démontré in vitro que la stabilité des RG4 n'était pas inhibée par la présence de m6A. Cependant, nous avons montré que la stabilisation des RG4 diminuait le niveau global de m6A dans les lignées cellulaires cancéreuses. Pour expliquer cet effet, nous avons étudié la capacité des RBP à se lier aux RG4, m6A ou aux RG4 contenant des m6A (RG4(m6A)). Nous avons découvert que les RG4s pouvaient agir comme des plateformes pour les protéines de liaison aux m6A et ainsi réguler leur présence sur les transcrits. Ce travail fournit des informations essentielles sur la régulation mutuelle de deux éléments cis majeurs de l'ARNm par les RBP. De futures analyses seront nécessaires pour découvrir l’effet de la colocalisation RG4(m6A) sur l’expression des gènes du cancer
Cancer development and response to treatments are associated to post-transcriptional rewiring which in turn modifies the cancer proteome qualitatively and/or quantitatively. Post-transcriptional regulation involves RNA binding proteins (RBP) interacting with cis-acting elements like RNA sequences, modifications or structures. Among the cis-regulators, non-canonical structures, called RNA G-Quadruplexes (RG4), and N6-methyladenosines modifications (m6A), play a critical role in shaping post-transcriptional expression of cancer genes and their targeting is currently investigated in pre-clinical studies. One major challenge in the field lies in understanding the mechanisms controlling selectivity in m6A deposition, reading and removal, as well as deciphering RG4 folding and regulators. Whether m6A and RG4 colocalize and regulate each other remains to be fully investigated. Another key challenge is to link RG4-protein interactions in transcripts to cancer-relevant biological functions by leveraging predictions of RG4 structuration and experimental data on RG4 and RBP.My thesis project tackled these two challenges centered on the cis- and trans- regulation of RG4s, using multidisciplinary approaches including bioinformatics, molecular and cellular biology. To globally map and characterize RG4 trans-acting regulators, we developed QUADRatlas (https://rg4db.cibio.unitn.it), a database of experimentally-derived and computationally predicted RG4 in the human transcriptome, linked with their biological function and disease associations (Bourdon et al, NAR, 2023). This work provides a broad access to a manually curated catalogue of known RG4-binding proteins, complemented with an extensive RBP binding sites dataset to discover new potential RG4-RBP interactions. Our study on the interplay between RG4 and m6A revealed their colocalization in the human coding transcriptome. We demonstrated in vitro that RG4 stability was not inhibited by m6A presence. However, we showed that the stabilisation of RG4 decreased global m6A level in cancer cell lines. To explain this effect, we studied the ability of RBP to bind RG4, m6A or RG4 containing m6A (RG4(m6A)) and found that RG4 could act as a platform for m6A binding proteins and thus regulate their presence on transcripts. This work provides insights on the co-regulation of two major mRNA cis-acting elements by RBP. Future analyses will then be needed to unravel the effect of RG4(m6A) colocalization on cancer gene expression

L’objectif de ces travaux de thèse était l’étude des interactions de petites molécules avec les multiples structures de l’ADN quadruplex via i) le développement et l’utilisation d’un test haut-débit pour l’analyse des interactions ligand-ADN quadruplex et le criblage de chimiothèques/ciblothèques et ii) la préparation de composés aux modes d’interactions multiples (empilement/sillon, covalent/non-covalent, etc.), sélectifs (quadruplex vs. duplex et intra-quadruplex) et éventuellement fonctionnalisés (biotine, fluorophore, etc.). La première partie des travaux a été centrée sur le développement du test G4-FID (G-quadruplex Fluorescent Intercalator Displacement) qui est une méthode semi-quantitative permettant l’évaluation de l’affinité et de la sélectivité de petites molécules pour l’ADN quadruplex par déplacement d’une sonde off/on, le Thiazole Orange (TO). Le test a notamment été transposé avec succès de la cuve vers la microplaque (HT-G4-FID). D’autre part, nous avons montré l’intérêt de fluorophores alternatifs, TO-PRO-3 et Hoechst 33258, aux caractéristiques spectrales complémentaires à TO. Cette méthode d’analyse a également été utilisée avec succès pour l’identification de nouveaux ligands sélectifs d’ADN quadruplex et la mise en évidence des relations structure-activité ainsi que des sélectivités structurales. La deuxième partie des travaux a été consacrée à la préparation et à l’étude de nouveaux ligands d’ADN quadruplex. Ces ligands possèdent des particularités, soit dans leur mode d’interaction (sillons, coordination) soit par leur bifonctionnalité (biotinylés, fluorescents). Nous avons ainsi préparé un ligand de quadruplex polyhétéroaryle acyclique (TOxaPy) possédant une sélectivité inattendue pour certaines structures de l’ADN quadruplex. D’autre part, nous avons montré que les complexes de dérivés de terpyridine peuvent être adaptés, en changeant le ligand organique et/ou la nature du métal, de façon à interagir avec l’ADN quadruplex par interaction covalentes et/ou non covalentes
The aim of this thesis work was to study the interactions of small molecules with multiple structures of quadruplex DNA via i) the development and use of a high-throughput test for the analysis of ligand-quadruplex DNA interactions and screening of chemical libraries and ii) the preparation of compounds with multiple binding modes (stacking/groove, covalent/non-covalent, etc..) selective (quadruplex vs. duplex and intra-quadruplex) and possibly functionalized (biotin, fluorophore, etc.). The first part of the work was focused on the development of the G4-FID (G-quadruplex Intercalator Fluorescent Displacement) assay, which is a semi-quantitative method for evaluating the affinity and selectivity of small molecules for quadruplex DNA by displacing an off/on probe, the Thiazole Orange (TO). The test has been implemented successfully with microplate (HT-G4-FID). On the other hand, we have shown the importance of alternative fluorophores, TO-PRO-3 and Hoechst 33258, with complementary spectral characteristics. This method of analysis has also been successfully used for the identification of new selective ligands of quadruplex DNA and the identification of structure-activity relationships and structural selectivities. The second part of the work was devoted to the preparation and study of new DNA quadruplex ligands. These ligands possess particular characteristics either in their mode of interaction (grooves, coordination) or by their bifunctionality (biotinylated, fluorescent). We have prepared an acyclic polyheteroaryle quadruplex ligand (TOxaPy) with an unexpected selectivity for certain structures of quadruplex DNA. Furthermore, we showed that complexes of terpyridine derivatives can be tailored by changing the organic ligand and / or the metal in order to interact with quadruplex DNA by covalent and / or non-covalent interaction

La traduction des ARNm et la production de protéines sont des phénomènes étroitement contrôlés dans la cellule. La séquence de l'ARNm et sa structure, auxquelles sont associées des protéines se liant à l'ARN, ainsi que la qualité de la protéine produite à partir de cet ARNm, évaluée notamment par la voie de contrôle qualité associée aux ribosomes, sont des éléments impliqués dans ce processus majeur pour la cellule. Dans ce domaine, le contrôle de la production de protéine EBNA1 (Epstein-Barr Nuclear Antigen 1) du virus d'Epstein -Barr est un exemple intéressant. La protéine EBNA1 est essentielle pour la survie du virus dans les cellules hôtes. Les niveaux cellulaires de protéine EBNA1 sont très faibles, bien que la protéine soit présente dans toutes les cellules infectées. Cette dernière est aussi extrêmement antigénique. Il est aujourd'hui admis que la quantité de protéine EBNA1 présente dans les cellules est suffisante pour assurer le maintien du virus dans la cellule, mais assez basse pour lui permettre d'échapper au système immunitaire de l'hôte. Un contrôle de sa production est nécessaire à cet équilibre. Des études précédentes ont montré que le domaine GAr (répétitions de glycine et alanine), présent dans la partie N-terminale de la protéine, déclenche un mécanisme conduisant à l'inhibition de l'initiation de la traduction de l'ARNm d'EBNA1 en cis, sans affecter la traduction des autres ARNm présents dans la cellule. L'équipe a montré précédemment que les structures G4s (G-quadruplex) peuvent être formées dans l'ARNm codant le GAr. De nombreuses études ont montré l'importance de ces structures secondaires de l'ARN dans la régulation de la traduction de l'ARNm d'EBNA1. La nucléoline, un facteur nucléaire, peut se lier aux G4s de l'ARNm du GAr. Cependant, il a aussi été montré que le peptide GAr, et non l'ARNm associé, est nécessaire au contrôle de la traduction de l'ARNm du GAr en cis. L'objectif principal de ma thèse est de mieux comprendre le mécanisme déclenché par l'ARNm et le polypeptide naissant conduisant au contrôle de la traduction de l'ARNm d'EBNA1 en cis. En accord avec le fait que les structures G4s de l'ARN sont extrêmement dynamiques, nous avons montré dans un premier temps que les fonctions associées au G4s de l'ARNm du GAr, à savoir la localisation de l'ARNm, sa traduction et sa capacité à se lier à certaines protéines, dépendent du contexte dans lesquelles ces structures se trouvent. Nous montrons ensuite que la traduction de l'ARNm d'EBNA1 est nécessaire à l'interaction nucléoline-ARNm, signifiant que la traduction de l'ARNm induit des changements dans les propriétés de l'ARNm. En parallèle, nous avons étudié le NACA, une sous-unité du complexe chaperon NAC (nascent polypeptide-associated complex). NACA se détache du ribosome lors de la synthèse du GAr et interagit avec le GAr. NACA est aussi capable de se lier aux ARN et est déterminant dans la suite des évènements liés à l'ARNm codant le GAr. Enfin, et de façon assez surprenante, les facteurs d'initiation de la traduction sont aussi des éléments clés dans l'inhibition de la traduction de l'ARNm d'EBNA1. Le facteur le plus impactant identifié jusqu'à maintenant est le facteur eIF4A1. Ces résultats indiquent que la séquence et structure de l'ARNm et le polypeptide naissant correspondant sont impliqués dans l'inhibition de l'initiation de la traduction de l'ARNm d'EBNA1. Cependant, cela n'enlève pas la possibilité que l'ARNm et le polypeptide naissant déclenchent chacun une voie d'inhibition de la synthèse d'EBNA1 distincte l'une de l'autre. Les virus utilisent des éléments déjà présents dans la cellule pour assurer leur maintien dans la cellule hôte. Ainsi, les principes de biologie cellulaire décrits ici peuvent apporter des indications importantes pour une meilleure compréhension d'autres pathologies en plus de celles liées au virus d'Epstein-Barr
MRNA translation and protein synthesis are tightly regulated events in the cell. Mechanisms describing these key cellular events involve the mRNA sequence and its structure with the association of RNA-binding protein to it, as well as the quality of the translation product encoded by the mRNA, assessed notably through ribosome-associated quality control. In this context, the Epstein-Barr virus EBNA1 (Epstein-Barr Nuclear Antigen 1) mRNA translation regulation is an interesting example. EBNA1 is known to be an essential protein for the virus survival in the host cells. Even though EBNA1 is present in every infected cell, its protein level is remarkably low. As EBNA1 is highly antigenic, it has been suggested that EBNA1 levels in the cells are low enough to escape the immune system of the host, but sufficient to maintain EBV infection. This balance requires a tightly controlled EBNA1 production. Further studies showed that the GAr (glycine-alanine repeat) domain, located in the N-terminal part of EBNA1, triggers an in cis mechanism leading to the inhibition of the translation initiation of its own mRNA, without affecting translation of other mRNAs in the cell. Thus, the GAr domain of EBNA1 is a unique tool to study selective mRNA translation control without affecting general protein synthesis. It was previously shown that RNA G4 (G-quadruplex) structures can be folded in the GAr-encoding mRNA. Numerous studies underlined the importance of these RNA structures in the regulation of EBNA1 mRNA translation, and the team previously showed that nucleolin can interact with these RNA G4 structures, interaction which can be competed by some G4 ligands. However, it was also formerly shown that the GAr peptide itself plays a role in controlling in cis the translation of EBNA1-encoding mRNA, rather than just the RNA sequence. The main focus of the study presented here is to shed light on how this translation event and the fate of the encoding mRNA are regulated in cis by the mRNA and the encoded nascent polypeptide. In line with the fact that RNA G4 structures are highly dynamic, we first showed that GAr RNA G4-associated functions, namely mRNA localisation, translation and ability to bind RNA-binding proteins, are dependent on the context they are in, i.e. their position in the mRNA, the structures in their surrounding or the factors binding the mRNA, such as G4 ligands. We next demonstrated that translation of the EBNA1 mRNA is necessary for nucleolin-binding to it, meaning that the translation event modifies some properties of the EBNA1 mRNA. In parallel, we showed that the NACA, a subunit of the NAC chaperone complex, is detached from the ribosome and interacts with the GAr polypeptide. Interestingly, the NACA is also an RNA binding protein in addition to its chaperone function, and is determinant for the future processing of the EBNA1 mRNA. Finally, and unexpectedly, we show that translation initiation factors are also key players in the downregulation of the EBNA1 mRNA translation, affecting also the mRNA nucleolin-binding capacity, the most effective translation initiation factor in the downregulation of EBNA1 mRNA translation identified so far being eIF4A1. These results support the idea that both the RNA sequence and structure and the corresponding nascent polypeptide are involved in the downregulation of EBNA1 mRNA translation. However, it does not rule out the possibility that both the RNA structure and the polypeptide sequence trigger also their own separated inhibitory pathway. As viruses use components already present in the cells to maintain themselves, the cellular biology elements brought out here can provide insights on many other pathologies in addition to EBV-associated diseases

Formés à partir de brins d’ADN ou d’ARN riches en guanines, les quadruplexes résultent de l’empilement de tétrades de guanines constituées chacune par l’auto-assemblage dans un même plan de quatre guanines, stabilisées entre elles par un réseau de liaisons hydrogènes. En s’inspirant de cet édifice naturel, il est présenté au long de ce manuscrit de thèse la synthèse et l’étude de molécules de type TASQ (pour template-assembled synthetic G-quartet) hydrosolubles capables de former de manière intramoléculaire une tétrade de guanines synthétique : les DOTASQ, le PorphySQ et le PNADOTASQ. La première application développée pour ces composés est le ciblage des quadruplexes d’ADN et d’ARN, présents dans des régions clefs du génome (télomères, promoteurs d’oncogènes) et du transcriptome (5’-UTR et TERRA), et dont la stabilisation par un ligand pourrait ouvrir de nouvelles perspectives en terme de thérapie antitumorale ciblée. Les résultats in vitro sont présentés et permettent de démontrer que les TASQ hydrosolubles développés sont des composés offrant une bonne sélectivité pour les quadruplexes mais surtout une excellente sélectivité grâce à un mode d’action bioinspiré basé sur une reconnaissance biomimétique. La seconde application mise au point est l’utilisation des TASQ comme catalyseurs pour des réactions de peroxydation : leur architecture même leur permet de mimer l’activité catalytique de l’ADN (ou DNAzyme) ainsi que celle de protéines (enzyme) comme la horseradish peroxidase. Ce processus est dépendant de la formation intramoléculaire de la tétrade de guanines synthétique et ouvre de nombreuses perspectives en terme d’utilisation en biologie ainsi qu’en nanotechnologie
Natural G-quartets, a cyclic and coplanar array of four guanine residues held together via Hoogsteen H-bond network, have recently received much attention due to their involvement in G-quadruplex-DNA, an alternative higher-order DNA structure strongly suspected to play important roles in key cellular events (chromosomal stability, regulation of gene expression). Besides this, synthetic G-quartets, which artificially mimic native G-quartets, have also been widely studied for their involvement in nanotechnological applications (i.e. nanowires, artificial ion channels, etc.). In contrast, intramolecular synthetic G-quartets, also named template-assembled synthetic G-quartet (TASQ), have been more sparingly investigated, despite a technological potential just as interesting.In this way, we designed and synthesized three series of innovative hydrosoluble TASQ: DOTASQ (for DOTA-Templated Synthetic G-Quartet), PorphySQ (containing a porphyrin template) and the most effective PNADOTASQ where PNA-guanine arms replace native DOTASQ alkyl-guanine arms. We report herein the results of both DNA and RNA interactions (notably their selective recognition of quadruplex-DNA according to a bioinspired process) and peroxidase-like hemin-mediated catalytic activities (either in an autonomous fashion as precatalysts for TASQzyme reactions, or in conjunction with quadruplex-DNA as enhancing agents for DNAzyme processes). These results provide a solid scientific basis for TASQ to be used as multitasking tools for bionanotechnological applications

Les lysosomes jouent un rôle clé dans divers mécanismes de résistance aux traitements anticancéreux, notamment en piégeant les médicaments à l’intérieur de leur structure et en activant des voies de stress adaptatives. Si cibler la transcription s’est révélée être une stratégie prometteuse contre les cellules cancéreuses, les mécanismes sous-jacents de résistance aux inhibiteurs de la transcription demeurent largement méconnus. Mon projet de thèse vise à explorer le rôle des lysosomes dans les mécanismes de résistance induits en réponse à deux inhibiteurs puissants de l’ARN POL I (composés A et B). Nous avons découvert que le composé A était séquestré dans les lysosomes où il induisait la perméabilisation de la membrane lysosomale (LMP). Mes résultats ont également révélé que l'induction de ce stress lysosomal entraîne l'activation à la fois du facteur de transcription TFEB et de l'autophagie, jouant des rôles cytoprotecteurs. De plus, cibler les lysosomes, en utilisant des dérivés de la chloroquine ou une excitation par lumière bleue, induit une augmentation importante de la LMP, suivie de la libération du composé A des lysosomes. D’un point de vue mécanistique, j’ai montré que l’induction de cette LMP massive amplifie à la fois l’inhibition de la transcription et la mort cellulaire induite par le composé A. Des effets similaires ont été observés quand les dérivés de la chloroquine sont combinés à l’autre inhibiteur de POL I (composé B). Enfin, nous avons confirmé l'effet bénéfique de l'association du composé A et du DC661 (un dérivé de la chloroquine) dans un modèle in vivo de xénogreffe de fibrosarcome chez des souris immunocompétentes. En conclusion, nous avons découvert des mécanismes liés aux lysosomes qui contribuent de manière inattendue à la résistance à deux inhibiteurs de la transcription médiée par la POL I (composés A et B). Cette étude suggère l'utilisation de combinaisons synergiques d'inhibiteurs de la transcription de POL I avec des agents ciblant les lysosomes tels que les dérivés de chloroquine ou avec la photothérapie pour lutter contre la résistance à ces traitements
Lysosomes have been known to contribute to the development of drug resistance through a variety of mechanisms that include the sequestration of drugs within their compartments and the activation of adaptive stress pathways. Although targeting RNA Polymerase I (POL I) has shown anticancer effects, the contribution of lysosomes to the efficacy and resistance of RNA POL I inhibitors remains largely unknown. In this study, we investigated this aspect in the context of two potent POL I transcription inhibitors (compounds A and B). We found that they were unexpectedly sequestered in lysosomes, causing lysosomal membrane permeabilization (LMP). This effect activated the transcription factor TFEB resulting in cytoprotective autophagy. Targeting lysosomes using chloroquine derivatives or blue light excitation induced substantial LMP, resulting in the liberation of the compound A from lysosomes. This effect amplified both the inhibition of DNA-to-RNA transcription and cell death induced by both POL I inhibitors. Moreover, combining compound A with the chloroquine derivative DC661 reduced the growth of fibrosarcoma established in immunocompetent mice more efficiently than did monotherapies with each agent. Altogether, our results reveal an unanticipated lysosome- related mechanism that contributes to resistance to POL I transcription inhibitors, as well as a strategy to combat this resistance

La maturation en 3' des pré-ARNm constitue une étape de régulation posttranscriptionnelle indispensable à l'expression d'un gène. Elle est composée de deux réactions : le clivage de l'extrémité 3' qui permet de libérer l'ARNm du site de transcription et l'ajout de la queue poly(A) qui contrôle à la fois l'export nucléaire, la stabilité et la traduction du transcrit mature. Plusieurs études montrent, d'une part, que la maturation en 3' est inhibée de manière globale lors de dommages à l'ADN et d'autre part, que des dérégulations de cette maturation peuvent contribuer au développement tumoral. Nos travaux se sont axés sur l'étude de la régulation de la maturation en 3' (i) du pré-ARNm de p53 lors de dommages à l'ADN (ii) du pré-ARNm de MSH6 impliqué dans le syndrome de Lynch. (i) Nos travaux ont mis en évidence que le pré-ARNm de p53 résiste à l'inhibition de la maturation en 3' due aux dommages à l'ADN, grâce au recrutement des protéines hnRNP H/F sur une structure appelée G-quadruplexe située en aval du site de clivage. (ii) Nous avons ensuite identifié une duplication des 20 nucléotides précédant le signal de polyadénylation du pré-ARNm de MSH6 dans deux familles distinctes atteintes du syndrome de Lynch. Cette duplication entraîne une diminution de l'efficacité de la maturation en 3' du pré-ARNm de MSH6 qui semble être à l'origine de la pathologie
Pre-mRNA3'-end processing is an essential post-transcriptional step. It is composed of two reactions: cleavage at the pre-mRNA 3' end that allows release of mRNA from the transcription site and addition of the poly(A) tail that controls nuclear export, stability and the translation of the mature transcript. Several studies show that 3'end processing is globally inhibited during DNA damage and that deregulation of this process may contribute to tumor development. Our work focused on 3'-end processing regulation of (i) p53 pre-mRNA during DNA damage (ii) MSH6 pre-mRNA involved in Lynch syndrome. (i) We have shown that p53 pre-mRNA resists to DNA damagedependent inhibition of 3'-end processing through the recruitment of the protein factor hnRNP H/F on a RNA structure called G-quadruplex located downstream of the cleavage site. (ii) We then identified a duplication of 20 nucleotides upstream of the polyadenylation signal at the MSH6 pre-mRNA in two distinct families with Lynch syndrome. This duplication causes a reduction of MSH6 pre-mRNA 3'-end processing efficiency and in consequence, it may be causal of Lynch syndrome

Des séquences compatibles avec la formation de G4 sont présentes au niveau de certaines régions clés du génome telles que les extrémités des chromosomes, mais également les régions de commutation de classe des immunoglobulines, les promoteurs de certains gènes dont des oncogènes et des séquences transcrites. Plus de 370 000 cibles potentielles ont été prédites lors des analyses bioinformatiques du génome humain. Cependant, ces prédictions ne sont pas exhaustives étant limitées par la formulation des algorithmes de prédiction utilisés. En effet, les séquences recherchées suivent la formule consensus suivante G3+N(1−7)G3+N(1−7)G3+N(1−7)G3+. Ainsi, en apportant plus de souplesse dans la description du quadruplex nous pourrons identifier et localiser plus de cibles potentielles. C’est pourquoi, nous proposons un nouvel algorithme G4-Hunter qui permettra l’identification la plus exhaustive possible de séquences cibles en prenant en compte la totalité de la région et non plus uniquement la cible potentielle. Par ailleurs, une étude expérimentale à grande échelle (sur une centaine de séquences cibles) a été menée afin de valider et tester la robustesse de G4-Hunter. A l’aide de ce nouvel outil, nous avons pu identifier de nouvelles séquences cibles non identifiées par les approches déjà existantes au sein des génomes humain, HIV et Dictyostelium discoideum
Biologically relevant G4 DNA structures are formed throughout the genome including immunoglobulin switch regions, promoter sequences and telomeric repeats. They can arise when single-stranded G-rich DNA or RNA sequences are exposed during replication, transcription or recombination. Computational analysis using predictive algorithms suggests that the human genome contains approximately 370 000 potential G4-forming sequences. These predictions are generally limited to the standard G3+N(1−7)G3+N(1−7)G3+N(1−7)G3+ description. However, many stable G4s defy this description and escape this consensus; this is the reason why broadening this description should allow the prediction of more G4 loci. We propose an objective score function, G4- hunter, which predicts G4 folding propensity from a linear nucleic acid sequence. The new method focus on guanines clusters and GC asymmetry, taking into account the whole genomic region rather than individual quadruplexes sequences. In parallel with this computational technique, a large scale in vitro experimental work has also been developed to validate the performance of our algorithm in silico on one hundred of different sequences. G4- hunter exhibits unprecedented accuracy and sensitivity and leads us to reevaluate significantly the number of G4-prone sequences in the human genome. G4-hunter also allowed us to predict potential G4 sequences in HIV and Dictyostelium discoideum, which could not be identified by previous computational methods

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