Human telomeric DNA consists of tandem repeats of the sequence 5'-d(TTAGGG)-3' assembled into a nucleoprotein complex that functions to protect the ends of chromosomes. Such guanine-rich DNA is capable of forming a variety of G-quadruplexes, which in turn, can have varying functional consequences on telomere maintenance. G-quadruplex stabilizing ligands have been shown to induce chromosome end-to-end fusions, senescence and apoptosis, effects similar to the expression of a dominant-negative TTAGGG Repeat Factor 2 (TRF2). With this in mind, we analyzed the effect of sequence and length of human telomeric DNA, as well as cation conditions on G-quadruplex formation by native polyacrylamide gel electrophoresis and circular dichroism. We show that K+ and Sr2+ can induce human telomeric DNA to form both inter- and intramolecular structures. Circular dichroism results suggest that the structures in K+ were a mix of parallel and antiparallel G-quadruplexes, while Sr2+ induced only parallel-stranded structures. We also found that TRF2, a protein essential for telomere maintenance, affects G-quadruplex structure. These structures serve as useful models to study the effects of G-quadruplexes on the activities of telomeric proteins, like TRF2, from human cells. The G-strand overhang at the ends of telomeres may periodically adopt at least some of these quadruplex conformations, which could subsequently affect protein binding and telomere function. TRF2, a protein essential for telomere maintenance, is not known to bind single-strand (ss) DNA, work performed in the lab suggested that the type of 3'-overhang in telomeric DNA ss/ds-junctions affects TRF2-binding. Specifically, preventing G-quadruplex formation by changing the overhang sequence from 5'-d(TTAGGG)4-3', to 5'-dTTAGGG(TTAGAG)2TTAGGG-3', reduced TRF2 recruitment to the ss/ds-junction from HeLa cell extracts. Using the same techniques as above, we show that the N-terminal basic domain of TRF2 in K+ induces a switch from the mixed parallel/antiparallel-stranded G-quadruplexes usually stabilized by K+-alone, to parallel-stranded G-quadruplexes. Interestingly, it also promotes intermolecular parallel G-quadruplex formation on non-quadruplex, single-stranded intermediates, but will not induce a switch from an antiparallel to a parallel G-quadruplex in Na+. These results are the first to demonstrate specific TRF2 G-quadruplex interactions, suggesting a novel mechanism for TRF2 recognition of the ds/ss junction of telomeres.

archives@tulane.edu / 1 / Pravin Pathak

No description available.

Des structures secondaires d’acides nucléiques atypiques, les structures G-quadruplex, peuvent se former autour d’un cation (K+ ou Na+) dans les régions riches en guanines, grâce à une association de type Hoogsteen. La formation de ces structures est impliquée dans de nombreux mécanismes biologiques, comme la réplication, la transcription ou l’épissage. Elles peuvent affecter l’architecture de l’ADN jusqu’au niveau de la chromatine et provoquer une instabilité importante, tant génétique qu’épigénétique. De nombreuses méthodes ont été développées afin de détecter ces structures in vivo et de comprendre leurs implications au niveau cellulaire. Cependant, le panel d’outils moléculaires disponible actuellement ne permet pas une exploration du génome complète et sélective. Nous avons souhaité développer des outils, capables de sonder efficacement un milieu biologique complexe à la recherche de structures G-quadruplex et d’évaluer le potentiel d’une stratégie thérapeutique anti-tumorale ciblant ces structures. Nous avons mis au point un panel de composés combinant des ligands d’ADN G-quadruplex (PDC, PhenDC3 et Métal-ttpy) avec une biotine et un groupement photoactivable, permettant la capture et l’extraction de structures G-quadruplex de milieux biologiques complexes. Les ligands ont été évalués grâce aux techniques de FID et de FRET-melting, et sélectionnés pour leur affinité mais aussi pour leur affinité pour l’ADN G-quadruplex, assurant un ciblage efficace. Il a également été possible de piéger directement une séquence G-quadruplex en utilisant un complexe de platine, formant un adduit métallique avec les bases de l’ADN. Grâce ce type de ligand d’ADN G-quadruplex, la liaison de coordination métallique joue le rôle de marqueur covalent. Nous avons déterminé sur gel d’électrophorèse la localisation des adduits formés par des complexes dérivés du tolylterpyridine-platine (Pt-ttpy) et étudié la cinétique de platination de l’ADN G-quadruplex. La fonctionnalisation du complexe Pt-ttpy par des groupements photoactivables a permis de réaliser un double-ancrage covalent dans une structure d’ADN G-quadruplex. Par ailleurs, la fonctionnalisation avec un fluorophore a conduit aux premières évaluations en milieu cellulaire.Enfin, notre panel de composés a été testé dans des conditions de capture supportée d’ADN G-quadruplex. Une mise au point de la technique de capture a été réalisée en utilisant des billes magnétiques recouvertes de streptavidine. Les expériences de capture sur billes ont montré que l’efficacité de nos outils varie en fonction de la topologie de la structure G-quadruplex ciblée et du ligand utilisé. Par ailleurs, le groupement photoactivable introduit sur certains de ces outils n’a pas permis d’améliorer la capture d’ADN G-quadruplex. Cependant, il a été possible d’utiliser ces outils en présence d’ADN génomique pour capturer efficacement de fragments d’ADN télomérique, par effet G-quadruplex. / Nucleic acids secondary structures may form in guanine-rich regions by Hoogsteen base-pairing around a cation (K+ or Na+) and stacking of guanine quartets. Those nucleic acid secondary structures called G-quadruplex are believed to play regulatory roles in the main functions related to DNA processing. However, although numerous sequences, potentially forming G4-structures are present in genomes, evidence concerning their in vivo formation and biological role remains limited. Primary aim of our research is to provide new chemical biology tools for evaluating the biological impacts of quadruplexes and the potential of our compounds for quadruplex-targeted anticancer therapy. We have synthetized a set of compounds equipped with biotin and cross linking moieties in order to trap and pull-down G4-structures in various cellular contexts. The G4-ligands (PDC, PhenDC3 and Metal-ttpy) were evaluated thanks to FID and FRET-melting assays, and carefully chosen to efficiently target G-quadruplexes but also to display enough selectivity for cellular assays. Direct trapping of a G-quadruplex structures can also be done by metal complexes, thanks to coordination with DNA bases. Platinum tolylterpyridine derivatives have been studied on gel electrophoresis to map the platination sites and to evaluate the kinetics of the phenomenon. By adding photo crosslinking moieties to Pt-ttpy, efficient double-anchoring has been done on DNA G-quadruplex structure. Moreover, first cellular imaging evaluations were done by adding a fluorophore to this platinum tolylterpyridine complex. To eventually probe quadruplex DNA at the genome-wide scale, full control of the trapping protocol is indeed a key step. Full development of the pull-down step has been done, using streptavidin-coated magnetic beads. On-beads experiments indicate that efficacy of trapping can vary dramatically depending on quadruplex and G4-ligand topologies. Moreover, photo crosslinking moiety, introduced on some compounds, has not shown any improvement of the trapping. However, the development of this method and the design of the capture compounds have led to an optimal isolation of telomeric G-quadruplex forming sequences, from genomic DNA.

Les G-quadruplexes (G4) sont des structures d'acides nucléiques non-canoniques formées par des séquences riches en Guanines (G) principalement localisées dans les telomères et les régions promotrices des oncogènes. Elles sont constituées de l'empilement de plusieurs tétrades de G en présence de cations. En utilisant la spectroscopie par RMN, nous avons caractérisé l'interaction entre le ligand TAP et le G4 télomérique humain constituée de la séquence d(AG3(T2AG3)3). CD et RMN 1D 1H ont été utilisés pour suivre l'interaction entre les deux partenaires. RMN 2D a été utilisé pour attribuer sans ambiguïté toutes les résonances de 1H dans le complexe et d'explorer le site d'interaction. Un modèle illustrant l'interaction de TAP avec 22AG au niveau des sillons et boucles a été généré. Une autre partie de ce travail consiste en l'étude du G4 tétra-moléculaire formé par TG4T et son interaction avec des ligands G4 par la RMN dans les cellules. Des spectres 1H-15N HMQC ont été effectués à l'intérieur de Xenopus laevis et les lysats des cellules HeLa et comparés avec ceux observés dans les conditions in vitro ce qui a montré une bonne stabilité de G4 à l'intérieur de la cellule. En outre, l'interaction de d [TG4T]4 avec des ligands spécifiques de G4 présentant trois différents modes d'interaction a également été étudiée. Le ligand 360A a montré un comportement prometteur. Enfin, dans la dernière partie, différentes séquences de promoteur Kras ont été criblés par RMN pour sélectionner des candidats pour la détermination de structure haute résolution. Deux séquences différentes ont été sélectionnées et caractérisées par spectroscopie CD. La stabilisation des structures G4 formées par ces séquences en interaction avec différents ligands a également été étudiée. Une titration RMN 1D 1H entre 22RT et le ligand Braco19 a montré un comportement intéressant de k-ras G4 par la formation d'espèces intermédiaires lors de l'addition de Braco19. / G-quadruplexes (G4) are non-canonical nucleic acid structures formed by G-rich sequences mainly localized in telomeres and promoter regions of oncogenes. They are built from the stacking of several G-quartets in the presence of cations. Using NMR spectroscopy, we have characterized the interaction between the TAP ligand and the human telomeric G4 formed by the sequence d(AG3(T2AG3)3). CD and 1D 1H NMR spectroscopy were used to follow the interaction between the two partners. 2D NMR was used to assign unambiguously all 1H resonances in the complex and to explore the binding site. A model depicting the interaction of TAP with 22AG in grooves and loops was generated. Another part of this work consists in the study of tetramolecular G4 formed by TG4T and its interaction with G4 ligands by in-cell NMR. 1H-15N HMQC spectra were performed inside Xenopus laevis and HeLa cell lysates compared to those observed in vitro conditions showing a good stability of G4 inside the cell. Furthermore, the interaction of d[TG4T]4 with three G4 specific ligands presenting different mode of interaction was also investigated. The ligand 360A showed a promising behavior. Finally, in the last part, different sequences of Kras promoter were screened by NMR to select good candidates for high resolution structure determination. Two different sequences were selected and characterized by CD spectroscopy. The stabilization of G4 structures formed by these sequences in interaction with different ligands was also investigated. A 1D 1H NMR titration between Braco19 and 22RT showed an interesting behavior of k-ras G4 by the formation of intermediate species upon the addition of Braco19.

Indiana University-Purdue University Indianapolis (IUPUI) / The G-quadruplexes (G4s) are guanine-rich four-stranded DNA/RNA structures, which have been found throughout the human genome. G4s have been reported to affect chromatin structure and are involved in important biological processes at transcriptional and epigenetic levels. However, the underlying molecular mechanisms and locating of G4 still remain elusive due to the complexity of G4s. Taking advantage of the development of high-throughput sequencing technologies and machine learning approaches, we constructed this comprehensive investigation on G4 structures, including discovery of a novel marker for functional human hematopoietic stem cells and gained interest in G4 structure, exploring association between G4 and genomic factors by incorporating multi-omics data, and development of a deep-learningbased G4 prediction tool with G4 motif. First, we discovered ADGRG1 as a novel marker for functional human hematopoietic stem cells and its regulation through transcription activities. Our interest in G4s was stimulated while the transcription-related investigations. Next, we analyzed the genome-wide distribution properties of G4s and uncovered the associations of G4 with other epigenetic and transcriptional mechanisms to coordinate gene transcription. We explored that different-confidence G4 groups correlated differently with epigenetic regulatory elements and revealed that G4 structures could correlate with gene expression in two opposite ways depending on their locations and forming strands. Some transcription factors were identified to be over-represented with G4 emergence. We found distinct consensus sequences enriched in the G4 feet, with a high GC content in the feet of high-confidence G4s and a high TA content in solely predicted G4 feet. As for the last part, we developed a novel deep-learning-based prediction tool for DNA G4s with G4 motifs. Considering the classical G4 motif, we applied bi-directional LSTM model with attention method, which captures sequential information, and showed good performance in whole-genome level prediction of DNA G4s with the certified G4 pattern. Our comprehensive work investigated G4 with its functions and predictions and provided a better understanding of G4s on multi-omics level and computational information capture riding the wave of deep learning. / 2023-04-03

Fragile X syndrome (FXS), the most common cause of inherited mental impairment, is caused by the loss of expression of the fragile X mental retardation protein (FMRP). As an RNA binding protein, FMRP has been proposed to regulate the transport and translation of specific message RNA (mRNA). It has been reported that FMRP uses its RGG box domain to bind mRNA targets that form a G-quadruplex structure, structure believed to be important for FMRP recognition of at least a subclass of its mRNA targets. We have hypothesized that the interaction of FMRP with selected relevant mRNA targets occurs in a G-quadruplex dependent manner. By analyzing the structure of two FMRP in vivo mRNA targets, Shank1 mRNA and BASP1 mRNA, and their interactions with FMRP, we showed a high-affinity interaction between Shank1 RNA G-quadruplex and FMRP. The other G-quadruplex forming mRNA BASP1, however, interacts with FMRP using other structural elements. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences; / Pharmaceutics / MS; / Thesis;

Aberrant expression of Platelet-derived growth factor A (PDGF-A) and PDGF receptor-β (PDGFR-β) play critical roles in the angiogenesis and proliferation of several malignancies. In this dissertation I explore the transcriptional regulatory role of the Gquadruplex- forming regions in the promoters of human PDGF-A and PDGFR-β, and identify new targets for developing small molecules to modulate their expression in tumors. For PDGF-A promoter, our studies focus on two essential nuclease hypersensitive elements, NHE(PDGF-A) and 5´-end far upstream 5´-SHS. The structural aspects of the intramolecular G-quadruplexes formed in NHE(PDGF-A) and the ligands to stabilize these secondary DNA structures have been investigated by using singlestranded and duplex DNA of the NHE(PDGF-A). We demonstrate that the G-quadruplexinteractive compound, TMPyP4, can selectively inhibit the basal promoter activity of PDGF-A, suggesting that the NHE(PDGF-A) G-quadruplex acts as a repressor in PDGF-A transcription. We also found that the 5´-SHS G-rich strand oligomer can invade the NHE(PDGF-A) and form a unique three-stranded complex in supercoiled plasmids, which is facilitated by potassium ions and TMPyP4. Therefore, we propose a novel molecular mechanism for transcriptional silencing of the NHE(PDGF-A) by 5´-SHS in the PDGF-A promoter, in that the formation of G-quadruplex in the NHE(PDGF-A) provides a platform for the G-rich strand of 5´-SHS to invade and form a partial duplex DNA with the C-rich strand of the NHE(PDGF-A), resulting in displacement of hnRNP K and thus transcription silencing. Prior to the studies describe here, the promoter of human PDGFR-β had not been identified. Herein, we have cloned and characterized the first functional promoter of human PDGFR-β gene. A crucial highly GC-rich region (NHE(PDGFR-β)) in the human PDGFR-β promoter has been identified by its hypersensitivity to the S1 nuclease. Further studies demonstrate that stable G-quadruplex structures can form in the G-rich strand of NHE(PDGFR-β). The G-quadruplex-interactive molecule, telomestatin, can selectively stabilize G-quadruplexes formed in the human PDGFR-β promoter and inhibit its expression in Daoy cells. On the basis of these results, we propose that ligandmediated stabilization of the G-quadruplex structure in the proximal promoter region of human PDGF-A or PDGFR-β can be used to modulate the expression of these protooncogenes.

Diarrheagenic Escherichia coli (E. coli) and Neisseria sp. are Gram-negative pathogens that cause high disease burden, especially in low-income countries.Enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) are a subset of E. coli that can cause disease. The sequence of E. coli genomes revealed the presence of at least 16 putative pili operons, it is still unknown if they encode functional pili. Several adhesins have been described in EPEC; however it is still an enigma if EHEC produces pili. In this dissertation the identification and characterization of a new pilus in EHEC is described. The main pilin subunit is encoded in the yagZ gene (renamed ecpA) and is present in all E. coli. We demonstrate ECP production in 137 (70%) of a total of 197 ecpA+ strains representing different categories of E. coli. Isogenic ecpA mutants of EHEC O157:H7 and fecal commensal E. coli showed significant reduction in adherence to cultured epithelial cells. Adherence levels were not hampered after single mutation of ecpA in EPEC. Only after the removal of the known EPEC adhesins such as BFP and intimin we were able to see significant reduction in adherence levels. In sum, ECP is the first pilus of EHEC O157:H7 with a potential role in host epithelial cell colonization. However, EPEC-ECP plays a secondary role in adherence.Since 2007 the CDC recommends only third generation cephalosporins as the elected treatment for Neisseria gonorrhoeae infections. There is an urgent need to search for new drug targets and to development new drugs. Regions rich in guanine in the DNA are able to form secondary structures known as G-quadruplexes. It has been shown that G-quadruplexes are involved in control of transcription, translation and telomere elongation in mammalian cells. G-quadruplex interactive compounds are being developed for cancer therapy. G-quadruplex motifs are also present in bacteria. The fact that G-quadruplex interactive compounds can impair cancer development leads us to hypothesize that these drugs can be used as antimicrobials. This work presents evidence for the potential of G-quadruplex interactive compounds as broad-spectrum antimicrobials.

Genomic complexity in non-Hodgkin’s Diffuse Large B-cell Lymphoma (DLBCL) leads to a treatment failure in ~40% of patients. Activation-Induced Cytosine Deaminase (AID), one of the enzymes involved in generating antibody diversity via class switching recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin (Ig) genes in activated B-cells is one mechanism for the introduction of genomic lesions. In previous studies, AID was shown to preferentially bind to super-enhancer (SE) regions within the genome, but 26% of AID targets were not within the SE regions. The mechanism by which AID interacts with SE elements and its off-target interactions still remains a mystery. Recent evidence suggests that AID may cause genomic lesions in DLBCL via interaction with oncogenes such as MYC and BCL2 resulting in mutations and translocations. Sequences within the MYC promoter contain the four-nucleotide AID target sequence (WRCY) and highly G-rich sequences known to form G-quadruplex DNA secondary structures. We hypothesize that key DNA secondary structures act as recruiting elements for aberrant AID activity at promoters and SEs of key genes involved in the development of DLBCL. Here, we first sought to determine whether known AID DNA targets have the potential to form G-quadruplex DNA secondary structures. The data collected from activated mouse B-cells showed 90% of the AID targets contained sequences that could potentially form G-quadruplexes and the data collected from the human Ramos cell line showed 100% of the sequences had the potential to form G-quadruplexes. To further study our hypothesis we used the techniques circular dichroism (CD) and the electrophoresis motility shift assay (EMSA) to explore the potential interaction between AID and the BCL2 and MYC G-quadruplexes. We observed no significant interactions between AID and these two G-quadruplexes, however further experimentation with different conditions and molecular techniques may show interaction. Additional studies will not only provide key insight into the genomic instability within DLBCL, but will also provide a potential mechanism by which AID is recruited to its DNA targets.