Academic literature on the topic 'DNA, G-quadruplex, structure'

AbstractAnalogous to the B- and Z-DNA structures in double-helix DNA, there exist both right- and left-handed quadruple-helix (G-quadruplex) DNA. Numerous conformations of right-handed and a few left-handed G-quadruplexes were previously observed, yet they were always identified separately. Here, we present the NMR solution and X-ray crystal structures of a right- and left-handed hybrid G-quadruplex. The structure reveals a stacking interaction between two G-quadruplex blocks with different helical orientations and displays features of both right- and left-handed G-quadruplexes. An analysis of loop mutations suggests that single-nucleotide loops are preferred or even required for the left-handed G-quadruplex formation. The discovery of a right- and left-handed hybrid G-quadruplex further expands the polymorphism of G-quadruplexes and is potentially useful in designing a left-to-right junction in G-quadruplex engineering.

G-rich telomeric DNA plays a major role in the stabilization of chromosomes and can fold into a plethora of different G-quadruplex structures in the presence of mono- and divalent cations. The reversed human telomeric DNA sequence (5′-(GGG ATT)4; RevHumTel) was previously shown to have interesting properties that can be exploited for chemical sensing and as a chemical switch in DNA nanotechnology. Here, we analyze the specific G-quadruplex structures formed by RevHumTel in the presence of K+, Na+, Mg2+ and Ca2+ cations using circular dichroism spectroscopy (CDS) and Förster resonance energy transfer (FRET) based on fluorescence lifetimes. CDS is able to reveal strand and loop orientations, whereas FRET gives information about the distances between the 5′-end and the 3′-end, and also, the number of G-quadruplex species formed. Based on this combined information we derived specific G-quadruplex structures formed from RevHumTel, i.e., a chair-type and a hybrid-type G-quadruplex structure formed in presence of K+, whereas Na+ induces the formation of up to three different G-quadruplexes (a basket-type, a propeller-type and a hybrid-type structure). In the presence of Mg2+ and Ca2+ two different parallel G-quadruplexes are formed (one of which is a propeller-type structure). This study will support the fundamental understanding of the G-quadruplex formation in different environments and a rational design of G-quadruplex-based applications in sensing and nanotechnology.

Four-stranded nucleic acid structures called G-quadruplexes have been associated with important cellular processes, which should require G-quadruplex–protein interaction. However, the structural basis for specific G-quadruplex recognition by proteins has not been understood. The DEAH (Asp-Glu-Ala-His) box RNA helicase associated with AU-rich element (RHAU) (also named DHX36 or G4R1) specifically binds to and resolves parallel-stranded G-quadruplexes. Here we identified an 18-amino acid G-quadruplex-binding domain of RHAU and determined the structure of this peptide bound to a parallel DNA G-quadruplex. Our structure explains how RHAU specifically recognizes parallel G-quadruplexes. The peptide covers a terminal guanine base tetrad (G-tetrad), and clamps the G-quadruplex using three-anchor-point electrostatic interactions between three positively charged amino acids and negatively charged phosphate groups. This binding mode is strikingly similar to that of most ligands selected for specific G-quadruplex targeting. Binding to an exposed G-tetrad represents a simple and efficient way to specifically target G-quadruplex structures.

Abstract G-quadruplexes are four-stranded nucleic acid structures involved in multiple cellular pathways including DNA replication and telomere maintenance. Such structures are formed by G-rich DNA sequences typified by telomeric DNA repeats. Whilst there is evidence for proteins that bind and regulate G-quadruplex formation, the molecular basis for this remains poorly understood. The budding yeast telomeric protein Rap1, originally identified as a transcriptional regulator functioning by recognizing double-stranded DNA binding sites, was one of the first proteins to be discovered to also bind and promote G-quadruplex formation in vitro. Here, we present the 2.4 Å resolution crystal structure of the Rap1 DNA-binding domain in complex with a G-quadruplex. Our structure not only provides a detailed insight into the structural basis for G-quadruplex recognition by a protein, but also gives a mechanistic understanding of how the same DNA-binding domain adapts to specifically recognize different DNA structures. The key observation is the DNA-recognition helix functions in a bimodal manner: In double-stranded DNA recognition one helix face makes electrostatic interactions with the major groove of DNA, whereas in G-quadruplex recognition a different helix face is used to make primarily hydrophobic interactions with the planar face of a G-tetrad.

Certain G-rich DNA repeats can form quadruplex in bacterial chromatin that can present blocks to DNA replication and, if not properly resolved, may lead to mutations. To understand the participation of quadruplex DNA in genomic instability in Escherichia coli (E. coli), mutation rates were measured for quadruplex-forming DNA repeats, including (G3T)4, (G3T)8, and a RET oncogene sequence, cloned as the template or nontemplate strand. We evidence that these alternative structures strongly influence mutagenesis rates. Precisely, our results suggest that G-quadruplexes form in E. coli cells, especially during transcription when the G-rich strand can be displaced by R-loop formation. Structure formation may then facilitate replication misalignment, presumably associated with replication fork blockage, promoting genomic instability. Furthermore, our results also evidence that the nucleoid-associated protein Hfq is involved in the genetic instability associated with these sequences. Hfq binds and stabilizes G-quadruplex structure in vitro and likely in cells. Collectively, our results thus implicate quadruplexes structures and Hfq nucleoid protein in the potential for genetic change that may drive evolution or alterations of bacterial gene expression.

Methods for stabilizing G-quadruplex formation is a promising therapeutic approach for cancer treatment and other biomedical applications because stable G-quadruplexes efficiently inhibit biological reactions. Oligo and polyethylene glycols are promising biocompatible compounds, and we have shown that linear oligoethylene glycols can stabilize G-quadruplexes. Here, we developed a new modified deoxythymine with dibranched or tribranched tetraethylene glycol (TEG) and incorporated these TEG-modified deoxythymines into a loop region that forms an antiparallel G-quadruplex. We analyzed the stability of the modified G-quadruplexes, and the results showed that the tribranched TEG destabilized G-quadruplexes through entropic contributions, likely through steric hindrance. Interestingly, the dibranched TEG modification increased G-quadruplex stability relative to the unmodified DNA structures due to favorable enthalpic contributions. Molecular dynamics calculations suggested that dibranched TEG interacts with the G-quadruplex through hydrogen bonding and CH-π interactions. Moreover, these branched TEG-modified deoxythymine protected the DNA oligonucleotides from degradation by various nucleases in human serum. By taking advantage of the unique interactions between DNA and branched TEG, advanced DNA materials can be developed that affect the regulation of DNA structure.

A cursory look at any textbook image of DNA replication might suggest that the complex machine that is the replisome runs smoothly along the chromosomal DNA. However, many DNA sequences can adopt non-B form secondary structures and these have the potential to impede progression of the replisome. A picture is emerging in which the maintenance of processive DNA replication requires the action of a significant number of additional proteins beyond the core replisome to resolve secondary structures in the DNA template. By ensuring that DNA synthesis remains closely coupled to DNA unwinding by the replicative helicase, these factors prevent impediments to the replisome from causing genetic and epigenetic instability. This review considers the circumstances in which DNA forms secondary structures, the potential responses of the eukaryotic replisome to these impediments in the light of recent advances in our understanding of its structure and operation and the mechanisms cells deploy to remove secondary structure from the DNA. To illustrate the principles involved, we focus on one of the best understood DNA secondary structures, G quadruplexes (G4s), and on the helicases that promote their resolution.

G-quadruplex is a special secondary structure of nucleic acids in guanine-rich sequences of genome. G-quadruplexes have been proved to be involved in the regulation of replication, DNA damage repair, and transcription and translation of oncogenes or other cancer-related genes. Therefore, targeting G-quadruplexes has become a novel promising anti-tumor strategy. Different kinds of small molecules targeting the G-quadruplexes have been designed, synthesized, and identified as potential anti-tumor agents, including molecules directly bind to the G-quadruplex and molecules interfering with the binding between the G-quadruplex structures and related binding proteins. This review will explore the feasibility of G-quadruplex ligands acting as anti-tumor drugs, from basis to application. Meanwhile, since helicase is the most well-defined G-quadruplex-related protein, the most extensive research on the relationship between helicase and G-quadruplexes, and its meaning in drug design, is emphasized.

G-quadruplex (G4) DNA secondary structures formed in human telomeres have been shown to inhibit cancer-specific telomerase and alternative lengthening of telomere (ALT) pathways. Thus, human telomeric G-quadruplexes are considered attractive targets for anticancer drugs. Human telomeric G-quadruplexes are structurally polymorphic and predominantly form two hybrid-type G-quadruplexes, namely hybrid-1 and hybrid-2, under physiologically relevant solution conditions. To date, only a handful solution structures are available for drug complexes of human telomeric G-quadruplexes. In this review, we will describe two recent solution structural studies from our labs. We use NMR spectroscopy to elucidate the solution structure of a 1:1 complex between a small molecule epiberberine and the hybrid-2 telomeric G-quadruplex, and the structures of 1:1 and 4:2 complexes between a small molecule Pt-tripod and the hybrid-1 telomeric G-quadruplex. Structural information of small molecule complexes can provide important information for understanding small molecule recognition of human telomeric G-quadruplexes and for structure-based rational drug design targeting human telomeric G-quadruplexes.

Abstract O 6-alkylguanine-DNA alkyltransferase (AGT) is a single-cycle DNA repair enzyme that removes pro-mutagenic O6-alkylguanine adducts from DNA. Its functions with short single-stranded and duplex substrates have been characterized, but its ability to act on other DNA structures remains poorly understood. Here, we examine the functions of this enzyme on O6-methylguanine (6mG) adducts in the four-stranded structure of the human telomeric G-quadruplex. On a folded 22-nt G-quadruplex substrate, binding saturated at 2 AGT:DNA, significantly less than the ∼5 AGT:DNA found with linear single-stranded DNAs of similar length, and less than the value found with the telomere sequence under conditions that inhibit quadruplex formation (4 AGT:DNA). Despite these differences, AGT repaired 6mG adducts located within folded G-quadruplexes, at rates that were comparable to those found for a duplex DNA substrate under analogous conditions. Repair was kinetically biphasic with the amplitudes of rapid and slow phases dependent on the position of the adduct within the G-quadruplex: in general, adducts located in the top or bottom tetrads of a quadruplex stack exhibited more rapid-phase repair than did adducts located in the inner tetrad. This distinction may reflect differences in the conformational dynamics of 6mG residues in G-quadruplex DNAs.

In this dissertation, the formation of unusual G-quadruplexes in the critical regions of the c-myb and hTERT promoters for control of promoter activity was investigated.The c-myb promoter contains three copies of an almost perfect (GGA)4 sequence. We demonstrate that the each (GGA)4 repeat forms a tetrad:heptad G-quadruplex and any two of the three can intramolecularly dimerize to form T:H:H:T G-quadruplexes. The three T:H:H:T G-quadruplex combinations are of differing degrees of stability and can be further stabilized by G-quadruplex interactive compounds. We also demonstrate that the c-myb G-quadruplex forming region is a critical transcriptional regulatory element and interacts with various nuclear proteins including MAZ (Myc Associated Zinc finger protein). The data from luciferase reporter assay show that the c-myb GGA repeat region plays dual roles as a transcriptional activator and an inhibitor by serving as binding sites for the activators and by forming G-quadruplex structures in the region, respectively. Furthermore, we show that MAZ is a transcriptional repressor of the c-myb promoter and binds to both the double-stranded and T:H:H:T G-quadruplex-folded conformations of the GGA repeat region of the c-myb promoter.The hTERT core promoter contains a G-rich region of 12 consecutive G-tracts, which includes three critical Sp1 binding sites. Although this G-rich region has the potential to form multiple G-quadruplexes, our investigation on the full-length G-rich sequence demonstrate that the G-rich region forms a unique G-quadruplex structure in which two tandem intramolecular G-quadruplex structures are present, consisted of one G-quadruplex formed by the G-tracts 1-4 and the other formed by the G-tracts 5, 6, 11, and 12. We also demonstrate that the latter unusual structure contains a 26-base middle loop that likely forms a hairpin structure and is more stable than the other conventional G-quadruplex. Significantly, the formation of this unusual tandem G-quadruplex structure in the full-length will disable all three critical Sp1 binding sites, which will dramatically downregulate hTERT expression. G-quadruplex formation in the hTERT promoter suggests that the effect of G-quadruplex interactive ligands on telomerase inhibition and telomere shortening may be exerted by the direct interaction between the hTERT G-quadruplex structure and the ligands.

Suppression of oncogenes transcription represents an ideal tool to integrate the currently available therapeutics to treat several cancer types and to overcome the potential occurrence of resistance. An experimentally validated mechanism of intervention is represented by the induction/stabilization of G-quadruplex structures in genes promoter by small molecules. G-quadruplexes are DNA non-canonical secondary structures consisting of stacked G-quartets, cyclic arrangements of four guanine residues held together by Hoogsteen hydrogen bonds and stabilized by a central cation. At the moment, none of the identified G-quadruplex ligands reached the clinic. Several reasons can contribute to this poor outcome comprising both the plastic structural features of nucleic acids and the multiple metabolic pathways which might be affected when a small molecule interacts with G-quadruplex structures. Thus, one of the major issues lies on the proper structural analysis of targeted G-quadruplex. To overcome this bias, in this Ph.D.’s thesis kinetic and thermodynamic behaviours of G-quadruplexes have been characterized to obtain an improved description of these structures as potential pharmaceutical targets. The study has been focused on G-rich sequences within c-KIT and EGFR oncogene promoters. By applying a set of complementary structural and biophysical approaches, the folding pathways of these G-quadruplexes and influence of flanking regions in terms of structural stability and folding rearrangement have been described. The obtained information indicates that the promoter architecture might be not properly derived by analysis of minimal G-quadruplex forming sequences at the thermodynamic equilibrium, commonly used for screening assayes. Indeed, reported data suggest the existence of different unique mechanisms/pathways involved in the regulation of these oncogenes transcription which comprise kinetically favored folding intermediates or unprecedented structural arrangements. The final outcome of Ph.D.‘s project is a deeper understanding of nucleic acid tridimensional arrangement of EGFR and c-KIT promoters which might help in setting up new drug-design programs based on models of G-quadruplex target more closely related to the physiological ones.

The present manuscript contains a report of the research activity conducted for the Ph.D. project regarding the study of DNA G-quadruplex structures in a rare type of human cancer (Liposarcoma) and in the genome of two world-wide spread viruses: HIV-1 and HSV-1.
Il manuscritto contiene una descrizione e discussione dei risultati ottenuti in ambito del progetto di ricerca di dottorato riguardante lo studio delle strutture G-quadruplex delDNA in un tumore umani raro (il liposarcoma) e nel genoma di due virus a diffusione mondiale, quali: HIV-1 e HSV-1.

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

The discovery of new anticancer targets is the key factor for the development of more efficacious therapies. Sequence selective binding of double stranded DNA in the classical B form has been extensively employed to target small molecules to defined polynucleotide portions. More recently, ligand recognition of non canonical DNA foldings has been additionally considered a useful approach to selectively target distinct genomic regions. In this connection, G-quadruplexes represent an interesting system since they are believed to be physiologically significant arrangements. These non-canonical DNA structures are found at the ends of the human chromosomes (telomeres) as well as at promoter regions of several oncogenes where there is a cluster of guanine-rich sequences and they are likely to play important roles in the regulation of biological events. The induction and stabilization of the G-quadruplex arrangement by small molecules can lead to the inhibition of the telomerase activity by interfering with the interaction of the enzyme and its single stranded template. A similar molecular mechanism is likely involved in the transcriptional control that leads to the suppression of the oncogene transcription and, ultimately, in the regulation of the gene expression. As a result, the quadruplex topic is very attractive for the development of a specific anticancer strategy defined by a dramatic reduction of side effects, typical of chemotherapy. The purpose of this work is to investigate the interactions between novel classes of small molecules and different quadruplex DNA sequences and conformations. These new molecules were properly designed providing systematic atom-wise substitutions based on rational evaluations of previous studied compounds in order to increase their selectivity for G-quadruplex structures and to reduce toxic effects. Biophysical and biological properties of all new derivatives are herein evaluated at molecular and cellular level. The thesis work is divided into three main sections based on the structural features of the compounds object of study. The first part focuses on heterocyclic dications: upon changing their molecular binding shape, a correlation with G-quadruplex binding have been drawn. In particular it was possible to rationalize a shift in the binding modes, in particular between end stacking and groove recognition. Nevertheless a correlation between biophysical (G-quadruplex affinity) and biological (telomerase inhibition and cytotoxicity) results was not always clear. This feature may suggest the involvement of cellular targets different from the telomere and that are now under investigation. In Chapter 3, the DNA binding properties of some phenantroline derivatives in presence and in absence of Ni(II) and Cu(II) are investigated. We confirmed that different complex geometries involving one, two or three ligands per metal ion can affect the pattern of DNA recognition by driving nucleic acid conformational changes. Finally, in Chapter 4 some transplatin derivatives are evaluated. We focused our attention on defining the compounds capability to form adducts, with the nucleic acids, the nature of adducts and the kinetics of adduct formation not only on double strand DNA but also using single strand as well as G-quadruplex as targets. The results showed how different structural modifications can cooperate to greatly affect the potential interaction of the compounds. Interestingly it turned out their preference to react on single stranded DNA portions than to double stranded ones. This is probably due to an unfavourable orientation of the reactive groups when the molecule interacts with the DNA substrate. As a result, they appear to crosslink unpaired strands. By extending these results at cellular level they can reflect distinct distribution of platination site along the genome in comparison to cisplatin and even transplatin. The results obtained increment the available knowledge of DNA-small molecules interaction. In particular it emerged that a conserved interaction mode is consistent with biological effects. On the other hand, a shift in the binding mode can drive to different cytotoxic effects. This can provide a rationale for subsequent drug structure optimization leading to the development of new efficient and selective anticancer agents.
La scoperta di nuovi target anticancro è il fattore chiave per lo sviluppo di terapie sempre più efficaci. Lo studio del legame selettivo a sequenze di DNA a doppia elica nella classica forma B è stato largamente impiegato al fine di direzionare piccole molecole verso porzioni polinucleotidiche definite. Più recentemente, il riconoscimento (da parte di ligandi) di porzioni non canoniche di DNA si può tradurre in un metodo vantaggioso per indirizzare questi composti verso regioni distinte del genoma. A tale proposito, le strutture G-quadruplex rappresentano un sistema interessante poiché sono ritenute fisiologicamente significative. Queste strutture “non-canoniche” di DNA si trovano alle estremità del cromosoma (telomeri) così come in varie regioni promotrici di oncogeni in cui vi è un’abbondante presenza di residui guaninici e sembrano coinvolte nella regolazione di importanti eventi biologici. Pare infatti che l'induzione e la stabilizzazione di strutture G-quadruplex dalle parte di piccole molecole porti all'inibizione dell'attività della telomerasi interferendo con l'interazione tra l’enzima e il suo substrato a singola catena. Un simile meccanismo molecolare è probabilmente coinvolto anche nel controllo della regolazione dell'espressione genica e può portare alla soppressione della trascrizione di un oncogene. Di conseguenza, “l’approccio G-quadruplex” si rivela molto interessante per lo sviluppo di una strategia anticancro specifica caratterizzata anche da una riduzione drammatica degli effetti collaterali, tipici della chemioterapia. Lo scopo di questo lavoro è lo studio delle interazioni tra nuove famiglie di piccole molecole e diverse conformazioni di DNA G-quadruplex. Queste nuove molecole sono state opportunamente progettate apportando sostituzioni di atomi o gruppi funzionali basate sulla valutazione di composti precedentemente studiati al fine di aumentare la loro selettività per strutture G-quadruplex e di ridurre gli effetti tossici. Le proprietà biofisiche e biologiche di tutti i nuovi derivati sono state valutate al livello molecolare e cellulare. Il lavoro di tesi si divide in tre parti in base alle caratteristiche strutturali dei composti. La prima parte è dedicata alla studio di dicationi eterociclici: si è cercato correlare modifiche nella conformazione molecolare con l’affinita’ verso strutture G-quadruplex. In particolare è stato possibile razionalizzare cambiamenti della modalità di legame in base alla struttura dei composti esaminati. Tuttavia una correlazione fra i risultati biofisici (affinità G-quadruplex) e biologici (inibizione della telomerasi e citotossicità) non è risultata sempre definita. Ciò può suggerire il coinvolgimento di bersagli cellulari diversi dal telomero umano. Nel capitolo 3, sono state studiate le proprietà di legame al DNA di alcuni derivati fenantrolinici in presenza ed in assenza di Ni (II) e Cu (II). Abbiamo confermato che complessi caratterizzati da diverse geometrie che coinvolgono una, due o tre molecole per ione possono compromettere o meno il riconoscimento del DNA o determinare cambiamenti conformazionali dell'acido nucleico. Per concludere, il capitolo 4 è dedicato allo studio di derivati del transplatino. In particolare ci siamo focalizzati nel definire la capacità dei composti di formare addotti, la natura dei complessi e la cinetica di formazione del complesso non solo con DNA a doppio filamento ma utilizzando anche substrati a singola catena come il G-quadruplex. I risultati hanno dimostrato come diverse modifiche strutturali possano avere un ruolo importante nell’interazione dei composti con gli acidi nucleici. E’ risultata interessante la loro preferenzialità a reagire con porzioni di DNA a singolo filamento rispetto a sequenze a doppia elica. Ciò è probabilmente dovuto ad uno sfavorevole orientamento dei gruppi reattivi quando la molecola interagisce con il substrato di DNA. Di conseguenza, i composti sembrano formare un cross-link tra due filamenti non appaiati. A livello cellulare, questi risultati riflettono una distinta distribuzione del sito di platinazione all’interno del genoma rispetto al cisplatino e perfino rispetto al transplatino. I risultati ottenuti incrementano la conoscenza disponibile sull’interazione tra DNA e piccole molecole. In particolare è emerso che la conservazione della modalità di interazione si correla con effetti biologici definiti. Al contrario, una variazione della modalità di legame può portare a effetti citotossici differenti. Ciò può fornire una spiegazione razionale per una successiva ottimizzazione della struttura dei composti finalizzata allo sviluppo di nuovi agenti antitumorali efficaci e selettivi.

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

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