Keywords: G-quadruplex, NMR, Ligands, Cancer, KRAS

List of figures

Abstract

KRAS gene codes for a highly mutated GTPase protein acting as a « switch » between an active and an inactive state, a mechanism found to be important in biological processes such as cell replication and proliferation. When misregulated, these processes are found to be at the origin many types of cancer. KRAS mutations are particularly implicated in lung (30%), colorectal (44%) and pancreatic (97%) cancers. Despite the fact that these mutations are well known, KRAS is still an undruggable target because all the actual strategies (RAS activator inhibitors, membrane association inhibitors, and so on) are not efficient enough as cancer therapies. That is why new strategies have emerged recently, such as directly targeting the KRAS promoter region and especially some specific structures called G-quadruplexes (G4).

Although we do not understand well the phenomena, there are plenty of evidence in the literature that these structures can assemble both in vitro and in cellular conditions. It was shown that G4 within KRAS promoter region can bind transcription related proteins and disturb transcription process acting as a block when transcription machinery is reading the genetic sequence. Stabilization of these structures, using small chemical ligands for example, could become a new area of therapy. In my thesis work, I am focused on a 32 nucleotide sequence (KRAS32R) which can form G4 and also corresponds to the minimal interaction domain of transcription proteins such as MAZ or hnRNP1. This last protein is capable of binding to KRAS32R G-quadruplexes and possibly unfolding it, favoring the transcription of KRAS. This project is divided into two major parts. One part is to understand the folding of this KRAS32R G-quadruplex at atomic level. In another part I want to understand how these DNA structures can interact with small organic molecules that prevent the interaction with transcription factors that have been associated with the G-quadruplex motifs found in the promoter region.

Résumé en français

L'oncogène KRAS code pour une protéine GTPasique hautement mutée qui agit comme un « interrupteur » entre des états actifs et inactifs, un mécanisme important dans les processus comme la réplication ou la prolifération cellulaire. Quand ils sont dérégulés, ces processus sont à l'origine des cancers. Les mutations de KRAS sont particulièrement impliquées dans les cancers des poumons (30%), colorectaux (44%) et pancréatiques (97%). Malgré le fait que ces mutations soient bien connues, aucune molécule ne cible KRAS car toutes les stratégies actuelles ne sont pas assez efficaces pour les thérapies contre le cancer. C'est pourquoi de nouvelles stratégies ont émergé il y a quelques années visant directement la région promotrice de KRAS et plus précisément des structures appelées G-quadruplexes (G4). Même si le phénomène n'est pas encore parfaitement compris, de nombreux exemples dans la littérature montre que ces structures peuvent se former in vitro et dans les conditions cellulaires. Il a été montré que les G4 formés dans la région promotrice de KRAS peuvent lier des facteurs de transcription et perturber le processus en agissant comme un bloc lorsque l'enzyme vient lire la séquence. La stabilisation ou la destruction des G4, en utilisant de petits ligands chimiques par exemple, pourrait devenir une nouvelle voie de thérapie. Ce travail se concentre sur une séquence de 32 résidus (KRAS32R) qui peut former un G4 et correspond également au domaine minimal d'interaction de certains facteurs de transcription comme MAZ ou hnRNP A1. Cette dernière est capable de lier les G4 de KRAS32R et de les défaire favorisant ainsi la transcription de KRAS. Ce projet est divisé en deux parties majeures. L'une est de comprendre la formation du G4 de KRAS32R au niveau atomique. Pour les autres, nous voulons comprendre comment il interagit avec de petites molécules organiques qui pourraient empêcher l'interaction avec les facteurs qui sont associés aux motifs G4 de cette région promotrice.

Résumé long en français

L'oncogène KRAS code pour une protéine GTPasique hautement mutée qui agit comme un « interrupteur » entre des états actifs et inactifs, un mécanisme important dans les processus comme la réplication ou la prolifération cellulaire. Quand ils sont dérégulés, ces processus sont à l'origine des cancers. Les mutations de KRAS sont particulièrement impliquées dans les cancers des poumons (30%), colorectaux (44%) et pancréatiques (97%). Malgré le fait que ces mutations soient bien connues, aucune molécule ne cible KRAS car toutes les stratégies actuelles ne sont pas assez efficaces pour les thérapies contre le cancer. C'est pourquoi de nouvelles stratégies ont émergé il y a quelques années visant directement la région promotrice de KRAS et plus précisément des structures appelées G-quadruplexes (G4). Les Gquadruplexes sont de structures inhabituelles des acides nucléiques. Ils sont formés via un empilement d'au moins deux tétrades qui sont composées de quatre guanines liées dans un réseau de liaisons hydrogène de type Hoogsteen et stabilisées le plus souvent par un cation monovalent. Même si le phénomène n'est pas encore parfaitement compris, de nombreux exemples dans la littérature montre que ces structures peuvent se former in vitro et dans les conditions cellulaires. Il a été montré que les G4 formés dans la région promotrice de KRAS peuvent lier des facteurs de transcription et perturber le processus en agissant comme un bloc lorsque l'enzyme vient lire la séquence. La stabilisation des G4, en utilisant de petits ligands chimiques par exemple, pourrait devenir une nouvelle voie de thérapie. Ce travail se concentre sur une séquence de 32 résidus (KRAS32R) qui peut former un G4 et correspond également au domaine minimal d'interaction de certains facteurs de transcription comme MAZ ou hnRNP A1. Cette dernière est capable de lier les G4 de KRAS32R et de les défaire favorisant ainsi la transcription de KRAS. Cette étude a pour but de déterminer et valider des cibles G-quadruplexe au sein de l'oncogène KRAS. De plus, elle vise à identifier les groupes chimiques caractéristiques de ligands pouvant interagir avec ces G4. Cependant, afin de créer de bons ligands et d'identifier des groupes chimiques clé, il est nécessaire d'avoir des informations structurales à un niveau atomique concernant les différentes conformations de G-quadruplexe. L'identification d'éléments structuraux spécifiques de ces G-quadruplexes permettraient de créer des ligands spécifiques de G4s comme ceux formés au sein de l'oncogène KRAS. Ce projet est donc divisé en deux parties majeures avec dans un premier temps la détermination de la structure des G-quadruplexes de KRAS32R pour obtenir des éléments structuraux et dans un second temps l'étude de l'interaction de ces G4s avec des protéines comme hnRNP A1 ainsi que plusieurs ligands provenant de différentes familles.

Comme expliqué précédemment, obtenir des informations structurales sur la cible est très important dans le concept de Drug Design. En effet, cela permet de créer des molécules qui sont capables de venir cibler spécifiquement les sites de liaison identifiés lors de l'obtention de la structure. Ensuite lorsque les ligands sont testés, la structure permet l'identification des groupements essentiels à l'interaction. Concernant la structure de KRAS32R, plusieurs études ont tenté de la résoudre sans y parvenir. Afin de comprendre comment s'organisent les séquences riches en guanine de la région NHE de la région promotrice de l'oncogène KRAS, nous avons commencé par étudier ce qui a été montré comme étant la plus petite séquence pouvant former des G4s aussi appelée KRAS21R. KRAS21R représente donc le plus petit G4 de la région NHE qui est capable, tout comme KRAS32R, d'interagir avec des facteurs de transcription comme MAZ. En utilisant la séquence KRAS22RT qui possède une adénine supplémentaire en 3' et en incorporant une mutation de la guanine 16 en thymine, nous avons réussi à garder la conformation principale de KRAS21R tout en améliorant la qualité des spectres RMN (notamment 2D) pour la résolution de la structure. Grâce à la détermination de cette structure et à de nombreux spectres RMN de la séquence KRAS32R, nous avons tenté de comprendre comment se formaient les G-quadruplexes de cette séquence. Plus d'une centaine de mutants ont été testés avant de comprendre la formation de ces G4s pour commencer la détermination de structure. A l'issue de toutes ces analyses, il était clair que la séquence KRAS32R n'adoptait pas une seule conformation mais qu'il y avait un mélange de deux conformations majoritaires. Ces deux conformations ont été identifiées en tant que G9T et G25T correspondant aux résidus mutés menant aux conformations correspondantes. D'un côté, G9T forme une structure unique dans laquelle G9 n'est pas impliqué mais le dernier résidu G32 l'est. De plus, elle est stabilisée par la formation d'une triade. De l'autre côté, G25T est très différente avec G9 impliqué dans la formation du G-quadruplexe sans la présence d'une triade. De plus, la structure reste toujours polymorphique dû à un glissement dans le dernier tract de guanines entre G26 et G29. Il est également possible que G9 soit responsable en partie du polymorphisme qui permet l'échange entre ces deux états. Dans cette étude, nous avons finalement compris la formation des G-quadruplexes au sein de la séquence KRAS32R et nous avons déterminé ces structures G-quadruplexe qui sont connues pour être hautement impliquées dans la transcription et pouvant interagir avec plusieurs protéines durant le processus. Ainsi nous avons pu apporter des informations cruciales concernant les structure de G-quadruplexes les plus probables au sein de la séquence KRAS32R de la région NHE. Ces structures tridimensionnelles pourraient représenter de nouvelles cibles essentielles pour le développement de nouvelles molécules spécifiques aux G-quadruplexes de la région promotrices de KRAS. En effet, elles pourraient interférer avec la régulation de la transcription en stabilisant ces G4s ou bien en entrant en compétitions avec des facteurs de transcription comme MAZ ou hnRNP A1. Grâce aux nouvelles informations concernant KRAS22RT et JRAS32R, nous avons ensuite testé un panel de ligands. En sélectionnant quelques candidats parmi les meilleurs testés et en étudiant leur interaction avec les G4s à un niveau atomique, nous pourrions comprendre comment les différents groupements chimiques participent à la stabilisation globale de la structure G4. En obtenant ce genre d'information nous voulons comprendre comment les ligands pourraient interagir de façon plus efficace avec les boucles, les sillons ou encore les tétrades. Comme les ligands pour G-quadruplexes sont souvent trop toxiques pour les cellules ou bien à l'inverse pas assez car ils ne peuvent pas passer les différentes barrières, c'est aussi un bon moyen d'identifier les groupements chimiques qui peuvent être remplacés pour surpasser ces limites. Comme ce travail a été réalisé en parallèle de la détermination de la structure de KRAS32R, la majorité des résultats des tests avec ligands a été obtenu avec KRAS22RT. Une fois les structures de KRAS32R G9T et G25T obtenues, nous avons également obtenu quelques résultats préliminaires. Dans le cas de KRAS32R, le but était de trouver des ligands capables de stabiliser les G4s mais également des composés qui seraient capables de déplacer l'équilibre entre les deux conformations G9T et G25T. Un autre objectif était de trouver des molécules capables de prévenir l'interaction avec UP1, une protéine contenant uniquement les deux domaines de liaisons à l'ARN de hnRNP A1. Parmi tous les ligands testés, aucun ne possédait toutes les propriétés que nous recherchions. Plusieurs d'entre eux ont cependant montré de bons résultats en terme de stabilisation, de protection contre la chaine complémentaire ou bien contre la protéine UP1 ou encore en terme de cytotoxicité et de capacité à rentrer dans la cellule. C8, un dérivé acridine, est probablement notre meilleur ligand notamment avec son effet stabilisant et sa capacité à protéger le G4 contre la chaîne complémentaire. Cependant, il n'est pas capable d'empêcher l'interaction de UP1 et n'est pas suffisamment spécifique des cellules cancéreuses en terme de toxicité. D'un autre côté, le ligand AG, un composé salphen, n'est pas notre meilleur ligand mais nous avons décidé de mener des études structurales plus poussées du complexe formé avec KRAS22RT car il était le plus indiqué pour cela. D'autres composés comme le ligand PhenDC3, de la famille des phénantrolines, ont montré qu'il était possible d'éviter l'interaction avec UP1. Cependant, les mécanismes impliqués n'ont pas encore été identifiés. En conclusion, ce travail de thèse a permis d'apporter de nouveaux éléments sur l'organisation des G-quadruplexes de la région NHE de l'oncogène KRAS avec la résolution de trois structures par RMN dont deux structures en équilibre dans le cas de KRAS32R. La résolution de cette

Introduction

I would like to start this chapter with a paramount statement that captured my imagination during the past three years or so: nucleic acids are considered the cornerstone of life. In this chapter I shall introduce the readers to the some of the concepts needed for the understanding of my results. One of the main reasons seems obvious and it is related to the fact that DNA holds the genetic information that each individual needs to survive, grow and reproduce. So it is without surprise that we expect a tight, sophisticated and intricate level of "management" of chromosomic DNA. During my early years in academic formation, especially during my master, I learnt that it was possible thanks to complex mechanisms and unusual structural motifs. For a long time, these unusual structures were largely unstudied and their role in all different biological processes seemed to remain rather unknown, especially at molecular level. In addition, thanks to their involvement in many metabolism processes, they became very attractive to be studied as possible targets for therapeutic purposes. Nowadays nucleic acids and their non-canonical structures represent one of the major fields in fundamental research. Hereafter, I will introduce you to this fascinating field of research that is the centrepiece of my dissertation.

I. Nucleic Acids

I.1. General information -History

Unlike public opinion, DNA double helix was not discovered by Watson et Crick in the 1950s.

Indeed, the DNA discovery is one century older thanks to Johann Friedrich Miescher, a young Swiss chemist [START_REF] Dahm | Friedrich Miescher and the discovery of DNA[END_REF][START_REF] Dahm | Discovering DNA: Friedrich Miescher and the early years of nucleic acid research[END_REF] . As he was trying to isolate and purify proteins from leucocytes in order to characterize them, he discovered a substance obtained from the nuclei being insoluble in acidic conditions. The substance could be dissolved again in alkaline conditions, so without surprise he called it "nuclein" and though that it represented some storage mechanism for cells. Although this discovery represented a major step forward in biological sciences, Miescher could not properly publish his results and it remained hidden from the public for several years. Then the composition of "nuclein" was discovered by Albrecht Kossel [START_REF] Dahm | From discovering to understanding. Friedrich Miescher's attempts to uncover the function of DNA[END_REF][START_REF] Xie | DNA and the brains behind its discovery[END_REF] who was working on the hypothesis that "nuclein" was participating in the formation of new biological tissue. A few years later he was able to identified the five nucleobases: Guanine, Adenine, Cytosine, Thymine only in DNA and Uracil only in RNA. to the one we know with ribose (RNA) or deoxyribose (DNA) linked at 5' -OH group to the phosphate group with a phosphoester bond and at 1'-OH to the bases A, G, C and T (DNA) or U (RNA) with an osidic bond [START_REF] Levene | THE STRUCTURE OF YEAST NUCLEIC ACID: IV. AMMONIA HYDROLYSIS[END_REF] (Figure 1).

In 1944, a huge discovery that shocked the nucleic acids world was published by Oswald Avery and colleagues [START_REF] Avery | Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types : Induction of Transformation by a Desoxyribonucleic Acid Fraction Isolated from Pneumococcus Type Iii[END_REF] in a seminal paper which proved that DNA is the carrier of genetic information. Inspired by this new breakthrough, Erwin Chargaff discovered the "Chargaff rules" which states that in DNA, the amount of purine bases is nearly equal to the amount of pyrimidine bases with amount of A similar to T and amount of G similar to C but at this point he did not know that A is paired to T and G to C [START_REF] Chargaff | Chemical specificity of nucleic acids and mechanism of their enzymatic degradation[END_REF] (Figure 2). Finally, in 1953 Watson and Crick, and also thanks to researchers such as Rosalind Franklin,

Maurice Wilkins or Linus Pauling, proposed their model of the DNA double helix [START_REF] Watson | Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid[END_REF] which leads to the actual knowledge about DNA (Figure 3). DNA which is the most common form in living cells. But there are also two other conformations which can be adopted by DNA double helix which are A-DNA also canonical corresponding to a smaller and wider form of the helix due to the fact that it is found in dehydrated environment and Z-DNA which is a non-canonical left handed helix know to be a transient form [START_REF] Shakked | The conformation of the DNA double helix in the crystal is dependent on its environment[END_REF] .

I.2. Nucleic acids based unusual structures

I.2.1. Hoogsteen hydrogen bonds

They have been discovered in 1959 by Karst Hoogsteen who wanted to study the structures of co-crystals containing 9-methyladenine and 1-methylthymine [START_REF] Hoogsteen | The structure of crystals containing a hydrogen-bonded complex of 1methylthymine and 9-methyladenine[END_REF] . He noticed some new type of hydrogen bonds between A and T where the adenine was flipped and even if one of the two hydrogen bond was identical to the one describes by Watson and Crick, the other one implicated different atoms on another face of the adenine (between N3 and N7 whereas it should be N3 and N1). Several nucleic acids secondary structures (hairpin, i-motifs, G-quadruplexes…) formations are possible because of the existence of these hydrogen bonds (Figure 4). [START_REF] Courtois | Protonated polynucleotide structures. 3. An optical rotatory dispersion study of the protonation of DNA[END_REF]

I.2.2. Stem-loop structures

Both DNA and RNA are capable of forming stem-loop structures even if they most of the time found in RNA sequences. Stem-loop which are also called hairpin or hairpin loop is formed in single-strand nucleic acid with a base paired part corresponding to the stem and an unpaired region forming a loop. Stem part can be formed if a part of the strand is complementary to another part in the sequence when read in opposite directions [START_REF] Swadling | Origins of biological function in DNA and RNA hairpin loop motifs from replica exchange molecular dynamics simulation[END_REF] (Figure 5). These structures can be found in ribozymes (RNA enzymes) and messenger RNA having a role in biological processes such as translation [START_REF] Paulus | RNA stem-loop enhanced expression of previously non-expressible genes[END_REF][START_REF] Darfeuille | An antisense RNA inhibits translation by competing with standby ribosomes[END_REF][START_REF] Balbontin | Expression of IroN, the salmochelin siderophore receptor, requires mRNA activation by RyhB small RNA homologues[END_REF] . Stem-loop structures can be implicated in long range interactions and participate in the diversity of RNA secondary structure by forming pseudoknots and kissing complexes via hydrogen bonds forming between a stem or a loop part from a stem-loop unit to the stem or the loop part of another stem-loop unit. In the case of pseudoknots formation (Figure 6), several conformations are possible but the most common form is called H-type fold where some base of a hairpin loop is going to establish intramolecular hydrogen bonds with other bases outside the stem.

This process lead to a pseudoknot with one more stem and one more loop formed [START_REF] Staple | Pseudoknots: RNA structures with diverse functions[END_REF] . Another kind of RNA-RNA dimer can be formed thanks to tertiary interactions which is called kissing complex (Figure 7). It is the result of the base pairing between the terminal loops of two hairpins. As simple stem-loop unit, pseudoknots and kissing complexes can be implicated in the catalytic core of ribozymes in the translation process but one of their main interesting role is that they can induce a ribosomal frameshifting during translational process especially in virus [START_REF] Michiels | Solution structure of the pseudoknot of SRV-1 RNA, involved in ribosomal frameshifting[END_REF][START_REF] Nixon | Solution structure of a luteoviral P1-P2 frameshifting mRNA pseudoknot[END_REF][START_REF] Brunel | RNA loop-loop interactions as dynamic functional motifs[END_REF] . This phenomenon is possible because of a higher energetic barrier to cross for the translating ribosome due to their structural geometry [START_REF] Shen | The structure of an RNA pseudoknot that causes efficient frameshifting in mouse mammary tumor virus[END_REF] . In the case of kissing complexes, one of their most known implication is during genome dimerization in HIV-1 [START_REF] Paillart | A loop-loop "kissing" complex is the essential part of the dimer linkage of genomic HIV-1 RNA[END_REF][START_REF] Ennifar | Crystal structures of coaxially stacked kissing complexes of the HIV-1 RNA dimerization initiation site[END_REF] . Indeed, for retroviruses, this phenomenon allows them to keep their RNA copies together by their 5' end and overcome immune system defenses [START_REF] Panganiban | Ordered interstrand and intrastrand DNA transfer during reverse transcription[END_REF] .

I.2.3. Triplex structure

DNA triplex is one of the most characteristic secondary structure adopted by nucleic acids in which Watson and Crick hydrogen bonds and Hoogsteen hydrogen bonds are involved. Triplex nucleic acids were first discovered in 1957 by Felsenfeld and Rich [START_REF] Felsenfeld | Studies on the formation of two-and three-stranded polyribonucleotides[END_REF] who found that a complex can be formed between polyuridylic acid and polyadenylic strands if they are put together in a 2:1 ratio. Then in 1986, by designing DNA-binding molecule [START_REF] Dervan | Design of sequence-specific DNA-binding molecules[END_REF] , Dervan discovered a DNA sequence capable of forming a stable specific triple helical DNA complex called triplex forming oligonucleotide (TFO). It binds to the major groove of the DNA duplex and then can form a DNA triplex if there are the needed requirements such as physiological conditions and especially Mg 2+ salt concentration [START_REF] Reither | Specificity of DNA triple helix formation analyzed by a FRET assay[END_REF] . TFO should be complementary to one of the two DNA strand but unlike DNA duplex it is not mandatory to have antiparallel formation. DNA triplexes can be intermolecular or intramolecular depending of the TFO's nature (Figure 8A). If TFO comes from a second DNA molecule, then an intermolecular triplex can be formed. But the third strand can be provided by one of the strands of the same duplex if there is some mirror repeat sequence and the resulting triplex will be intermolecular and is known as "H-DNA" [START_REF] Demidov | Sequence-specific targeting of duplex DNA by peptide nucleic acids via triplex strand invasion[END_REF][START_REF] Vasquez | Triplex-forming oligonucleotides: principles and applications[END_REF] . Depending on the bases implicated in the triad formation (from the TFO and from the duplex), two types of DNA triplexes can be formed: parallel and anti-parallel [START_REF] Simon | Targeting DNA with triplex-forming oligonucleotides to modify gene sequence[END_REF] . Generally, parallel triplexes would form in case of pyrimidine rich triad such as TAT or CGC where the TFO bring pyrimidine base and anti-parallel would form in case of purine rich triad such as GGC or AAT where base from TFO Is purine (Figure 8B). Several studies about association and dissociation of DNA triplexes showed that the association can simply be described with a simple model Duplex + TFO  Triplex, and is highly depending on pH essentially due to protonation of cytosine which can lead to one-step formation or sequential formation [START_REF] Sugimoto | pH and cation effects on the properties of parallel pyrimidine motif DNA triplexes[END_REF] . For dissociation, a model was proposed with two different steps with; first a dissociation step of TFO from DNA duplex followed by the dissociation of the duplex : Triple  Duplex + TFO  2 Single Stand + TFO. Indeed, studies revealed that DNA triplex is thermodynamically less stable than bound DNA duplex [START_REF] Plum | Thermodynamic characterization of the stability and the melting behavior of a DNA triplex: a spectroscopic and calorimetric study[END_REF] . DNA triplexes are not only some kind of unusual DNA secondary structure because they also have some important implication in biological process especially because over 2 million TFO binding sites have been identified in mammalian genome [START_REF] Wu | High-affinity triplex-forming oligonucleotide target sequences in mammalian genomes[END_REF][START_REF] Gaddis | A web-based search engine for triplex-forming oligonucleotide target sequences[END_REF] .

I.2.4. I-motif structure

The i-motif also called i-tetraplex is a four-stranded structure that can be formed within C-rich strand with intercalated C-C base pairs at slightly acidic pH. It has been first described in 1993 by Guéron and co-workers [START_REF] Gehring | A tetrameric DNA structure with protonated cytosine.cytosine base pairs[END_REF] but formation of C-C pairs base in cytosine-rich polymer was already demonstrated more than 50 years ago [START_REF] Akinrimisi | Properties of helical polycytidylic acid[END_REF][START_REF] Langridge | Molecular structure of helical polycytidylic acid[END_REF] . However, i-motif is the only know nucleic acid structure which can be formed via a system of intercalated base pairs. This structure is formed with two parallel duplexes linked via hemi-protonated cytosine-cytosine base pairs that are intercalated in an antiparallel orientation [START_REF] Gueron | The i-motif in nucleic acids[END_REF][START_REF] Zhou | Formation of i-motif structure at neutral and slightly alkaline pH[END_REF] (Figure 9). Formation of i-motif depends on several factors such as the number of implicated intercalated cytosine [START_REF] Leroy | Intramolecular folding of a fragment of the cytosine-rich strand of telomeric DNA into an i-motif[END_REF] , loop length [START_REF] Mergny | Kinetics and thermodynamics of i-DNA formation: phosphodiester versus modified oligodeoxynucleotides[END_REF] , or environmental condition. Indeed, it has been shown that imotif can even be formed at neutral pH conditions [START_REF] Fojtik | The fragile X chromosome (GCC) repeat folds into a DNA tetraplex at neutral pH[END_REF] and supports the idea that i-motif are dynamic structure over a wide range of pH from a folded structure to a more disorganized conformation especially at higher pH values. Moreover, i-motif can be formed in a tetramer with four strand containing at least a stretch of cytosine [START_REF] Leroy | Solution structures of the i-motif tetramers of d(TCC), d(5methylCCT) and d(T5methylCC): novel NOE connections between amino protons and sugar protons[END_REF] , in a dimer of a DNA strand containing two cytosine stretches [START_REF] Nonin | Solution structure and base pair opening kinetics of the i-motif dimer of d(5mCCTTTACC): a noncanonical structure with possible roles in chromosome stability[END_REF] or even in a monomer by forming an intramolecular imotif with a single strand of four cytosine stretches [START_REF] Phan | Intramolecular i-Motif Structures of Telomeric DNA[END_REF] (Figure 10).

The i-motif arrangement is quite stable and generally shows a high melting temperature in acidic conditions, nevertheless stability is highly dependent on the conditions especially pH conditions. Concerning kinetics, i-motif often show a very slow association and dissociation constants depending again on the conditions and the sequence [START_REF] Collin | Stability of Chimeric DNA/RNA Cytosine Tetrads: Implications for i-Motif Formation by RNA[END_REF] . Even if the role of i-motif remains unclear, there a lot of C-rich sequences such as promoter regions and human telomeric DNA which could be capable of forming i-motif [START_REF] Brooks | Making sense of G-quadruplex and i-motif functions in oncogene promoters[END_REF] . Other C-rich regions can be found in minisatellite such as human insulin minisatellite or in the human centromeric α satellite [START_REF] Catasti | Cystosine-rich strands of the insulin minisatellite adopt hairpins with intercalated cytosine+.cytosine pairs[END_REF][START_REF] Gallego | Centromeric pyrimidine strands fold into an intercalated motif by forming a double hairpin with a novel T:G:G:T tetrad: solution structure of the d(TCCCGTTTCCA) dimer[END_REF] . It has also been reported that some proteins such as the heterogeneous nuclear Element) promoter region [START_REF] Takimoto | Specific binding of heterogeneous ribonucleoprotein particle protein K to the human c-myc promoter, in vitro[END_REF] .

I.2.5. G-quadruplex structure

I. 2.5.1. From a nuisance to a molecule of interest G-quadruplexes also known as G4 are nucleic acids secondary structures that can be formed within DNA or RNA guanine rich strands. Interestingly, their history is older than the double helix discovered by Watson and Crick in 1953. In 1910, Bang [START_REF] Bang | Untersuchungen über die Guanyksäure[END_REF] discovered the that guanylic acid is capable of forming a gel at high concentrations suggesting that G-rich sequences could form auto associate and form high order structures. Around fifty years later, Gellert and colleagues [START_REF] Gellert | Helix formation by guanylic acid[END_REF] used X-ray diffraction and found that guanylic acids can assemble into tetrameric structures. After several studies [START_REF] Zimmerman | X-ray fiber diffraction and modelbuilding study of polyguanylic acid and polyinosinic acid[END_REF][START_REF] Howard | Stable and metastable forms of poly(G)[END_REF] , the G-quartet (or G-tetrad) (Figure 11) was finally described as the basic structural motif of G-quadruplexes composed of four guanines associated in a planar square with Hoogsteen hydrogen bonds. Each guanine is associated to the two adjacent ones. Stacked G-quartets lead to the formation of a G-quadruplex. For years, even after being described, G-quadruplexes were seen as a nuisance first and then as an in vitro artefact until studies show that G-rich sequences at the ends of human telomeric DNA were capable of forming this kind of structure.

I.2.5.2. G-quadruplexes polymorphism

G-quadruplexes cannot be described as only a simple four-stranded structure. Indeed, Gquadruplexes are well known to be highly polymorphic structures. This polymorphism has several explanations such as the sequence [START_REF] Cang | 3rd, Explaining the varied glycosidic conformational, G-tract length and sequence preferences for anti-parallel Gquadruplexes[END_REF] , the number of strands implicated in the formation and their orientation [START_REF] Bochman | DNA secondary structures: stability and function of G-quadruplex structures[END_REF] , the loops size, orientation and connectivity [START_REF] Cevec | Role of loop residues and cations on the formation and stability of dimeric DNA G-quadruplexes[END_REF][START_REF] Risitano | Influence of loop size on the stability of intramolecular DNA quadruplexes[END_REF][START_REF] Guedin | How long is too long? Effects of loop size on G-quadruplex stability[END_REF] , the syn or anti conformation of the guanines [START_REF] Karsisiotis | Structural probes in quadruplex nucleic acid structure determination by NMR[END_REF] , the nature of the stabilizing cation [START_REF] Smargiasso | G-quadruplex DNA assemblies: loop length, cation identity, and multimer formation[END_REF][START_REF] Li | Potassium-lead-switched G-quadruplexes: a new class of DNA logic gates[END_REF][START_REF] Dai | Structure of the Hybrid-2 type intramolecular human telomeric Gquadruplex in K+ solution: insights into structure polymorphism of the human telomeric sequence[END_REF] and some other factors depending on the environment.

Number of strands and orientation (Figure 12)

G-quadruplexes formed with only one strand are called unimolecular or intramolecular Gquadruplexes. They can be simply described with this kind of model [GnLxGnLxGnLxGn] where

Gn is the number of guanines in the tract that can be implicated in the G-tetrad and Lx is the number of residues which are implicated in the loops and can also be guanines if they are not part of a tetrad. Generally, Gn will contain at least three guanines because even if Gquadruplexes with only two stacks are possible, they are less stable [START_REF] Chen | Sequence, stability, and structure of G-quadruplexes and their interactions with drugs[END_REF] . G-quadruplexes can also be formed with several strands with bimolecular G-quadruplex (two strands) or tetrameric G-quadruplex (four strands). The strand sequence is not necessary the same and could be different but in most of the cases, sequences are identical. Among this three types of G-quadruplexes, the most polymorphic one is the bimolecular because the association of two strands increase topology variation. Recently an example of a triple stranded G- quadruplex has been published [START_REF] Singh | A triple stranded G-quadruplex formation in the promoter region of human myosin beta(Myh7) gene[END_REF] showing that polymorphism due to the number of strand in the G-quadruplex formation is not totally unsolved. In addition to the number of strands, polymorphism is also due to the orientation of these strands. Indeed, G-quadruplexes can be divided into three different groups [START_REF] Hansel | The parallel G-quadruplex structure of vertebrate telomeric repeat sequences is not the preferred folding topology under physiological conditions[END_REF] . When all strands are oriented in the same direction, Gquadruplex is called parallel [START_REF] Yang | Visualization of Parallel G-Quadruplexes in Cells with a Series of New Developed Bis(4-aminobenzylidene)acetone Derivatives[END_REF] . If there are two strands in a direction and the two other in the opposite direction, then the G-quadruplex is anti-parallel. In the case of anti-parallel conformation, the two strands in the same direction can be on the same side in a "chairlike" [START_REF] Geng | The crystal structure of an antiparallel chair-type G-quadruplex formed by Bromo-substituted human telomeric DNA[END_REF] conformation or on opposite sides in a "basket-type" [START_REF] Lim | Structure of the human telomere in K+ solution: a stable basket-type G-quadruplex with only two G-tetrad layers[END_REF] conformation. Finally, the last case corresponds to only one strand in the opposite direction compared to the others strands and the G-quadruplex is called hydrid [START_REF] Sengar | Structure of a (3+1) hybrid G-quadruplex in the PARP1 promoter[END_REF] also knowns as "3+1" mixed conformation.

Loops diversity and bulges (Figure 12)

Loops diversity also participates to increase G-quadruplex polymorphism. Loops can only be formed in bimolecular and intermolecular G-quadruplexes because there is no connection between strands in tetramolecular G-quadruplexes. Three different types of loops can be distinguished in G-quadruplexes [START_REF] Cang | 3rd, Insight into G-DNA structural polymorphism and folding from sequence and loop connectivity through free energy analysis[END_REF] . First, the propeller type [START_REF] Havrila | Structural dynamics of propeller loop: towards folding of RNA Gquadruplex[END_REF][START_REF] Peng | Probing the propeller-like loops of DNA G-quadruplexes with loopedout 2-aminopurine for label-free switchable molecular sensing[END_REF] which corresponds to a loop linking adjacent parallel strands from a bottom G-tetrad to a top G-tetrad. This kind of loop can be formed in all type of G-quadruplexes. The two other types of loop that can exist only in anti-parallel and hybrid G-quadruplexes are known as lateral and diagonal loops. Lateral loops [START_REF] Fadrna | Molecular dynamics simulations of Guanine quadruplex loops: advances and force field limitations[END_REF] connect two anti-parallel strands on the same side and can be separated in two types:

"head-to-head" and "head-to-tail" [START_REF] Lech | Guanine base stacking in G-quadruplex nucleic acids[END_REF][START_REF] Lavery | A comprehensive classification of nucleic acid structural families based on strand direction and base pairing[END_REF] . "Head" and "Tail" refer to the opposite faces of a guanine base. Diagonal loops [START_REF] Kuryavyi | A double chain reversal loop and two diagonal loops define the architecture of a unimolecular DNA quadruplex containing a pair of stacked G(syn)-G(syn)-G(anti)-G(anti) tetrads flanked by a G-(T-T) Triad and a T-T-T triple[END_REF] joins also two anti-parallel strands but on opposite sides.

Another type of connectivity between guanines of tetrad which participate to G-quadruplexes polymorphism but not considered as loop is bulge [START_REF] Pan | Crystal structure of a bulged RNA tetraplex at 1.1 a resolution: implications for a novel binding site in RNA tetraplex[END_REF][START_REF] Mukundan | Bulges in G-quadruplexes: broadening the definition of G-quadruplex-forming sequences[END_REF] . Bulges consist in, like loops, connecting guanines of G-tetrad but unlike loops which connect guanines of opposite sides, bulges connect two adjacent guanines of the G-core belonging to the same strand.

Guanine glycosidic conformation

In G-quadruplexes, guanine cap adopts two different conformations depending on the glycosidic angle χ: anti and syn [START_REF] Yang | Free energy landscape and transition pathways from Watson-Crick to Hoogsteen base pairing in free duplex DNA[END_REF] (Figure 13). The glycosidic bond links the sugar and the base (A, T, C, G, U if RNA) between the C1' and N9 in the case of purine or N1 in the case of pyrimidines. The corresponding torsion angle χ may be really different depending on the rotation of this bond. Torsion angle χ can be described as the angle between O4′-C1′-N9-C4

for purines and O4′-C1′-N1-C2 for pyrimidines. If χ is comprised between -120 and 180° then the corresponding conformation is anti whereas syn correspond to a torsion angle between 0 and 90°.

Parallel G-quadruplexes have all guanine in anti conformation but anti-parallel Gquadruplexes can both anti and syn conformation. There is no rule to predict if it would have formed one or the other conformation because glycosidic conformation will be unique for a given topology. The only exception concern RNA G-quadruplexes which generally have anti oriented guanines and harbor a parallel conformation due to the 2'-hydroxyl group [START_REF] Malgowska | Overview of the RNA G-quadruplex structures[END_REF][START_REF] Ruggiero | G-quadruplexes and G-quadruplex ligands: targets and tools in antiviral therapy[END_REF] .

Nature of the cation

As described before, G-quadruplexes can be stabilized by monovalent cations and their role in G-quadruplexes stability and polymorphism have been deeply studied. Cations can contribute to neutralize the global negative charge of DNA but in the case of the Gquadruplexes their role become central [START_REF] Wlodarczyk | Effect of ions on the polymorphism, effective charge, and stability of human telomeric DNA. Photon correlation spectroscopy and circular dichroism studies[END_REF][START_REF] Bhattacharyya | Metal Cations in G-Quadruplex Folding and Stability[END_REF] . Indeed, during G-quadruplexes formation, the implication and orientation of guanines in the tetrad allow the creation of a central channel which is highly negatively charged. Due to the orientation of the O6, a strong negative electrostatic potential is created inside the channel which needs to be compensate by cations positive charge. Coordination by monovalent cations is possible due to the geometric arrangement of the guanine O6 pairs of electron. As the most biological relevant cations are K + and Na + , they are the most known and studied cations in G-quadruplexes formation but there are several other cations, monovalent or divalent, that can stabilize G-quadruplexes, sometimes in a better way. As a result, cations have been identified depending on their stabilizations potential: K + , Na + , Rb + ,NH3 + , Ca 2 +, Mg 2+ , Pb 2+ [START_REF] Hardin | Cation-dependent transition between the quadruplex and Watson-Crick hairpin forms of d(CGCG3GCG)[END_REF][START_REF] Venczel | Parallel and antiparallel G-DNA structures from a complex telomeric sequence[END_REF][START_REF] Nagesh | Ammonium ion at low concentration stabilizes the Gquadruplex formation by telomeric sequence[END_REF][START_REF] Miyoshi | Effect of divalent cations on antiparallel G-quartet structure of d(G4T4G4)[END_REF] for the most known of them, K + being the one with the higher stabilization effect [START_REF] Zaccaria | The role of alkali metal cations in the stabilization of guanine quadruplexes: why K(+) is the best[END_REF] . Depending on the nature of the cations, position within the G-quadruplex channel can be different. For example, Na+, which is smaller compared to K+ can be in the same plane as guanines O6 in some case or it can adopt a configuration similar to K+. Potassium cation which is larger than sodium does not fit in a planar configuration and has to be equidistant between each tetrad plane be in a tetragonal bipyramidal electrostatic configuration. The nature of cations can be very important in G-quadruplexes polymorphism because in some cases, with the same sequence, different cations can lead to the formation of different conformations, such as in the case of human telomeric sequence 5' A(GGGTTA)3GGG 3'. In presence of Na + it forms an antiparallel G-quadruplex [START_REF] Wang | Solution structure of a parallel-stranded G-quadruplex DNA[END_REF] but with K + it forms a parallel structure [START_REF] Gray | Kinetics and mechanism of K+-and Na+-induced folding of models of human telomeric DNA into G-quadruplex structures[END_REF] .

High order G-quadruplexes structures Even of most of the studies have been focused on monomeric structures, G-quadruplexes can adopt a variety of multimeric forms from the smallest represented by a dimer to very large structures containing hundreds of G-quadruplexes monomers called G-wires [START_REF] Bose | High-resolution AFM structure of DNA G-wires in aqueous solution[END_REF][START_REF] Marsh | G-wires: self-assembly of a telomeric oligonucleotide, d(GGGGTTGGGG), into large superstructures[END_REF] . Regarding the potential high G-quadruplexes concentration availability in eukaryotes, formation of Gwires structures is particularly interesting. Different types of multimeric structures can be distinguished to monomeric G-quadruplexes [START_REF] Kolesnikova | GTP-Dependent Formation of Multimeric G-Quadruplexes[END_REF][START_REF] Kolesnikova | Structure and Function of Multimeric G-Quadruplexes[END_REF] : intramolecular or intermolecular; parallel, antiparallel or hybrid based structures depending on the conformation of G-quadruplex units.

Moreover, each unit can be stacked in three different orientations: 5' to 3' "head-to-tail" [START_REF] Chaires | An improved model for the hTERT promoter quadruplex[END_REF] , 5' to 5' "head-to-head" [START_REF] Do | Stacking of G-quadruplexes: NMR structure of a G-rich oligonucleotide with potential anti-HIV and anticancer activity[END_REF] or 3' to 3' "tail-to-tail" [START_REF] Kato | Dynamics and thermodynamics of dimerization of parallel Gquadruplexed DNA through pi-pi stacking interaction[END_REF] . Formation of these higher structures is essentially possible thanks to the π-π stacking of the terminal faces of each G-quadruplex unit [START_REF] Smargiasso | G-quadruplex DNA assemblies: loop length, cation identity, and multimer formation[END_REF] . An example of this kind of structure has been described by Neidle and coworkers [START_REF] Haider | Molecular dynamics and principal components analysis of human telomeric quadruplex multimers[END_REF] who described consecutive parallel G-quadruplexes connected by a TTA loop in a "head-totail" configuration.

I.2.5.3. G4-motif localization

In vitro, G-quadruplexes are considered as highly stable structures with melting temperature much higher than that of duplex DNA [START_REF] Lane | Stability and kinetics of G-quadruplex structures[END_REF]100] . Due to their stability, their existence in vivo is still a critical point in G-quadruplexes studies. However, computational studies revealed more than 375,000 G-quadruplexes forming motifs in the human genome [101,102] suggesting that they may form in vivo. Moreover, these motifs are not randomly located within the genome but are concentrated in several regulation key regions [103][104][105] . G-quadruplexes motifs are essentially present at the end of the chromosome telomeres [106] , in promoter regions [107] ,

within UTRs (Untranslated Regions) of messenger RNA [108] and in several other regions such as mitotic and meiotic DSB (Double Strand Break) sites. All these localisations suggest that Gquadruplexes could have a biological role in several biological processes such as replication or transcription acting as a regulator. Several other studies have been performed in other organism especially in Saccharomyces cerevisiae [109,110] where more than 1,400 Gquadruplexes forming motifs have been identified which remain quite high regarding the entire genome of Saccharomyces cerevisiae. Even if G-quadruplexes forming sequences have been identified in all over the genome, two localisations have been particularly studied:

telomeres and gene promoter's regions.

G4 in telomeres

Telomeres are specialized DNA nucleoprotein complexes that cap the end of chromosomes [111- 113] . Their primary role is to protect against gene erosion at cell divisions and attacks from nuclease [114] . For human (and vertebrate) telomeres consist of a tandem repeat of the hexanucleotide 5' d(TTAGGG)n 3' with a length of 5 to 10 kb terminating with a G-rich single strand capable of forming G-quadruplexes [115,116] . In normal cells, each cell replication leads to the loss of 50 to 200 base in telomeres. This phenomenon is related with cancer, aging and genetic stability as the loss of bases directly corresponds to an information loss [117,118] . In somatic cells, each cell replication cycle leads to the loss of 50 to 200 base in telomeres. After a critical shortening of the telomeres the cell undergoes apoptosis [119] . In most organisms, the telomerase, a reverse transcriptase enzyme, is capable of increase the length of telomere by using its RNA subunit as a template [120] . Telomerase is inactive is most somatic cells except when its functions are needed but it is upregulated in most cancer cells leading to immortal cancer cells [121] . It has been shown that the G-quadruplexes formation could influence the telomerase activity with different effects depending on the conformation: intramolecular antiparallel G-quadruplexes block telomerase whereas intermolecular parallel ones could favor telomerase activity [122,123] . Moreover, in human, telomeres are associated with several related proteins such as Pot1 (protection-of-telomeres 1) which has been shown to avoid G-quadruplexes formation by interacting and trapping the G-rich single strand [124,125] . As a consequence, Pot1 can enhance telomerase activity. As the potential role of G-quadruplexes in telomerase activity is known for almost 40 years, several different of studies employing different methods have been performed in order to determine the structure adopted by telomeric G-quadruplexes (Figure 14) [126][127][128] .

Surprisingly, the variety of methods and conditions tested produced a variety of structures.

The telomeric sequence can form different G-quadruplexes much depending on the salt composition. In Na + buffer seems to form parallel G-quadruplexes or hybrid G-quadruplexes even if the predominant form seems to be the hydrid one. The parallel conformation may have been induced by crystallization condition. Thus, G-quadruplexes forming motifs are particularly present in promoter regions and suggest a potential function in transcription. Indeed, at least one G-quadruplexes motif have been found near (1000 nucleotides upstream) TSS (Transcription Starting Sequence) in more than 50% of the human genes [129] . This phenomenon is quite conserved across species because it can also be observed in yeast, plants or bacteria [130,131] . During transcription process, there is only one strand used as a template and the other is the non-template strand. Depending on the situation, G-quadruplexes motifs can be located in one or in the other strand and two hypotheses can be proposed about the role of these structures. The presence of Gquadruplexes within the sequence can be compared to supercoiling [132] which is a well-known phenomenon that is thought to affect transcription [133] . Supercoiling can have both positive and negative effects on transcription that is why two hypotheses are possible (Figure 15). In the case where the G-quadruplex motif is located on the template strand, it could inhibit the transcription by acting as a roadblock and as a result disturb the transcription machinery.

G4 in promoter regions

But, if the G-quadruplex motif is located on the other strand (non-template), it could enhance the transcription by maintaining the transcribed strand in a single-strand conformation. The main criticism of this model is that the G-quadruplex formation is too slow and the stability of these structures is too high to be considered as regulation elements. In addition to the two previous hypotheses, transcription may also be altered by protein binding to G-quadruplexes especially transcription-related proteins such as transcriptional enhancers or repressors [134] .

These proteins could affect formation or unfolding of quadruplexes. If these proteins could act as chaperones [135,136] , then the time scale for G-quadruplexes formation could be adapted for a regulatory role. Several transcription-related proteins are known to bind to Gquadruplexes [137,138] and make their role in transcription more relevant. One of the G-quadruplexes most exhaustively studied is the mammalian MYC (or c-MYC) oncogene which codes for a transcription factor associated with cell proliferation [139] . In more than 80% of human cancer cells, there is increased level of MYC expression [140,141] . In MYC promoter, there is a sequence called Nuclease Hypersensitive Element III1 (NHE III1) which is essential for MYC transcription and capable of forming G-quadruplexes in vitro. Several structures adopted by sequences within the MYC NHE III1 and within several other oncogenes have already been studied (Figure 16) [142][143][144] . The role of the G-quadruplex has been studied by looking at the levels of expression of the gene with a wild type sequence containing the motif and compared to a mutated sequence without it and the results showed that the motif in NHE III1 represses transcription [145] . Several other oncogenes have been studied such as c-KIT [146] , VEGF [147] , BCL-2 [148] or KRAS with similar results.

I.3. Use in therapeutics

In the last decade's we have seen some major developments in the field of therapeutics such as the development of monoclonal antibodies, immune modulators or replacement enzymes to target over 20,000 "druggable" targets which have been identified [149,150] both by academia and industrial researchers all over the world. Unfortunately, a lot of those targets remain "undruggable". In order to overpass these limitations new approaches and strategies are necessary. It is without surprise that some actors in the field have turned to nucleic acids as possible targets, to the point of creating a new field by itself. This is due to the "explosion" of fundamental research performed in the field of nucleic acids in the past 30 years. Nucleic acids have many advantages for building artificial nanostructures in "drug Design" concept [151][152][153] :

(1) their secondary structure could be determined; (2) they are easy and rapid to design; (3) their production is less costly involving chemical synthesis without variability of biologics; (4) they are stable and ( 5) they can be combined to increase drugs flexibility. A large variety of strategies have been developed based on nucleic acids and several applications in therapeutics and biotechnology have emerged with several patents being accepted both in Europe and in the USA.

I.3.1. Examples of nucleic acids applications

I.3.1.1. Antisense Oligonucleotide (ASO)

Antisense Oligonucleotides (ASOs) (Figure 17) are generally single-stranded nucleic acids (DNA, RNA or chemical analogs). They are chemically modified to have enhanced stability, affinity and cell penetration augmented and improved specificity to their target in order to use them as drugs. But, as they need to be recognized by cellular mechanisms, their modifications need to be carefully chosen and sometimes restricted which limited the improving of their properties [154] . ASO are usually used to bind pre-messenger RNA in order to modulate the splicing, cause a sequence-specific degradation or even to block polyadenylation to accelerate RNA decay [155,156].

Depending on the purpose of the ASOs, their chemical modifications will be different. For example, for a sequence specific degradation, ASO need to bind to the selected part of premessenger RNA and need to be recognized by RNase H which will cleave the targeted sequence. A good ASO candidate would be around 20 nucleotides long (corresponding to the classical length of ASOs) with phosphorothiate linkages between nucleosides to form the backbone. Several bases can be added to the flacking order to increase stability in vivo by protecting ASO from exonuclease and to improve binding to pre-mRNA. For slicing modulation or polyadenylation block, it is necessary to avoid degradation by RNase H that is why ASO would contains bases with other modifications. ASO can also be designed to target mRNA in order to inhibit or enhance translation. The main problem with this strategy is the ASO intracellular uptake remaining quite poor that is why only a few drugs antisense-based have been approved by FDA (USA): fomivirsen [157] , an antiviral drug briefly marketed before effective anti-HIV drugs emerged and mipomersen [158,159] which is used to treat familial hypercholesterolemia. where the delivery of a long, double-stranded RNA (dsRNA) silenced a gene expression by the degradation of the messenger RNA [160] . The mechanism was described as the degradation of this long double-stranded RNA into small (around 20 nucleotides) double-stranded RNA called siRNA which is capable of interacting with a multi-protein RNA-Induced Silencing Complex (RISC). Then, in the RISC, siRNA is unwound and the antisense strand binds to mRNA. AGO2, a component of RISC recognizes the complex and cleaves the mRNA around 10 nucleotides downstream from the 5'-end of the antisense strand [161] (Figure 18).

I.3.1.2. siRNAs (short interfering RNA)

This mechanism was thought not existing within mammalian cells but later studies showed that they just lack the degradation system of dsRNA and synthetic siRNA can be used to interact with RISC and degrade mRNA in mammalian cells [162,163] . siRNAs were rapidly used in therapeutics with very encouraging results in vitro by inhibiting HIV replication [164] by depleting viral genes and in vivo by protecting mice from autoimmune hepatitis [165] . The principal problems found with siRNA strategies were similar to those found in ASO applications: they are rapidly degraded and the uptake into the respective targets is quite poor. In addition, unlike ASOs, chemical modifications are more limited and that is why in clinical trials, the development of siRNA strategies are focused on the delivery system part.

Two major delivery systems have been studied: (1) using lipid nanoparticles to introduce siRNAs and ( 2) by conjugating siRNAs to trivalent N-acetylgalactosamine (GalNac) [166,167] .

These two systems target the liver which is the primary sites of several circulating proteins. The next generation of siRNA delivery system is focusing on the second system as the lipid nanoparticles can be difficult and the system has side effects due to the immune system response.

I.3.1.3. CRISPR-Cas9 system

CRISPR is a defensive mechanism adopted by bacteria against viral infections [168,169] . CRISPR is the abbreviation for Clustered Regularly Interspaced Short Palindromic Repeats. These clusters are separated by non-repeated DNA sequences corresponding to copies of foreign DNA encountered by the bacteria which are called CRISPR arrays and act as memory. When encountering a foreign genetic element again, bacteria produced RNA segments from CRISPR arrays to target directly pathogen and then use adaptive immune response thanks to nucleases coded by CIRSPR-associated (Cas) genes. CRISPR cluster has been first reported in 1987 by Yoshizumi Ishino [170] , biotechnological application remained unexploited until 2013 when CRISPR-Cas9 was formerly applied for human gene editing [171] . CRISPR/Cas system has been classified into six main types (I-VI) and two main classes (1 and 2). Cas9 nuclease belongs to type II and class 2. By designing a guide RNA (sgRNA), it is possible to target genes and having an excised site at the correct location in the gene and then by triggering a cellular DNA repair mechanism having a precise sequence alteration. Once again, CRISPR-Cas9 technology (Figure 19) relies on the antisense pairing of sgRNAs but on DNA (chromosomal) instead of RNA for ASO and siRNA. CRISPR/Cas9 system lead to gene editing field which could allow to correct mutations in coding or regulatory regions of genes in therapeutics [172,173] . However, as CRISPR-Cas9 modifies the genome, ethical and safety concerns remain a major problem depending in the effects of direct mutation in the genome.

In order to use CRISPR-Cas9 efficiently, a sgRNA need to be designed to target the selected sequence and then add in the Cas9 plasmid construction. CRISPR system including sgRNA and Cas9 need to be delivered at a chosen target that is why delivery system have to be developed to allow gene editing only where it is needed. This technology remains very interesting especially in the case of cancers which are genetic diseases and progress for the last 5 years

show that it can be rapidly developed.

I.3.1.4. Triplex DNA

Secondary nucleic acid structures also have applications in therapeutics. Triplexes DNA for example and more precisely intermolecular triplexes (with external TFO as described before)

attracted attention because of their potential role in inhibiting gene expression [174] . Thus, they can be implicated in therapeutics concerning cancer or other genetic diseases. The idea is to design a TFO sequence specific to the target to allow the formation of triplex DNA leading to gene inactivation, stimulating DNA repair or homologous recombination. TFO represents a good candidate to bond to duplex DNA with high affinity and specificity as it can be perfectly complementary to the targeted strand and, similar to ASO, it can be chemically modified or link to a delivery cargo [175] . An advantage of using DNA rather than RNA is that there are fewer copies to target. Moreover, unlike ASO and siRNA, it is possible with TFO and triplexes formation to mutate or inactivate gene. Different approaches can be considered with triplex formation. The first application for DNA triplexes in therapeutics is the inhibition of transcription which could be very interesting in the case of oncogene (Figure 20). This has been shown two decades ago by Cooney and colleagues [176] by using a TFO targeting c-MYC promoter region and since a lot of studies have been performed with several other targets [START_REF] Wu | High-affinity triplex-forming oligonucleotide target sequences in mammalian genomes[END_REF] .

Another way to use triplex DNA is for DNA repair. Indeed, thanks to its properties, it is possible to design a TFO that can target damage DNA and then the triplex formation will induce DNA repair to restore the sequence.

This approach has been applied to c-MYC gene [177] in presence of gemcitabine, an antitumor antimetabolite, and the results were encouraging to inhibit growth of human breast cells.

Finally, the last strategy is to simply use TFO as a probe to deliver drug to its target [178,179] . The TFO has a high affinity for DNA and can be chemically modified to be linked to the drug and to increase stability, specificity or uptake. Limitations for this strategy remains quite the same compare to ASO or siRNA especially for cellular uptake but also some problem concerning triplexes formation. As cytosine N3 need to be protonated, triplex formation induced by TFO can be prevented by physiological pH which can in G-rich sequence promote G-quadruplexes formation [180,181] .

I.3.2. G-quadruplexes

Unlike strategies described before, G-quadruplexes are structures that are already present and formed in DNA sequences that is why there is no need to insert DNA or RNA material to promote their formation. However, some compounds, especially small chemical molecules (<1 kDa), will be used in most of the cases to promote G-quadruplexes formation and stabilize them. Considering that G-quadruplexes are implicated in several important biological processes, interfering with them could be worth exploring in order to find new possible therapeutic targets. Depending on the biological process and the G-quadruplexes (G4) implication, several strategies can be considered. Most of the time, the aim is to stabilize these structures to influence cells in different ways. In the case of transcription, especially for oncogenes, stabilizing G-quadruplexes would increase their implication in transcription regulation [182] .

I.3.2.1. Overview of G-quadruplexes therapeutic strategies

Indeed, there is a lot of proteins capable of interacting with G-quadruplexes that can unfold them and make G-quadruplexes more transient structures. Concerning their implication in telomeres, the aim is similar in order to reduce telomerase action [183,184] . As G-quadruplexes in promoter, proteins can bind to them and affect their formation such as Pot1 which forces the single strand conformation. Another application to consider is the destabilization of Gquadruplexes which makes sense in the hypothesis of G-quadruplexes favoring transcription when formed on the non-template strand [185] . If the G4s are present and stable in the nontemplate strand, this favors the single strand conformation of the template strand and consequently increases the transcription. For these examples, destabilizing G4s would reduce the efficiency of transcription. Another way to use ligands to influence G-quadruplexes formation is to act directly on their interaction with proteins. The idea would be to avoid binding of proteins capable of unwinding G4 structures [186] and to increase interaction with proteins promoting G-quadruplexes formation, either by interacting with G-quadruplexes or the proteins. In the last case, the major issue remains in the fact that ligands could influence the activity of the protein which can lead to the defect of its functions or at the opposite an undesirable increase of their activity. All these strategies need ligands to be designed in ways adapted to G4 biophysical and structural properties, features that need to be designed case by case.

I.3.2.2. G-quadruplexes and G-quadruplex-Protein complex ligands

G-quadruplexes ligands are usually small chemical molecules that are supposed to bind to them with high affinity (binding constant which is generally lower than 10 -6 mol.L -1 ). Several ligands from different families have been designed over the years in order to interact and stabilize G-quadruplexes (Figure 21) [187,188] . These ligands have specific features in common for G-quadruplex interaction. First, a polycyclic heteroaromatic core favouring π-π interaction with the planar aromatic faces of G-core and then, even it is not always the case, some charged hydrophilic groups can be added for electrostatic interaction with DNA negative charges and also for water solubility.

These small molecules can bind to the planar quartets, the grooves, the loops or a combination of these possibilities. For example, adding positive charges will lead to improve grooves and loops interaction. As these chemical ligands are created to be used as drugs, they need to be stable under physiological conditions and their druggability need to be considered. Beside ligands designed for G-quadruplexes stabilization only, some other are used to block the interaction between proteins and G-quadruplexes (Figure 22) by interaction with the quadruplex such as PIPER or directly with the protein such isaindigotone (SYSU-ID-01) [189] derivative 37 [190] . This ligand is capable of inhibiting interaction between NM23-H2 protein and G-quadruplexes. Protein NM23 (NME/nm23/NDPK) was discovered in 1988 [191] and it is known for its implication in transcriptional regulation of gene expression. The original compound was known to interact with the protein but displays little binding affinity to Gquadruplexes [192] and the modification has been done to reduce the quadruplexes stabilization. A lot of studies have been performed with ligands and some of them show promising results that can be used in therapeutics.

I.3.2.

Examples of G-quadruplexes ligand applications

Braco-19 is a ligand optimized from the acridine derivative family. It is generally known as telomeric G-quadruplex binder that can interact by stacking and groove binding [193] . Braco-19

is mostly known to inhibit telomerase activity which lead to anti-tumor activity on human epidermoid carcinoma cells [194] or human uterus carcinoma cells [195] . The mechanism explaining this inhibition has been identified and Braco-19 is capable of uncapping 3' telomere ends [196] . Recently Braco-19 has been further studied as anti-HIV agent [197,198] . Results showed that the ligand stabilized the viral RNA quadruplexes and during infection, was able to reduce virus genome copy in cells. The main limitation in using Braco-19 concerns its poor permeability across biological barriers that is why even if the ligand is soluble in aqueous environment and considered as a reference in G-quadruplexes research, pharmacological applications are quite restrained [199] . TMPyP4 is another well studied ligand for Gquadruplexes [200] . It has been designed and optimised from the porphyrin family which has a similar scaffold to the G-quartet and regroup strong quadruplexes ligands especially by π-π stacking. Several studies have shown that TMPyP4 have good affinity for G-quadruplexes and can inhibit telomerase and expression of several oncogenes such as c-MYC [201] or BCL-2. It has also shown good anti-tumor activity in several tumor cells including retinoblastoma cell lines [202] or leukaemia cell lines [203] . The main criticism about this ligand concerns the fact that it can bind both quadruplexes and duplexes DNA and lacks specificity. Based on the knowledge acquired with G-quadruplexes ligand studies, ligands such as pyridostatin (PDS) have been designed [204] . The properties of this ligand are relying on a rich aromatic surface, a flat conformation and an ability to form hydrogen bonds by adding donors and acceptors groups.

Even if this ligand is not so well studied compared to the ones described before, it shows interesting results by increasing telomere stability [205] . The last ligand is CX-3543 also called quarfloxin is one of the most important ligands for G-quadruplexes therapeutics. Indeed, this ligand is the first quadruplexes ligand to enter human clinical trials, even if it did not finish Phase II. It is a fluoroquinolone derivative which can bind to G4 DNA and disrupt their interaction with the nucleolin protein [206] . A derivative compound CX-5461 has been designed and show similar mechanism by inhibiting DNA replication and protein translation implicating

Pol I [207] . This compound is now in advanced phase I clinical trials (Canadian trial, NCT02719977, started in May 2016).

II. KRAS oncogene

II.1. Generalities

As indicated before, G-quadruplexes motifs can be particularly enriched in several oncogene promoter regions including KRAS (Kirsten Rat Sarcoma 2 viral oncogene homolog). KRAS is a proto-oncogene that codes for a small GTPase protein. This protein belongs to RAS superfamily also called RAS-like GTPases which regroups a large number of triphosphate (GTP)

binding proteins [208] . KRAS is one of front-line sensors that controls the activation of several signaling pathways by allowing the transmission of transducing signals from the cell surface to the nucleus. The most implicated cellular processes are cell differentiation, growth, chemotaxis and apoptosis. Understanding KRAS function, mechanism of action, regulation, partnership interaction, desensitization, and so on, is off upmost importance in order to tackle many forms of cancers

II.1.1. KRAS and RAS superfamily

In humans, the RAS superfamily consists of more than 100 members which can be divided in several subfamilies based in their structure, sequence and functions. The 5 main families are:

RAS, RHO, RAN, RAB and ARF [209] . All proteins among the RAS superfamily share very similar molecular structures and a common ability to bind guanine nucleotides and hydrolyse them thanks to G domain. Three RAS genes codes for highly homologous Ras proteins: KRAS, HRAS and NRAS [210] . Expression of these three genes is highly conserved across species but their expression levels depends on the tissue [211] . For example, KRAS will be mainly expressed in pancreatic, colon or lung cells where NRAS and HRAS expression is quite low. In mice, the level of HRAS transcripts is highest in the brain muscle and skin but lowest in the liver. For KRAS, they are more abundant in gut, lung and thymus similar to humans and are in skin and skeletal muscle. Finally, NRAS transcripts are more present in testis and thymus. These three genes encode for four proteins: Hras, Nras, Kras4A and Kras4B [212] . The last two proteins are the result of alternative splicing of exon 4 but the dominant form is Kras4B commonly known as Kras [213] . KRAS oncogene is located on chromosome 12 at position 12.1 [214] . Kras protein contains four different domains [215] : a N-terminal domain which is common to all Ras proteins and another domain which show low identity. These two regions are very important for the GTPase function because they form the needed G-domain. This domain includes the GTP-binding pocket. The last important region is at C-terminal domain and it is implicated in posttranslational modifications and membrane anchoring. Contrary to Hras, Nras and Kras4A which traffic through the Golgi to the plasma membrane, Kras4B traffics through the cytosol [216] . Even if four Ras isoform proteins are highly homologous in their primary acid sequence (around 80%) the differences among them are more predominant in the hypervariable region (HVR) on the C-terminal domain [217] .

II.1.2. KRAS GTPase mechanism

Newly synthesized Kras protein is a cytosolic and inactive protein. Localization and function of Kras, and more generally the Ras proteins, are regulated by several post-translational modifications in the C-terminal "CAAX" motif [218] . This tetrapeptide contains a Cysteine, two

Aliphatic amino acids and a terminal X amino acid. The different modifications include a farnesylation of the cysteine by a farnesyltransferase, as well as a methylation by ICMT (Isoprenylcysteine Carboxyl MethylTransferase) and the proteolytic removal of the last three residues by RCE1 (Ras Converting Enzyme 1). Adding a methyl group prevents the plasma membrane repulsion by negating the negative charge. In addition, the plasma membrane localization of Kras requires a basic poly-lysine region which is located upstream of the Cterminus [219] . Unlike the Kras4A variant, Kras4B does not need palmitoylation by palmitoyl transferase to the targeting to the membrane. Once bound to the membrane, Kras cycles between an active state when bound to GTP and an inactive state when bound to GDP due to the hydrolysis of the GTP. Switches between these two different states are mediated by two different classes of proteins [220] : GEF (Guanosine nucleotide Exchange Factors) and GAP (GTPase-Activating Proteins). GEF proteins mediates the exchange between bound GDP with GTP and GAP proteins stimulates the hydrolytic ability of Kras to convert bound GTP to GDP.

Among the GEF proteins, one of the most known is SOS (Son of Sevenless) which controls exchange between GDP with GTP within Kras. Under physiological conditions, Kras is mainly bound to GDP. Upon external stimuli, nucleotide binding of GEF is disabled and nucleotide is released. When bound to GTP, Kras GTPase activity is increased (around 100,000 times) because of changes in Kras interactions with GAP and the release of GTP is promoted by affecting interactions with GEF. These changes are mainly due to conformational changes when Kras binds GTP. One the most well-understood model for studying Kras activation is activation implicating epidermal growth factor receptor (EGFR) [221,222] , part of the receptor tyrosine kinases (RTK) family (Figure 23).

The binding of EGF to its receptor will induce its phosphorylation and the fixation of GRB2

(Growth-Factor-Receptor-Bound protein 2) which will recruit SOS in the cytosol. Then Kras is bound to GTP in an active state. Kras mediates a lot of downstream pathways of kinases including RAF (Rapidly Accelerated Fribrosarcoma)/MEK/ERK and PI3K (PhosphoInositide 3-Kinase)/AKT cascades. The first cascade will propagate the growth signal and the second one will control apoptosis suppression [223] . Studies have shown that GTP-bound Kras could directly bind RAF protein recruiting another RAF protein to dimerize allowing activation [224,225] . Then activated RAF induce phosphorylation cascade to activate MEK and ERK.

II.1.3. KRAS mutations and implications in cancer

RAS is one of the most frequently mutated oncogenes in human cancer but the mutations are not uniform [226] . Indeed, 85% of RAS mutations correspond to KRAS mutations [227] . Kras4B is [228] . All mutations lead to a reduced GTPase activity maintaining Kras in an active state but depending on the implicated mutation, the mechanism could be different. For example, mutation of glycine 12 affects GAP binding affecting GTP hydrolysis and mutations of residue 13 sterically clash with arginine with the same consequences.

Glutamine 61 is directly implicated in the catalytic site by positioning a water molecule to facilitate GTP hydrolysis and that is why G61 mutation directly affects the reaction. Beside the localization of these mutations, the mutation type is also important [229,230] . As an example, mutation of glycine 12 by a valine has been shown to have a worse prognosis in lung cancers than the mutation by aspartic acid. In addition of the maintained active state by mutation, there is another important fact to consider in KRAS-driven cancer. Indeed, it has been shown

for years that the association between wild-type and mutant Kras has in important role in malignancy [231] . Kras wild-type exhibits a restraining function of tumor growth in KRAS mutant cancers because it is capable to antagonize oncogenic Kras resulting in reduced activity.

Unfortunately, this inhibitory effect is overcome during tumor progression because of KRAS gene allele loss and increasing copy number of the oncogenic form. It has been recently shown that this effect of Kras wild-type is due to its capacity of dimerization with the mutant form.

Finally, the last type of mutations concerns the tumor microenvironment which increase the cancer malignancy [232] . Indeed, cancer cells that expressed mutated Kras are capable of inducing production of chemokines, cytokines and growth factors implicating in stroma reprogramming.

II.2. KRAS oncogene therapeutics

For therapies, the common strategy is to used chemotherapy that has important cytotoxic side effects. It generally uses pyrimidine analogs such as 5-fluorouracil (5-FU) which inhibits thymidylate synthase required for nucleotide synthesis or other cytotoxic drugs [233,234] .

Regarding the huge side effects of this method and its efficiency which is often limited, the need for more efficient therapies have been pursued by private to governmental institutions with dozens of billions of dollars spend annually, and hundreds of billions in indirect social costs (World Health Organization). As the central role of KRAS oncogene has been broadly studied and better understood in the last past decades, there were also a tremendous attempt to target it through different ways. All studies covered the multiple aspects of KRAS activation in order to finally find a possible Achilles's heel in order to treat efficiently Kras implicated cancers (Figure 24).

II.2.1. Directly targeting Kras

The first approach to be considered is to directly target Kras and its function. In principle, designing small molecules that directly compete with GTP for Kras interaction in order to decrease Kras activity. However, considering different factors difficult to bypass such as targeting Kras G12C mutation which represents 10 to 20% of all G12 mutations [235] . In Kras structure, this mutation is close to the nucleotide binding pocket and the switch regions implicated in effector interactions. Several compounds have been designed to covalently bind with the cysteine. Studies show that these compounds bind to the GDP state of Kras, impair action of SOS, decrease the affinity for GTP and block Kras-RAF association. Among all the designed compounds, ARS-853 gives good results in vitro but not in vivo [236] and another last generation designed ARS compound ARS-1620 shows in vivo anticancer activity [237] . Both compounds are actually in preclinical trials. Several limitations can be found in this strategy.

The first one is that it is limited to Kras G12C mutations but recent studies also reported Kras G12D inhibitors [238] . There are also strategies aiming to reduce KRAS expression by using siRNA as previously described. The major part of the successful compound is in preclinical trials except one called AZD-4785 which is studied in Phase I.

II.2.2. Membrane association

Kras requires membrane association for the oncogenic activity that is why several strategies aim to directly targeting post-translational modifications that modulate Kras membrane association. One of the first drugs discovered concerns farnesyl-transferase inhibitors (FTIs) that inhibit the prenylation of Kras required for membrane attachment [239] . FTIs triggered growth suppression in cancer cells in pre-clinical studies but phase II and phase III trials were disappointing because FTIs did not exhibit clinical efficiency as single agents [240] . It was explained by Kras4B prenylation through alternative mechanisms involving geranyl transferase I (GGT I) [241] . Geranylgeranylation of Kras is an alternative 20-carbon isoprenylation that can help Kras bioactivity when farnesylation is impaired. It has been proven by simultaneous genetic inactivation of farnesyl-transferase (FTase) and geranyl transferase (GTTase) which reduced KRAS-driven lung tumorigenesis in mice [242] . Dual inhibitors of FTase and GTTase have been developed (L778, 123) showing efficiency in KRASmediated cancers but also exhibiting high toxicity leading to the prohibition of further clinical development [243] . Another strategy is to inhibit the interaction between Kras and cyclic GMP phosphodiesterase δ (PDEδ) required for the correct localization and signalling of farnesylated Kras [244] . PDEδ increase Kras signalling by enriching Kras at the plasma membrane. PDEδ binds and solubilizes farnesylated Kras enhancing diffusion in the cytoplasm. Recent inhibitors [245] such as deltarasin have been designed to interfere with binding of mammalian PDEδ to Kras and avoid Kras localization to endomembrane. They showed inhibition of oncogenic Kras signalling and supress in vitro and in vivo proliferation of human pancreatic cells. Then a new generation of compounds has been designed named daltazinon 1 showing high selectivity and less unspecific cytotoxicity but also less stability [246] .

II.2.3. Acting on metabolism pathways

KRAS-mediated cancers are highly connected to several metabolic pathways [247,248] . As examples, KRAS-mediated colorectal cancers are associated with increased expression of glutamine metabolic proteins and in human pancreatic cancers, KRAS mutation lead to the reprogramming of the glutamine metabolism [249,250] . These cancer cells use a pathway using aspartate derived from glutamine metabolism. Aspartate is transported into the cytoplasm and converted into oxaloacetate by aspartate transaminase (GOT1). This pathway is essential for cancer cells growth by maintaining a favourable cellular redox state. KRAS mutation such as Kras G12D has also been recently reported to reprogram lipid homeostasis which supports tumorigenesis [251,252] . All these implications related to metabolism create a new way to treat KRAS-driver cancers by studying metabolic inhibitors. Several compounds have been test in preclinical trials and one of them CB-839 which is a glutaminase inhibitor is a compound in Phase II trial.

II.2.4. Synthetic lethality

An emerging strategy for targeting oncogenic mutations, including KRAS mutations, which shows great promise in specifically targeting cancers cells is the exploitation of synthetic lethality [253,254] . This mechanism also known as conditional genetics is based on the interaction of two genes that both contribute, even in a nonlinear way, to essential processes [255] . When only one gene is mutated the cell is viable but if mutations occurs in the two genes, it leads to cell death. This process is known as synthetic lethality because cells with both gene mutations are not viable and it is not possible to directly isolate these cells. In the case of cancer therapeutics, it is very interesting because cells without the cancer-inducing genotype are unaffected by such targeting as only one gene exhibit mutations. This approach can obviously be envisaged for KRAS-mediated cancers (Figure 25). Several tools have been used such as siRNA, shRNA (small hairpin RNA) and CRISPR library to screen synthetic lethal interactors with the KRAS oncogene [256,257] .

Several agents have been found to have synthetic lethality effects. For example, in a recent study, a shRNA which screened KRAS-driven pancreatic cells identified several synthetic lethal interactions with mutant KRAS including depletions of the mitotic protein polo-like kinase-1 (PLK1), anaphase-promoting complex subunits and other components of the proteasome [258] .

There is increasing number of studies focused on synthetic lethality for several reasons. One of them concerns combinational therapy. Indeed, even if synthetic lethality agents could be used as single method, it seems that the best solution is to used them in combination with cytotoxic chemotherapy and radiotherapy [259] . In some cases, the effect of synthetic lethal interaction will be increased when combined with a genotoxic stress. It would be possible to accentuate the efficiency of cytotoxic therapies at lower doses and as a consequence to reduce side effects and increase therapeutic indices.

II.2.5. Immunotherapy

Cancer immunotherapy is the strategy drawing the most attention in cancer treatment.

Indeed, several immunotherapy strategies using antibodies have been performed several years ago. For examples, several antibodies have been used as anti-EGFR antibodies known as cetuximab and panitumumab. These anti-EGFR antibodies can inhibit ligand binding and its dimerization which is necessary for its auto-phosphorylation for its full activation [260,261] .

Cetuximab is a chimeric human-murine antibody of the IgG1 isotype and panitumumab is a humanized antibody of the IgG2 isotype [262,263] . In addition to their role against the ligand binding, these antibodies are also capable of inducing EGFR internalization and its degradation resulting in a decrease level of EGFR at the cell surface. In clinical trials, these two antibodies have shown some therapeutic benefit as stand-alone agent and in combinational therapies.

However primary and secondary resistance to this therapy has led to the end of the trials.

Indeed, KRAS mutations lead to resistance to anti-EGFR antibodies even if the reason remains unsolved [264,265] . However, this failed attempt did not stop efforts in immunotherapy field applied to KRAS mutations. Recently, antibodies against anti-programmed cell death protein 1 (PD-1) [266] named pembrolizumab have been designed and tested in KRAS mutant NSCLC (Non-Small Cell Lung Cancer). They have been used in combination with trametinib which is a MEK inhibitor but it is too early to conclude about some therapeutic benefit in clinical trials.

For the next antibodies generation, studies may focus on FBP1 (Fructose-1,6-BisPhosphatase 1) which has an aberrant expression in natural killer (NK) cells and inhibits glycolysis and impairs viability. Indeed, it has been shown that KRAS-driven cancer lung cancer are closely associated with the dysfunctional state of NK cells [267] .

II.3. G4 within KRAS promoter region

Despite all strategies used in KRAS-driven cancers, there is actually no good strategies passing to late stages of clinical trials. As previously described, in the last decades G-quadruplexes became a possible target at different levels to develop new cancer therapies. Bioinformatics and other genomic analysis tools showed that promoter regions of several oncogenes contains G-rich sequence which are capable of forming G-quadruplexes. The most studied oncogene is c-MYC but KRAS promoter region has also been examined, although with less attention, probably due to its more complex DNA-sequence. The promoter region contains a nuclease polypurine-polypyrimidine hypersensitive element (NHPPE or simply NHE) [268,269] which is essential for transcription and capable of forming secondary structures such as Gquadruplexes. The NHE sequence is comprised from -285 to -341 from the TSS in human and from -273 to -332 from the TSS in mouse. It has been shown that the deletion of this NHE region led to a significant downregulation of KRAS transcription. Moreover, KRAS promoter has been targeted by TFO in the triplex strategy for therapeutics [270,271] . Unfortunately, the obtained results were not satisfying and the main hypothesis was that the triplex formation encountered a serious obstacle which could be the formation of G-quadruplexes. Gquadruplexes forming sequences have been investigated in the promoter region around 300 bases from the TSS [272] . Three main regions have been identified: a region near the TSS with a length of around 30 bases; a mid-region of around 50 bases and a far region of around 35

bases (Figure26).

The near and mid regions form stable G-quadruplexes. Moreover, the near region is being part of the KRAS NHE region and the absence of G-quadruplex formation within this sequence (by inserting mutations G into T) leads to an increase in transcription efficiency [273] . That is why this sequence became the main target of studies for the formation of G-quadruplexes. It can form G-quadruplexes via five tracts of guanines including an another sequence called KRAS21R

was also identified as G-quadruplex forming sequence via four tract of guanines [274] . It has been shown that this near sequence name KRAS32R can fold into a dynamic intra-molecular G-quadruplex with two conformations in equilibrium. Xodo and colleagues [275] show that this KRAS32R G-quadruplex is capable of competing double strand DNA from NHE sequence for the binding of several nuclear proteins. They used nuclear extract of pancreatic adenocarcinoma (Panc-1) against KRAS32R G-quadruplex and dsNHE sequence and they identified several proteins which bind to quadruplex such as Poly [ADP-Ribose] Polymerase 1

(PARP-1), subunits of ATP-dependent DNA helicase 2 (Ku70 and Ku86) and heterogeneous nuclear RibonucleoProtein A1 (hnRNP A1). For the last protein, it has also been shown that it can, with its derivative, destabilize KRAS32R G-quadruplexes and facilitate hybridization to the complementary NHE strand. This point will be discussed later in the manuscript. Considering the failure of numerous strategies for cancer therapy and all evidences for G-quadruplexes roles, we thought that to understand and study if KRAS G4s were a possible new target for developing new cancer molecules, I had to understand its possible structure, dynamics and its polymorphism in the NHE region.

III. Project overall

This thesis project aims at participating in Drug Design for anticancer therapy applied to KRASdriven cancers by targeting G-quadruplex forming sequences within the KRAS promoter region. The principal goal is to identify a compound capable of becoming a drug in anticancer therapy by screening and testing several ligands from different families. To this end, we need to evaluate and validate possible G4s targets in the NHE region of KRAS. My project fits in this category. This study aims at determining and validating a G-quadruplex target and identify the best chemical molecules to interact with and stabilize G4 from KRAS NHE region. However, in order to design good ligands and identify chemical moieties implicated in the interaction, it is necessary to have structural information at atomic level about the KRAS G-quadruplexes conformations. Identification of characteristic structural elements of these G-quadruplexes can allow researchers to design optimized ligands against a specific G-quadruplex such as KRAS G-quadruplexes. This project is divided in two major parts: the first one is devoted to the determination of KRAS32R G-quadruplexes structure to obtain atomic structural elements, and the second one is focused on the study of their interactions with proteins such as hnRNP A1 and several ligands from different families.

III.1. Determination of KRAS32R G-quadruplexes structure

As explained before, obtaining structural information on the target is very important in the concept of Drug Design. Indeed, it can help designing molecules that target specific binding sites identified in a resolved structure. Then, when ligands are tested against the target, the structure allows to determine the chemical moieties that are important in the interaction. For KRAS32R G-quadruplexes, structural determinations have been attempted but there is no structure of the wild type KRAS32R. However, Plavec and colleagues [276] determined the structure of a modified sequence from KRAS32R which is called KRAS32R-3n corresponding to the removal of the three first residue at 5' end and the addition of three residues at the 3' end [277] . It corresponds to a shifts within the NHE sequence. They showed that this sequence can form a stable dimeric G-quadruplex giving important structural information for ligand design. The main criticism concerning this work is that the shift of three residues could lead to major changes in the G-quadruplex formation compared to KRAS32R wild type. It has been

shown by Xodo and colleagues [275] via DMS footprinting experiments that guanines 2 (G2) and 3 (G3) are implicated in KRAS32R G-quadruplexes conformations. We can assume that the Gquadruplex formation from 32R-3n could be different from the wild type sequence. Thus, the first objective of this work is to determine the structure of the major scaffold adopted by KRAS32R.

III.2. KRAS G-quadruplexes interaction

The second part of this work is focused on KRAS G-quadruplex interactions. The first goal is to perform interactions between our structure of KRAS32R quadruplex and the hnRNP A1 protein which has been shown to unfold KRAS32R G-quadruplexes it. We did use a truncated version of hnRNP A1 to study the interaction called UP1, a shorter version containing both RRM motifs but without the unfolded domain Gly-rich region. In parallel, I performed ligand binding assays to find good candidates that can efficiently stabilize KRAS32R G4. These ligands could be used to increase transcription disturbance or to compete with proteins such as hnRNP A1 to avoid G4 unfolding and reduce KRAS transcription. Once the best ligands identified, we tried to obtain structural information to identify the most important chemical moieties in interaction to design or modify ligands which can overcome common limits of G-quadruplexes ligands.

Part 1: Determination of KRAS32R Gquadruplexes structure

I. Introduction

To understand how are organized the guanine rich sequences in the NHE region in KRAS promoter we started by studying what is thought to be the smallest sequence that can form a stable G-quadruplex. Described previously by L. Xodo and coworkers, the 21R sequence represented the smallest G4 from the NHE region that interact with the transcription factor MAZ [277] . we continued the studies from previous work in the laboratory where the structure of KRAS 22RT, a mutated version of 21R, was determined at 20 °C (Annexes Article 1). The first step was to determine the structure at 37 °C in order to have more biological relevance (structure deposited in the protein data bank with code 6T51). By analyzing this sequence in a G-quadruplex algorithm such as G4Hunter [278] which gives a score depending on the probability of forming G-quadruplex, it showed a similar score compared to KRAS21R (1.45 for KRAS22RT versus 1.41 for KRAS21R) known to form G-quadruplexes. Then, I started acquiring NMR and CD spectra of the complete sequence 32R, and I tried to understand how both the longer and smaller sequence could form G-quadruplexes. Several dozens of mutants were analyzed and I believed I could solve the structure of the longer sequence. By the end of the preliminary analysis, it was clear that KRAS32R WT did not adopt a single conformation but rather a mixture of two main conformers. Discovering how the 32R sequence would organize and how both conformers 3D structure looks like (at 37 °C) could be important to understand G4s structures in KRAS promotor region and their interplay with transcription factors. This become then the central core of my research project.

II. Determination of KRAS22RT G-quadruplex

II.1. KRAS22RT studies at physiological temperature (Article 1)

The article below represents the studies that we conducted in order to determine the structure of KRAS22RT at physiological temperature. What we found out was that the increasing of temperature from 20 to 37 °C did not affect much the overall folding of the structure. Nevertheless, even minor changes are important if we want to use the structure for rational drug design. In next section I will describe in details the findings.

II.2. Comparison between KRAS22RT G-quadruplexes at 20°C and 37°C

Based on the results obtained in our KRAS22RT studies at 37°C we determined the Gquadruplex structure of KRAS22RT with PDB deposition with code 6T51 and BMRB deposition with code 27173 (Figure 27).

This 3D folding was determined after structure calculations with ARIA/CNS software based on distance retrains obtained by NMR. Usually 750 final structures were calculated and the ten lowest energy were selected. At the end, an NMR refinement protocol was performed with AMBER molecular dynamics software and the ten best structures based on energy deposited in the PDB. A more detailed protocol concerning structure calculation and refinement is described in the KRAS32R G-quadruplex structure determination part. As there is a difference of 17°C degrees between the previous determined structure and the new one, we compared these two structure features. Indeed, the additional energy brought to the system by this temperature difference did allow an increased motion, especially in the most external parts such as G-quadruplex ends or loops. This phenomenon could lead to the creation of new containing restrains used in structure calculation and statistics about the calculated structures binding sites or difficulties for ligands by changing the pockets accessibility or increasing the size of grooves and the azimuthal dispersion of loops. In the case of KRAS22RT G-quadruplex (Figure 28) the change in temperature did not trigger major changes in the structure but there are several minor changes that can be interesting for ligands studies.

These minor changes essentially concern the loop between G13 form the bottom tetrad and G19 from the top tetrad. The bases in the two loops seem to be oriented at opposite directions leading in the 37°C temperature structure to a slightly narrower loop. The other difference that can be observed concerns the 3' end of KRAS22RT G-quadruplex. Indeed, 3'end extremities are oriented once again in opposite directions. At 20°C, A22 is oriented toward the center of the G-quadruplex whereas A21 is oriented toward the outside. At 37°C, both of the residues are oriented toward the inside of the structure leading to a partial obstruction of a potential binding site. There is no other noticeable change between these two structures.

Based on the results we obtained with KRAS22RT G-quadruplex structures, especially at 37°C, we then started to study structure KRAS32R G-quadruplex.

III. Determination of KRAS32R G-quadruplex

III.1. Structure of Two G-quadruplexes in equilibrium in the KRAS promoter (Article 2)

This article was submitted on November 6 th (2019) where I am the first author. The main results indicated that two main conformations can be formed within KRAS32R. I determined the NMR solution structure of these two conformers called KRAS32R G9T and G25T. These results were obtained after analyzing over 10 NMR 2D spectra and more than 100 1D spectra.

Several of them have been recorded at CEITEC (Central European Institute of Technology)

where I went in order to perform NMR experiments and learn more about this technique. I would particularly like to thank Radovan FIALA for all these advices and the time he dedicated to me.

III.2. Additionnal NMR dynamics relaxation experiments

III.2.1. Introduction

I used NMR solution state relaxation to understand the dynamics of exchange between the two conformers reported as G9T and G25T. In NMR, relaxation corresponds to the changes occuring to the signal with time. Generally, time causes deterioration of the signal which becomes weaker and broader. The deterioration of the signal can be splitted into two separate process T1 and T2. T1 (or longitudinal relaxation) is responsible for the loss of signal intensity, whereas T2 (or transverse relaxation) is associated with the brodening of the signal. The analysis of these two parameters allows to obtain site specific information about motion in Gquadruplexes, especially in the case of base exchange or formation of intermediate structures.

The longitudinal and transverse relaxation rate constants (R1 and R2) and [START_REF] Balbontin | Expression of IroN, the salmochelin siderophore receptor, requires mRNA activation by RyhB small RNA homologues[END_REF] N-1 H steady-state nuclear Overhauser effect (NOE) of a given imino [START_REF] Balbontin | Expression of IroN, the salmochelin siderophore receptor, requires mRNA activation by RyhB small RNA homologues[END_REF] N are influenced mainly by dipolar interaction with its attached proton and by [START_REF] Balbontin | Expression of IroN, the salmochelin siderophore receptor, requires mRNA activation by RyhB small RNA homologues[END_REF] N chemical shift anisotropy. The strength of the dipolar interaction is determined by the motion of the N-H bond vector.

III.2.2. Experimental parameters

Imino 15N T1, T2 relaxation and NOE/noNOE measure-ments data were acquired on an 850MHz spectrometer at 310 K with a fully labelled [START_REF] Paulus | RNA stem-loop enhanced expression of previously non-expressible genes[END_REF] C and 15 N KRAS32R (isotopic purity is >98 %) wild type sample produced by enzymatic synthesis. Full labelled KRAS32R WT was purchased from Silantes. Delays for relaxation were chosen based on the estimation of the relaxation time from the first increment (0.85 seconds for T1 and 170 ms for T2) so the intensity would drop by 10% in each of the 10 data sets to uniformly sample the relaxation curve. For T1, delays are : 10, 90, 190, 300, 430, 590, 780, 1020, 1370 and 1960 ms. For T2, delays are: 17, 34, 51, 85, 102, 136, 170, 220, 288 and 407 ms.

III.2.3. NMR dynamics results

In these experiments, we looked at the the imino region with signals corresponding to the guanines implicated in the KRAS32R wild type G-quadruplex structure. As we prevoously performed NMR experimentd using 5% 15N, 13C site-specific low-enrichment labelling, we had imino protons assignments. Even if we had several peaks for several guanines we were capable of assigning peaks corresponding to the major conformation within wild type that we determined later as G9T. We analysed evolution of T1 and T2 for each residue as well as the value of their NOE. Results from our NMR relaxation experiments are presented in Figure 29.

When we looked at T1 and T2 relaxations, it was pretty clear that G9 is the most involved residue in KRAS32R G-quadruplex dynamics. This result is also confirmed in NOE experiments because it has the more important variation in its NOE. Another residue which seems to be quite dynamic is G28. Indeed, even if it is not so clear by looking at T1 relaxation, its T2 relaxation is far less important compared to the other residue. However, it seems similar to the others in NOE experiments. The other residue we could identify implicated in KRAS32R dynamics is G25 especially in NOE experiments but results are not so clear compared the the previosu residues. As a conclusion, these NMR relaxation experiments supported the model we proposed with our two main conformation G9T and G25T. Indeed, the important motion of G9 is in favour of the idea that it could be implicated in only one conformation and its exclusion of the structure coud lead to the other conformation. Relaxation results for G28 could be explained by its role in the triad formation in G9T conformation and not in G25T.

Concerning G25, even it is pretty unclear, we could assume that its motion is due to the fact that it is implicated in G9T conformation and also in G25T with the slide betwwen the last guanines we observed in this conformation. However, we could observe that the results we had were not so clear as the results we expected. Indeed, we are clearly looking at a wrong time-scale here. The rates of T1, T2 and heteronuclear NOE can reveal the motions on the nanosecond time-scale or faster while the exchange between the conformers is likely to occur at time-scales slower by at least three orders of magnitude. As a conclusion, we need to think about other methods allowing us to observe dynamics in the ms range such as CPMG and XESY (Figure 30). Unfortunately, I han no more time to perform these experiments and bring new elements for the understanding of KRAS32R G-quadruplex dynamics.

IV. Conclusion

In this part, we wanted to determine the major scaffold adopted by the G-quadruplex formed within KRAS32R sequence. As this G-quadruplex seemed to be highly polymorphic, the structure determination, especially by NMR, represented a huge challenge. Despite the fact that KRAS22RT and KRAS32R G-quadruplexes shared common characteristics especially in the implication of the first and third tracts of guanines, the overall structure remained quite different. Indeed, in KRAS22RT, the last tract implicated in the G-quadruplex formation is G18-G19-G20 but in KRAS32R, these residues are not implicated in none of the conformations leading to the existence of a loop with a quite important size. Our study about KRAS32R Gquadruplexes showed that this sequence can adopt two major conformations that we identified as G9T and G25T corresponding to the mutated residue leading to the corresponding conformations. On one hand, G9T is a unique conformation stabilized by the formation of a triad by the 3'end residues in which the residue G9 is not implicated. On the other hand, G25T is very different as G9 is implicated in the G-quadruplex formation and there is no triad formation. Moreover, the structure is still polymorphic due to a slide in the last tract of five guanines between G26 to G29 residues leading to different implications of the guanines such as G26-G27-G28 or G27-G28-G29 in the G-quadruplex. It is also possible that G9 is also responsible for a part of the polymorphism by exchanging between two states. In the wild type structure, G9T conformation represents around 70% of the structure and G25T around 25% looking at the intensities in KRAS32R wild type two dimensional 1 H-1 H NOESY spectrum.

Unfortunately, it seems that these two conformations are not the only ones formed by KRAS32R. Indeed, when we looked at the results with selectively labelled guanine samples in KRAS32R wild type we found that several peaks show more than the two expected peaks in the case of two conformations. (Figure 31). In this study, we finally understand the Gquadruplex formation within KRAS32R sequence and we resolved the structure of this Gquadruplex which is known to be highly implicated in transcription and interact with several proteins during transcription. We brought some crucial information concerning the possible selecting few good candidates and study their interaction at the atomic level, we might be able to identify how chemical groups contribute to the overall stabilization of the G4 structure.

Our goal is to obtain such structural information and find how ligands could interact more strongly with certain loops, grooves or tetrad sites. As G-quadruplexes ligands often have cytotoxicity or cellular uptake issues, it is also a good way to identify and replace chemical groups that are not needed for interaction but implicated in these problems. In parallel to KRAS32R G-quadruplex structure determination, we also studied ligands against KRAS22RT

structure to obtain structural features and performed a first screen against KRAS Gquadruplexes (Figure 32). Recently, when the structures become available, we started using these data for studies against KRAS32R G-quadruplex conformers G9T and G25T as well as the wild type. The main goal of this project was to find ligands to stabilize G-quadruplexes but also to shift the equilibrium between conformers of KRAS32R G-quadruplexes. We also wanted to identify ligands able to inhibit interactions with proteins such as hnRNP A1, shown to unfold KRAS Gquadruplexes [279][280][281] . hnRNP A1 belongs to the hnRNP family that are ribonucleoproteins. This family is composed of 20 majors hnRNPs including hnRNP A1 in the family named A. This protein shares common structural elements found in hnRNP family with two RNA recognition motifs (RRM) that are the most common RNA-binding domains (RBDs) found in hnRNP family.

It also has another common RBD that is a RGG box consisting of repeats of Arginine-Glycine-Glycine. In addition to these RBD domains, hnRNP A1 also has an auxiliary domain that is a glycine-rich domain whose role is to mediate protein-protein interaction or subcellular localization. hnRNP A1 is involved in RNA transport (thanks to its capacity to bind mRNA by interacting with the nascent transcript), is implicated in alternative mRNA splicing and has also a role in the transcription process (by associating with multiple promoter sequences and also by interacting with G-quadruplexes and unwinding them to facilitate transcription).

Unwinding Protein 1 also called UP1 is an acid proteolytic product of hnRNP A1 of 196 amino acids which contains only the two RRM domains (Figure 33). It has been shown that this protein is also capable of interacting with KRAS G-quadruplexes and unwinding them. As a consequence, we wanted to confirm that the G-quadruplexes formed by our sequences (22RT, 32R wild type and conformers) could interact with UP1 and assess if these structures can be unfolded. The structure of UP1 has already been deposited in the PDB [282] (PDB code 1L3K) making the study of the interaction straight forward. The last part of this chapter will be focused on the preliminary experiment we performed using the best ligands we identified by FRET melting and 1D NMR, in order to assess whether they can compete and prevent binding of UP1 to different KRAS G4s. As we determined 22RT and 32R

G4s structures at physiological temperature, all experiments with ligands have been performed at 37°C as well.

II. Material and methods

II.1. Oligonucleotides samples preparation

Non labelled samples were purchased from Eurogentec (Belgium) and IDT (Integrated DNA Technologies; USA). They were synthesised with 250 nmoles or 1 µmole scale and purified by reverse HPLC. Oligonucleotides solutions were prepared in 1X buffer (10 mM K2HPO4/KH2PO4; 50 mM KCl; pH 6,5). In order to fold G-quadruplexes, samples were heated at 95°C during 5 minutes then quickly cooled in ice. This process is repeated 3 times and sample are conserved at 4°C.

II.2. Labelled and Non-labelled UP1 Production

Recombinant protein UP1 tagged to with GST were expressed in Escherichia coli BL21 using plasmids pGEX-Up1. After transformation, the bacteria were grown for the night in Lysogeny Broth (LB) medium (10 g/L Yeast Extract, 10 g/L Tryptone, 10 g/L NaCl) at 37°C with 100 μg/ml ampicillin. Then bacteria were transferred in Terrific Broth (TB) medium (24 g/L Yeast Extract, 20 g/L Tryptone, 4 mL/L Glycerol, 100 mM Phosphate Buffer (KH2PO4/K2HPO4)) for nonlabelled production and in M9T medium (300µM CaCl2, 1mM MgSO4, 6g Na2HPO4, 3g KH2PO4, 0,5g NaCl, 1mg Vitamin B1, 1g NH4Cl 15 N, 2g Glucose 13 C) for 15 N, [START_REF] Paulus | RNA stem-loop enhanced expression of previously non-expressible genes[END_REF] C labelled production with the same concentration of ampicillin and grown to an A600 of at least 2.0 prior to induction with IPTG (1 mM final concentration). Cells were allowed to grow for the night before harvesting. The cells were centrifuged at 5000 RPM., and 4°C. After centrifugation the supernatant was removed. The pellet was re-suspended in a solution of PBS with PMSF 100 mM and DTT 1 M. The bacteria were lysed by sonication (40%: 45sec ON, 45sec OFF for 4 min and 30 seconds). The lysate was then centrifuged for 10 min at 4°C at 10 000 r.p.m.

Glutathione Sepharose 4B (GE Healthcare) (50% slurry in PBS) was added to the supernatant and incubated for 30 min at 4°C on a shaker. The mix was centrifuged for 5 min at 500 g and the pellet was washed 5 times in PBS and eluted with elution buffer containing 20 mM NaCl, 20 mM reduced glutathione, 200 mM Tris-HCl, pH 9.5 for A1 elution and pH 7.5 for Up1 elution. Alternatively, to re-move the GST tag, the mix was centrifuged for 5 min at 500g, washed with PreScission Cleavage buffer (GE Healthcare) and centrifuged 5 min at 500g. The pellet was incubated for 4 h at 4°C with PreScission protease to cleave the GST tag from the purified proteins. After PreScission cleavage, the UP1 moieties were detached from GST which remained bound to the Glutathione Sephadex beads. The reaction mixtures were centrifuged for 5 min at 500g, 4°C, and the untagged proteins collected from the supernatant. Finally, the purification of tagged and un-tagged UP1 protein was checked by SDS-PAGE.

II.3. Ligands origin

All the ligands used in this work had different origins with some of them commercially available, but the majority was issued from ongoing collaborations. Their structures are all presented in Figure A1 (Annexes) with their corresponding chemical families. Braco19, PhenDC3 and Quarfloxin were purchased from commercial companies such as Sigma Aldrich.

Salphen ligands [283] were provided by the laboratory of Fabrice Thomas (Prof.) from Grenoble (France). Phenantroline ligands (except PhenDC3), IP5 and NaphTrip were provided by Carla Cruz group in Beira Interior (Portugal) and JL205 [274] by Alexandra Paulo group in Lisboa (Portugal). Naphthalene DiImide (NDI) ligands [284][285][286] were provided by Mauro Freccero from Pavia (Italy). 20A was synthesised by chemists from our unit INSERM U1212.

II.4. FRET melting experiments

II.5. Circular Dichroism

All Circular Dichroism experiments were realized with Jasco J-1500 CD spectrometer using Spectra Manager software. Quartz cuve were used and contains 500 µL of 5 µM oligonucleotide samples in 1X buffer. The CD spectra were measured in the region between 220 and 320 nm with a scan speed of 50 nm/min and a response time of 1 s. Three scans were collected and averaged. All experiments were done at 37°C degrees.

II.5.1. Titrations

Ligands were diluted from their stocks to 1 mM with water and kept on ice during the titration.

Some ligands where previously diluted in 10-20% D6 DMSO before diluted in water. Before starting the titration experiments a spectrum without ligand is recorded to check the G4 signal. Ligands were added starting at 0.5 equivalent and incubated for 10-15 min after which CD spectra were collected. Then addition was done at 1, 1.5, 2, 2.5, 3, 4 and 5 equivalents (total volume change for the G4 sample was ≤ 10 %).

II.5.2. Melting experiments

CD melting studies were performed with 5 µM DNA sample alone or in the presence of different equivalent of ligand. CD melting was performed using either a full wavelength or a single wavelength mode. In the former case, the data were collected in the wavelength range 350-220 nm with 0.5 sec averaging (DIT) time, 2 nm bandwidth, 100 nm/min scan speed, two accumulations and 0.2 nm step. Temperature was changed from 4 to 90 °C with 1 °C interval, and 0.4 °C/min rate. These parameters lead to the overall acquisition time of 1h per 10 °C temperature change. All data collected in this manner were examined for the presence of possible intermediates during the melting process. However, the overall shape of the CD signature remained unchanged for every sample examined. Thus after completion of the full wavelength scan for each sample additional melting data were collected at specified wavelengths of 264 nm (characteristic for a parallel G4) and of 330 nm (used as a reference to factor out instrument fluctuations), the averaging time 32 s, and the bandwidth was 2 nm.

Temperature was changed from 4 ° C to 95 °C and back at 1 °C/min rate. This set of experiments allowed us to test the reversibility of the melting process and to obtained thermodynamic parameters.

II.6. Native gel experiments

Native gel experiments were performed using 8% acrylamide/bisacrylamide (19:1 ration) gel containing 10 mM KCl and 1X TBE buffer with a running buffer containing also 10 mM KCl and 1X TBE buffer in native conditions by cooling down the system at 4°C. A DNA ladder containing polythymine samples with different lengths (9 to 90 nucleotides) have been used. Each sample contains 25 µM of DNA and different equivalents of UP1 and ligands. Before loading samples, a migration was performed in order to eliminate impurities. Then samples were loaded and migrated for 3H hours. Stains all coloration was used to revealed oligonucleotides bands.

II.7. Isothermal titration calorimetry (ITC)

Isothermal titration calorimetry (ITC) experiments were performed using a Microcal VP-ITC instrument (GE Healthcare) at 37°C. All samples were thoroughly degassed while being stirred prior to use. Titrations were conducted with the different DNA sequences (KRAS32R WT, G9T, G25T and double mutant G9TG25T. For the ITC titrations, the sample cell was filled to capacity with a dilute solution of UP1 at 10 μM and titrated with DNA at 100 μM. A typical titration involved the injection of 16 (6-12 μL) [START_REF] Paulus | RNA stem-loop enhanced expression of previously non-expressible genes[END_REF] C site-specific lowenrichment labelling for imino protons. The resonances of the KRAS22RT G4-ligand complex were assigned through the peak assignments of the free G-quadruplex by using exchange cross-peaks (in NOESY) between the free and ligand-bound species obtained at 2 equivalents of ligand. Interproton distances were measured by using NOESY experiments.

II.8.1. NMR ligand titrations

Concentration of oligonucleotide samples used were 350 µM. Ligands were added at different equivalent increments depending on the ligand studied. Then 1 H 1D spectra were recorded after mixing oligonucleotide with ligand.

II.8.2. NMR ligand competition experiments

Samples contained 350 or 500 µM of KRAS22RT oligonucleotide (depend on the experiment).

For the competition assay without ligand, 500 µM of 22RT complementary strand were added and sample put immediately in the spectrometer to measure spectra at different period times after ligand addition and follow kinetics of duplex formation and G4 disappearing. For completion with ligands, before adding complementary strand, we included 2 equivalent of ligand. Then, we selected an unchanged peak, usually an isolated aromatic peak (before and after addition of complementary strand) to scale all different spectra and integrate the duplex region. We converted the integration data into concentration using the initial concentrations of 22RT and its complementary strand. Then we plot duplex concentration in function of time.

To fit the data, we use a simple model for hybridization of two DNA independent chains in an exponential model equation ( 1):

𝑦 = 𝑎 * (1 -exp(-𝑘 * 𝑥))
where a is the maximal concentration of duplex formed and k the speed constant of the duplex formation.

We also determined t1/2 duplex using a second order equation ( 2):

t = 1 2 * k * C 0

II.8.3. 2D 1 H-15 N HSQC NMR UP1 titrations

Kinetics for the interaction between UP1 and KRAS32R conformers G9T and G25T have bene followed by using 2D 1H-15N HSQC NMR with 15N, 13C isotopically enriched samples of UP1 allowing us to identify each residue with its backbone -NH connection. Assignment was done by using the deposited data from PDB structure 1L3K. Then we added increasing amounts of G4 samples (0.25, 0.5, 0.75, 1 equivalent of 32R G9T or G25T) and we followed peaks shifting.

We calculate shift for several peaks using the equation ( 3):

𝛿 = √(𝛿𝐻²+(( 𝛿𝑁 5 )²)
with δH the shift in 1 H dimension and δN the shift in [START_REF] Balbontin | Expression of IroN, the salmochelin siderophore receptor, requires mRNA activation by RyhB small RNA homologues[END_REF] N dimension. Shifts were then plotted in function of the corresponding residue in Origin 8.6

II.9. Cell Viability Assay and Cellular Imaging

II.9.1. Cell Viability Assay

HeLa, NHDF non-malignant cells (normal human dermal fibroblasts) and KRAS-mediated cancer cells were seeded in 48-well plates (5×104 cells/well) and exposed to various concentrations of compounds. After 72 h, 50 µL of 1 mg/mL methyl thiazolyl tetrazolium (MTT) solution was added to each well, and the cells were further incubated for 4 h in the dark at 37 °C. At the end of the incubation period, dark purple formazan crystals were formed. These crystals were solubilized in dimethyl sulfoxide (DMSO) (250 µL per well for 10 min), and the absorbance was measured at 570 nm using Biorad xMark™ microplate reader. The experiment was repeated three times. The IC50 values were derived from the curves of the cell viability against the compound concentration. The extent of cell death was expressed as the percentage of cell viability in comparison with control cells.

II.9.2. Cellular Imaging

The HeLa cells were grown on μ-Slide 8-well plates from ibidi at a density of 3 × 104 cells/well for 24 h at 37 °C in a 5% CO2/air incubator. Cells were then incubated with 10 µM of AG and 1 µM of TriPropil for 3 h. Following incubation, cells were washed three times with PBS and stained with 1 µM of Hoechst 33342 (nuclear stain) for 15 min. Confocal images were recorded using a Zeiss AxioObserver LSM 710 confocal laser scanning microscope and ZEN software (Carl Zeiss Microimaging, Gottingen, Germany), using 4,6-diamidino-2-phenylindole (DAPI), green fluorescent protein (GFP) and Rhodamine/Texas Red channels at 405-, 488-and 514nm laser excitation, respectively. An appropriate emission band was selected for the three fluorescent channels by using a Plan-Apochromat 63×, 1.4 N.A. oil immersion objective (Carl Zeiss) to ensure high-resolution images. Images were processed with LSM Image Browser (Zeiss) and Adobe Photoshop.

III. KRAS22RT G-quadruplex ligands study

Besides the study that we conducted with 22RT sequence, other studies have been performed by different laboratories using our deposited structure [287] , including our collaborators with Carla Cruz lab (Annexes Article 2). In this study, a novel panel of acridine orange (AO) derivatives was tested against KRAS22RT G-quadruplexes. These ligands have an AO moiety with an aliphatic side chain with a different number of ethylene groups and an iodobenzene moiety. Overall, all ligands from the AO series, i.e., C3, C5 and C8 named according to the number of carbons in the arm between the acridine and the iodobenzene group, show good results in term of thermal stabilization: 18, 29, and 40°C for C3, C5 and C8 respectively, obtained using CD melting experiments. However, their specificity for KRAS G-quadruplex when tested against DNA duplex was very modest. In cytotoxicity assays, these ligands show high toxicity against HeLa cancer cells but also against non-malignant cells which has to be improved for the next generation of AO ligands. A positive point observed by confocal microscopy, is their good capacity to cross the cellular membrane and localize in the cell nucleus. This series of ligand and especially C8 seems to be good candidates for KRAS22RT Gquadruplexes as several other ligands we also tested.

III.1. FRET melting screening

The first step in ligand studies against G-quadruplexes is to screen a large number of ligands from different families with FRET melting assay (Figure 34). For a matter of simplification in the analysis we decided that the temperature cut-off will be the following: < ≈5 °C, low stabilization properties, not worth investigating; 5 -12 °C, average stabilization effect, worth pursuing studies; > 13°C good stabilization properties and worth testing by 1D NMR experiments). From the figure 34 we can observe that the lowest stabilization effects have been observed with 38Phen, 20A, NaphTrip and IP5 (0, 2, 1.5 and 3.5°C respectively, at 2 molar equivalents of ligand). As a consequence, these ligands cannot be considered as good stabilizers for 22RT as well as JL205 which gave a stabilization temperature of 5.5°C. Braco19 can be considered as a good stabilizer (20°C) for 22RT. Among the salphen, family we tested several modifications such as aliphatic chain length, the nature of the coordinated metal, and the presence of additional chemical substituents. The ΔTM for AG, BG, DG and HG are respectively 9.5, 6.5, 14.5 and 3.5°C at 2 ligand equivalents. Regarding the results, the most effective modification if we consider AG as the reference compound, is the addition of a dimethyl imidazole group (Figure A1). With this modification, an extra planar conjugated system is available to interact via π-π stacking and can explain this increased stabilization. We also tested three different phenantroline ligands from the same family as PhenDC3 which is known as one of the best ligand for G4 [288,289] . All of them show relatively low stabilization temperature (9°C for 32Phen which is the best one) compared to PhenDC3 which means that this kind of modifications are not beneficial for 22RT G4 stabilization. These low stabilizations could be explained due to the size of 32Phen and 38Phen ligands with their long aliphatic chains. In the case of 16Phen, the absence of positive charges is probably the main reason for the low ΔTM observed. We finally used ligands from NDI family. They all gave good stabilization results as they all have a ΔTM superior to 15°C, the best one is TriPropil with a stabilization of 25°C which is better than PhenDC3. Among this family we chose TriPropil as the reference ligand on which modifications have been done such as addition of tertiary amines or side chains. Regarding the results, these modifications decreased the stabilization and did not increase specificity toward duplex DNA (Figure A2). All the other ligands did not show significant DNA duplex stabilization except DG.

After this first screening, the most promising ligand for 22RT G-quadruplex are PhenDC3, AG and DG from salphen family, TriPropil from NDI family and Braco19. Considering the DNA duplex stabilization obtained for DG we decided to use AG as principal ligand from this family to pursue further NMR studies.

III.2. Ligands effect on KRAS22RT G-quadruplex by CD titrations

In parallel to FRET melting experiments, we also perform Circular Dichroism titrations to observe the effects on the conformation of 22RT G4. We looked at the modifications on the two characteristic peaks at 260 and 243 nm of parallel conformation obtained for 22RT. Figure 35 is showing spectra of the best ligands identified in FRET melting experiments. The remaining spectra are presented in Figure A3. All CD titrations with all the ligands are showing similar results. Indeed, it is clear from observing the spectra that all the different ligands tested did not change 22RT G4 conformation. However, we could notice some slightly change with a decrease of the signal at 260 nm and also at 243 nm in spectra which could be a hint for an interaction but it is only a qualitative observation. The only exception is PentaNMe2 ligand because in the spectrum, we can observe a more pronounced decrease in intensity and a slight shift at 260 nm and 243 nm than the other ligands, together with an increasing at 290 nm, a characteristic signal of antiparallel conformation. This result means that this ligand seems to be able to change part of 22RT conformation. We also performed NMR titrations with ligands against KRAS22RT G-quadruplex. These experiments could provide useful information. First, as the assignment of KRAS22RT imino region is known, we could follow the peaks shifting by increasing the amount of ligand. Then, NMR titrations could be analysed to determine the exchange between the G-quadruplex free state and ligand bound state (Figure 36).

III.3. NMR titrations

In the case of slow exchange indicating a tight binding, transition is quite difficult to observe in the NMR signature with only initial and final states. Signals are disappearing and reappear only when saturation state is almost reached. In the case of fast exchange indicating a weak binding, we can easily follow the peaks when other factors such as aggregation do not broaden the peaks of interest. Intermediate exchange is characterised by a mix between the two others with peaks that almost disappear in a non-resolved region and that reappear faster than slow exchange. Another crucial information that can be obtained from the NMR titrations is the binding affinities for each ligand. Unfortunately, many of the tested ligands do not allow to correct follow individual peaks due to the exchange nature of the ligand complex, but also due to severe ligand-induced aggregation when interacting with DNA in a nonspecific manner.

Finally, the last information we can extract from NMR titrations is the possibility of performing structural studies on the complex in 2D dimensional NMR depending on the quality of the spectrum during titrations. Among all the tested ligands, we selected the ones with the best ability to stabilize G4 and also some other criteria such as solubility, their chemical family, cytotoxicity and imino peak dispersion. Without a clear imino peak dispersion we would not From combining good stabilization with best 1D NMR peak dispersion, AG had the best results.

All 12 imino peaks were well resolved indicating a single conformation in complex with 2 ligands molecules, probably with two independent binding sites. Looking at the profile of the transition, it seems that AG induces an intermediate exchange of 22RT. For the case of both TriPropil and Braco19 the imino profile did not had the 12 well resolved peaks as expected, Figure 37. NMR titrations performed with the best stabilizers with increasing amount of ligands, depending of the saturation in each complex only a few peaks could be distinguished (around 6). Looking at the different transitions, we also have intermediate exchanges. For TriPropil, JL205 and Braco19 saturation is respectively reached at 2, 3 and 2 equivalents of ligand suggesting the corresponding number of binding sites. Among the other tested ligands, PhenDC3 which has one of the highest ΔTm values, did not produce a profile that could be properly analysed as the imino region became quite broad with no resolved peak. To the exception of AG that was a limiting factor for pursuing 2D NMR studies with different ligands. We could observe similar results with PentaNMe2. For the case of NaphTrip and IP5 we can observe sharp peaks up to 4 molar equivalents, and then an aggregation phenomenon where most probably the oligo desolvates and precipitates.

Nevertheless, those ligands have not good ΔTm values and will be used in further studies. In the case of 20A and 16Phen, we could assume that ligands could partially unfold the Gquadruplex looking at the imino region but it is also possible that it is still in a transitional state with an unfinished titration, so the results need to be completed. As a results of these titration experiments, together with melting studies, we found that the best ligand for NMR structural studies in complex with 22RT was AG.

III.4. NMR competition experiments

In G-quadruplexes studies, it is not unusual to use competitors such as duplex DNA to probe the ligand efficiency to prevent hybridization of the G4 sequence with the complementary sequence. That is why we thought about performing NMR competition experiments in order to see the G4 protection from the complementary chain for each ligand and at same time get information about the first and the last tetrad to be unfolded. The complementary sequence represents the best competitor as it is capable of unfolding G-quadruplex by forming the corresponding double stranded DNA in cells. NMR would allow us to follow the disappearance of G-quadruplex along with the appearance of the corresponding duplex (new peaks in the region of 12.5-14 ppm). We wanted to know if the ligands we studied were also able to maintain the G4 integrity despite the presence of the complementary chain. By integrating the peaks corresponding to the duplex region at different periods of time we were able to determine the association constant of 22RT with its complementary strand to form a duplex.

We also determined duplex formation half-life time (t1/2 duplex) by using second order reaction equation. NMR competition results without ligand used as control are presented in In the case of competition assay without ligand, the duplex formation is a very fast event since after 32 minutes the G4 is almost totally unfolded and hybridized. As a result of the data fitting using equation ( 1), we obtain a t1/2 of 19 minutes without ligand, which can be used as reference to assess the efficiency of a ligand to protect G-quadruplexes against complementary strand. Figure 39 shows the results we obtained with AG and TriPropil ligands.

Even if TriPropil is a better stabilizer than AG in terms of Tm, in NMR competition experiments, AG had the best protector properties and the longest with a t1/2 of 170 min (k = 5.87x10 -3 ±8.12x10 -4 mM.min -1 ) whereas TriPropil had a half-life time of 46 min (k = 31.0x10 - 3 ±3.27x10-3 mM.min -1 ). Compared to the control experiment, TriPropil only slightly increases the protection of the G-quadruplex meaning that it can well stabilize 22RT G-quadruplex but it is not able to avoid duplex formation in presence of complementary strand. AG is not the best stabilizer but increase the protection by almost a factor 10. We also tested several other ligands presented in Figure A5 JL205 and NaphTrip. For NaphTrip, the obtained half-life is even worst compared to TriPropil which means that this ligand is no good in stabilization or protection neither. JL205 gave an average result with a t1/2 of 90 min increasing the protection of the G-quadruplex by a factor 4.5. Although we expected the hybridization profile (corresponding to duplex signal) to be similar with the different ligands, it was obvious that it was influenced and target by each ligand because the peaks profiles were quite different. concentration, the cell viability is 59 % suggesting a modest selectivity for cancer cells. As a conclusion, none of these ligands is specific to cancer cells.

III.5. Cytotoxicity assays and cellular imaging

Indeed, we studied G-quadruplex formed within KRAS promoter region that is why we decided to test again our ligands but in KRAS-driven cancers cell lines A549, H23, H358 and H460 respectively with mutations in Kras G12S, G12C, G12C and Q61H. The last one has been chosen in order to study another mutated residue which is not G12. In this assay, we tested as before AG and TriPropil but also PhenDC3 which is a good stabilizer and C8 the best ligand we tested (Figure 43). These experiments have been done with our collaborators at IECB. AG and TriPropil showed similar results that the ones obtained in HeLa cancer cells with TriPropil which is too toxic with IC50 under nanomolar range and AG which is not enough toxic with IC50 show interesting results with IC50 around 1 µM for PhenDC3 and C8 with an IC50 in the nanomoles range which corresponds to the desired value for an IC50. Unfortunately, C8 also showed similar toxicity for NHDF cells in the previous studies. However, another critical point to assess whether a molecule is a good candidate considered as a potential drug is the cellular uptake and localization. Among all the G-quadruplexes ligands that have been tested cellular uptake remain a major issue and need to be improved. That is why we tested AG and TriPropil to see their localization by cellular imaging in HeLa cancer cells with confocal microscopy (Figure 44).

We were able to study localization of these two compounds thanks to their fluorescence properties. AG emits green and TriPropil red fluorescence, upon excitation at 488 and 514 nm, respectively. Even if AG is a poor fluorophore compared to TriPropil, the emission is sufficient to clearly identify their cellular localization by merging with nuclear staining fluorescent compounds. As a results, we could identify TriPropil localization in the nucleus, whereas AG is Looking at the structures of the two ligands, we can hypothesize that the cause can be related to both the aliphatic chain which makes AG longer and the Ni 2+ cation. Taken together, the biophysical preliminary studies with different ligands allows to select a few ligands worth solving the structure in complex with 22RT. Among them, we identify AG, TriPropil and C8 as the most interesting ligands. We started the 2D NMR experiments with AG since it had the best peak dispersion in the imino region. To unambiguously identify the position of each imino we proceeded with 1 H-15 N 1D HMQC experiments in different individual samples containing an isotopically labelled guanine at each position.

III.6. Structural studies of KRAS22RT G4 in complex with AG ligand

In order to directly find interactions between both molecules, we first needed to assign proton peaks from each molecule. I was able to identify all 12 imino peaks in presence of AG from the with AG, was the fact that some peaks disappear and reaper later on. So, the 1D HMQC experiment was crucial to identify slow exchange peaks that underwent large chemical shift displacements such as G9 residue which had a chemical shift near 11.2 ppm and moved to 10.7 ppm, experiencing a difference of 0.5 ppm. Then, I proceeded to record 2D NMR spectra such as 1 H-13 C HSQC, 1 H-1 H TOCSY and 1 H-1 H NOESY in order to characterize and assign all possible peaks and compare with free 22RT. Once assignments were completed from an exhaustive observation of different 1 H-1 H NOESY spectra acquired at different temperatures, we were able to assign (as much as possible) cross-peaks between 22RT and AG. In order to be able of doing that it was also necessary to assigned the individual protons of isolated AG samples. Fortunately, imidazole protons such as the methyl group, imid-H2, -H3 and -H4, together with aromatic protons (aro1 and aro2) and the aliphatic chain (-CH2 protons) were simple to identify from 2D NMR spectra. The last proton is located near to the cationic ion Ni 2+

and called HJV (Figure 46). The most important interacting peaks that we could unambiguously assign were imid-H2 and imid-H4 with H1' protons from G3, C5, G6, G7, G11, G12, A14, G18 and G19. Some cross peaks of imid-H2 and imid-H4 were also identified interacting with H3' of C5, G11, G18. Since AG is a symmetric molecule, we only named half of the protons because they are isochronous with the second half. So it is without surprise that we have several cross peaks from AG with so many different bases.

Considering the interaction pattern and the number of ligand used until saturation, AG is likely interacting in two different binding sites that I was not able to solve. Further studies need to be performed before we could decipher the exact location of both binding sites. After validating the list of cross-peaks with AG a molecular dynamics run over several hundreds of ns would probably be necessary to understand correctly the binding mode of the different chemical moieties.

IV. KRAS32R G-quadruplex ligands study

By studying ligands against 22RT structure, we identified good candidates that could be used for ligand development. But before that we need to test them against longer sequences from KRAS that have also a propensity to fold into G4 structures. So we studied the same ligands against 32R, which is also a parallel G4 but contains a large propeller loop with 11/12 bases, depending on the conformer. We were expecting that the interaction behaviour could also be different. We tested the previous ligands against 32R wild type, G9T and G25T conformers. By looking at the results with the three different sequences, we wanted to know if we could find the same trend in the WT as in both G9 and G25 conformers or simply if any of the ligands could shift the equilibria of WT towards a single conformation. Similarly to what we did before, we tested a panel of ligands against all three sequences in order to obtain ΔTM values from FRET and finally 1D NMR titrations. Then AG and DG gave similar results (between 10 and 15°C for G9T and G25T and between 15°C and 20 and between 15°C and 20°C for WT at 5 equivalents). Still from the salphen family, BG is the ligand that gave the poorest results. Finally, the last ligand we tested so far in FRET melting, JL205 reported poor results for G9T and G25T and gave correct results for WT (around 15°C at 5 equivalents). After analysing the results in figure 48, a major observation that can be made is that all ligands gave a better stabilization with the wild type sequence. We can hypothesize that we were measuring the stabilization of the multiples conformations that could form aggregates in presence of ligands making some sort of macromolecular complex difficult to interpret. Results obtained with 32R G-quadruplexes are similar to 22RT for PhenDC3, TriPropil and AG ligands. As we obtained similar results with these ligands, we also wanted to assess the stabilization of C8 ligand which was the ligand with the best results in terms of ΔTM with 22RT. As we could not use FRET melting we performed CD melting experiments (Figure 49). Results from this experiment show that C8 is capable of stabilizing 32R G-quadruplexes but not as efficiently as 22RT. Even if the stabilization is lower than the one observed with 22RT (around 40°C at 3 equivalents) it could stabilize 32R WT, G9T and G25T with a respectively temperature of 14, 10 and 19°C at 5 ligand equivalents. It seems that C8 could stabilize with a better efficiency WT and G25T compared to G9T and this seems to be inversely proportional to TM values of each sequence without ligand. This phenomenon was also observed in FRET melting experiments but the difference is that in CD melting the stabilization for WT at 5 equivalents seemed to be an average between G9T and G25T values.

IV.1. Stabilization assays against KRAS32R G-quadruplexes

Considering the differences between FRET and CD melting experiments, we could assume that our hypothesis concerning aggregation in FRET melting wells may be a valid point. Despite the fact that C8 had lower stabilization effects on 32R G-quadruplexes, it remains a good stabilizer.

In addition, these results also reinforce the hypothesis that C8 ligand interacts in a tight binding pocket in 22RT made by a short propeller loop composed by 4-nucleotide (A14, A15, T16, A17), located near G11 which imino had an important cross-peak with C8 ligand (Figure 5, article 2, Annex). The 32R sequences do have a longer propeller loop that most probably does not make such tight binding pocket. As previously reported, NMR titrations provide useful information by monitoring the chemical shifts of isolated peaks, inspecting possible exchange events and determine if samples have enough quality to be involved in further NMR structural studies. In order to have robust results about the interaction between 32R G-quadruplexes and ligands, we continued to study WT, G9T and G25T in the same conditions. Based in the FRET results and cellular cytotoxicity we decided to use PhenDC3 and C8 ligand as a starting point for the new set of NMR titrations resumed in Figure 50.

IV.2. NMR titrations

From this figure it can be concluded that the interaction with 32R WT sequence induced the formation of multiple indistinguishable peaks. The presence of the two ligands does not induce the appearance of a single conformer and lead to small aggregates. In the case of G9T and C8, the titration depicts a nice profile with well-resolved peaks in which we could clearly

WT G9T G25T

identify the isolated peaks even at 4 molar equivalents of ligands. At ratios off 1:1 (C8:G9T), it seems clear that intensities of the three imino peaks (G2, G6, G11) belonging to the 5' side tetrad together with G25, are severely diminished compared to the remaining residues (G25 is not unambiguously detected in 1D spectra). The behaviour is characteristic of an intermediate exchange processes (µs-ms) and could be indicative of a preferred binding site region. It is difficult to extract more information from 1D spectra, but it shows good quality to proceed with 2D NMR characterization. At the same amount of ligand, G25T showed a better imino profile, with a completely set of unresolved imino peaks, hinting that multiple conformations are in exchange in the NMR time scale. G25T shows severe peak coalescence, which is lower limit of intermediate exchange with multiple unspecific binding events.

Unfortunately, PhenDC3 ligand interaction were not completed up to 3 or more equivalents of ligand. We can observe that for all 3 sequences the co-existence between the free and bound states of the G-quadruplexes that are shifted upfield. In G9T profile, a new set of well resolved peaks is appearing around 10-11 ppm. In G25T profile, even if not so well resolved compared to G9T, we also could distinguish a second set of peaks in the same region whereas WT presents multiple small and undistinguishable peaks. At 1 molar equivalent of PhenDC3, the free state of the G-quadruplex cannot be seen anymore indicating the saturation and as a consequence one preferred binding site. Regarding the evolution of the imino profile, the exchange is at intermediate regime, indicating a medium binding mode in the order of µM.

Considering future NMR structural studies, WT will be almost impossible to follow, G9T could be studied with both ligands but for G25T, only PhenDC3 seems to be a valid candidate.

V. KRAS G-quadruplexes interaction with UP1

In the ligand interaction studies, we were able to identify good candidates that can be used as G4s stabilizers for further structure preliminary studies. Next we wanted to know if the same ligands were capable of interfering with transcription in cancer cells. But before that we need to observe if the same ligands can be used to inhibit the interaction with proteins such as hnRNP A1 in vitro. If ligands could have both stabilization and capping properties, i.e., could prevent transcription factors from binding and unwinding the G4 structures. Before directly using the ligands against KRAS addicted cancer cells, we performed several experiments in order to characterized the interaction between all KRAS G-quadruplexes we studied so far (22RT, 32R, G9T and G25T). Indeed, the role of hnRNP A1 and UP1 has been shown for 32R

WT and 21R [279,281] but we did not have any data concerning the isolated conformers G9T and G25T. In this part, I describe the tests concerning binding and functional properties in presence of UP1, and a possible unfolding mechanism, using PAGE, ITC and NMR.

V.1. Interaction studies between KRAS G-quadruplexes and UP1 by native gel experiments

I performed native gel experiments to assess the interaction between KRAS G-quadruplexes and UP1. This assay would directly allow us to see if there is any interaction with a shift of the band corresponding to G4 in case of interaction. Moreover, by using a ladder including a panel of different sizes we could also have an idea of the stoichiometry of the complex formed. I performed the native gel experiment with 22RT, 32RWT, G9T and G25T. I also included a sequence of 32 residues corresponding the wild type sequence but with several mutations (G into T) in all guanines tract to avoid G-quadruplexes formation (32R mut) (Figure 51). band almost disappeared. For the 32R G4 sequences, we observe similar results with additional complex bands with lower mobility in the gel than those observed for 22RT. We could also notice that UP1 is not specific for G4 sequences. Indeed, with the addition of UP1, the 32R mutated sequence is also forming a complex with the protein with the highest band appearing above the T90 mark. We can conclude that all G4s sequences have a pattern that is compatible with the formation of 1:1 and 1:2 complexes as observed before and the negative control seems to form predominantly a 1:1 complex. The nonspecific interaction of UP1 makes sense as it corresponds to two RRM domains which are known to bind to nucleic acids and not only to G-quadruplexes. Moreover, no sufficient differences can be seen between interaction with 22RT and 32R G4s despite their structural differences. Concerning the unfolding role of UP1, this experiment did not allow us to conclude about the capacity of UP1 to unfold G4s.

V.2. Interaction parameters obtained by ITC

After the confirmation that 32R WT and conformers are capable of interacting with UP1, we wanted to obtain interaction properties such as stoichiometry, KD and other thermodynamics parameters in order to better characterize and understand this phenomenon. Due to low yields in expressing UP1, titrations were conducted with UP1 in the sample cell and DNA in the injection syringe as a reverse titration. In addition to WT, G9T and G25T, we also tested the double mutant G9TG25T known not to form a stable G4 but rather multiple different conformers in slow exchange. So, G9TG25T cannot be used as a pure negative control. The results (Figure 52) showed that UP1 has a moderate binding affinity for the KRAS quadruplexes in the order of µM, i.e., 1.1, 0.5, 0.79 and 1.1 µM for WT, G9T, G25T and G9TG25T respectively. The differences are not significant in view of the experimental errors.

Interestingly, the double mutant G9TG25T has the same affinity for UP1 as the WT. Although there are in the literature examples with both 1:1 and 2:1 ratio (protein: DNA), in our case G9T displays a 2:1 stoichiometry. In the case of WT, G25T and G9TG25T the results deviate from this model and we relate that to the apparent extra degree of flexibility that those sequences seem to have from NMR spectra. The ΔG results have mild differences between all four sequences and the more structural-flexible sequences have a better enthalpic component.

The entropic component seems to be far more important for the better folded model, G9T.

With ITC experiments, we also expected to have information concerning the unfold of KRAS G4s by UP1. However, the ITC experimental data did not allow us draw a definitive conclusion.

Regarding the results, we cannot clearly see a difference between interaction with the different conformers maybe because the way the titrations have been performed (reverse way) cannot reveal the diversity of the interactions with different conformers. I think this work needs to be repeated with the oligonucleotide in the cell compartment and the protein in the syringe.

V.3. Interaction studies by NMR

Next, we were interested in following the capacity of using previously identified ligands to disrupt and prevent UP1 binding to the KRAS G4s. It is more difficult and expensive in terms of preparing [START_REF] Balbontin | Expression of IroN, the salmochelin siderophore receptor, requires mRNA activation by RyhB small RNA homologues[END_REF] N isotopically enriched oligonucleotides so we decided to proceed and express UP1 in E. coli. Isotopically labelling the protein also permits to follow the folding state, i.e., we can observe the formation of the complex. We were only able to do part of the experiments involving each G4 and UP1 to characterize their binding properties. After forming the complex at 1:1 molar equivalents, the sample started to precipitate and the ligand experiments were not conducted.

then we measured again after each addition of DNA. Finally, I superimposed all the spectra to assess shift of the most implicated residues in the interaction (Figure 53 and54).

The spectrum of UP1 alone showed peaks which were separated and well resolved. Upon addition of DNA G-quadruplexes it became more complex and we cannot clearly see all peaks dimension. Among the remaining peaks that could be analysed, some peaks do not shift or disappear. The first ones are probably not involved in the interaction but for the second set of peaks, it is difficult to conclude about their role. Then, when possible, we measured shifts in each dimension for peaks and calculated a global shift with an equation described in Material and methods. All shifts where plotted with the corresponding residue in a graph in Figure 55.

Despite the fact that we missed several residues shifts due to the overlapping of peaks in the spectra, we had enough data to observe that the main regions interacting with G9T and G25T

G4s corresponded to the two RRM domains of UP1. This result is not surprising considering that RRM domain is known to interact with a variety of nucleic acids sequences, and for the case of G9T and G25T with almost a perfect match between them. There is only one exception in the peaks we identified in the interaction which corresponds to Leucine 9. Indeed, this G25T G-quadruplexes residue does not belong to RRM domains but it seems to be highly implicated in UP1 binding and can be coloured-visualized in UP1 structure in Figure 56.

By looking at the UP1 interactions identified with G9T and G25T, we could see that in both cases most of the residues that belong to the RRM domains but the residues involved are not exactly the same. Concerning the peak shift observed for Leucine 9, even it is not part of RRM domain 1, it is structurally close to this motif. Although we obtained some structural information about the interaction, especially in UP1 structure, this experiment does not provide direct observation that UP1 unfold the G4. For this end, we have to perform the same experiments looking at the G-quadruplexes structure instead of UP1, using labelled DNA sequences. Concerning the mechanism of interaction, if we considered a 1:1 stoichiometry, it means that two RRM domains from one UP1 molecule are needed which has to form a kind of "sandwich" with the G-quadruplex. In the case of a 2:1 stoichiometry then four RRM domains from two UP1 molecules are necessary for the interaction thereby for the unfolding process.

V.4. NMR preliminary studies of the unfolding by UP1

As we did not have any information about the unfolding process of G9T and G25T G4 by UP1

we tried to follow the event by 1D NMR experiments, similarly to the experiments we The unfolding process of UP1 is not clear. Ninety minutes after the addition of UP1, we have the impression that very mild or almost no unfolding process did occur with G9T sample. In the case of G25, almost all G4 peaks disappeared, with the exception of the peaks of the central tetrad around 11.3 ppm. Visible changes were 24h after including UP1 in samples. In this case, the G9T spectra remain virtually the same but some peaks in G25T seemed to have reappeared. Even the peaks around 11.3 ppm look more resolved. After several days of incubation at 37°C both G9T and G25T samples looks to start losing peak intensity and resolution. These results indicate that further unfolding is occurring and at same time samples are precipitating. The precipitation can be induced by both protein and DNA unfolding. I did not explore the conditions further due to a lack of UP1. For example, I would like to repeat the same experiment but in conditions where UP1 would be in excess (2:1) molar ration of oligonucleotide. Nevertheless, I was more encouraged with these experiments because G25T in presence of UP1 really looks that was partially unfolded. Since I was expecting a refolding back event it is not surprising the reappearance of some imino peaks after a certain amount of time.

VI. Preliminary results from ligands studies in UP1 interaction

Even without conclusive results about the unfolding role of UP1, assessed the action of ligands against the complex formed between KRAS G4s and UP1. We were obtained preliminary results with native polyacrylamide gel experiments. In these experiments I chose to use PhenDC3 and C8 ligands because they were tested with 22RT and 32R G4s giving good overall results. I included 2 equivalents of each UP1 and ligand (Figure 58). As described before, I observed that a complex is formed with 22RT upon addition of UP1. Upon addition of PhenDC3, the band corresponding to this complex becomes less intense and new bands appeared with higher MW. This means that the ligand induced formation of a high-order complex with the protein and is also capable to protect the G4 by interacting with it. Similar results were obtained with 32R WT and the conformer G9T. For G9T we observed a band with a lower mobility compared to the absence of ligand meaning that PhenDC3 is interacting with G9T. For G25T, it seems that PhenDC3 is not capable of protecting the G4 from the interaction with UP1. In the case of the mutated 32R sequence, PhenDC3 was also capable of partially avoid the interaction with UP1 and looking at the new band corresponding to the sequence in presence of PhenDC3, it is also capable to induce the formation of a more compact structure.

For C8 ligand, I observed that the addition of ligand did not have any effects with oligo/UP1 interaction. However, the ligand seems to enhance the interaction between UP1 and Gquadruplexes probably by interacting and stabilizing the formed complex. In conclusion, PhenDC3 seems to be a good ligand to inhibit UP1 interaction whereas C8 is not a good candidate in this end. We also tested the effect of PhenDC3 on the UP1 interaction by Circular Dichroism (Figure 59). I first recorded the spectrum of 32R WT and the two conformers G9T and G25T. I obtained a parallel signal for all sequences. I then added 2 equivalents of UP1. I observed that the signal corresponding to the protein which appears as a negative signal around 225 nm. However, there were no sign of the G-quadruplex unfolding since the parallel signal remained unchanged. We finally mixed 2 equivalents of PhenDC3. G-quadruplex and the protein signals were severely modified with the disappearance of protein CD signature and the reduction of the G4 signal (about one third of the signal intensity). This observation could mean that the ligand has probably an effect on G4-UP1 interaction by interacting and modifying G-quadruplex structure. the characterization of UP1 interaction with KRAS G4s, we confirmed that this protein is capable of interacting with them and we obtained some structural information about UP1 binding and G25T partial unfolding. Our preliminary studies with ligands showed that it is possible to avoid interaction with UP1 as demonstrated with PhenDC3. However, the mechanism of its action remains unclear and additional experiments such as 2D NMR or ITC need to be performed.

Part 3: Discussion and Perspectives

The overall aim of this thesis was to understand how G-quadruplex from the NHE promoter region of KRAS organizes and folds into a stable 3D structure. Knowing about the 3D highresolution of structures fits the global objective, understanding how molecular machines work. In addition, my project also aims identifying and characterizing new targets related to cancer, where numerous private and public laboratories do huge efforts to develop new drugs against cancer. Since no FDA-approval drugs that directly bind G4s are yet available, Gquadruplexes still need to be validate as important targets. So a global effort must be pursued to assess the relevance of nucleic acid unusual structures and at same time work at the level of drug development since both studies complement each other and help to achieve a steadyprogress in the field. The goal is to design or find existing molecules that are capable of increasing the effect of G-quadruplexes which are known to reduce the efficiency of the transcription process. As G-quadruplexes have multiple putative roles in transcription by directly disturbing the process acting as a roadblock or indirectly by recruiting effectors (activators or repressors), we have two major possibilities. The first one is to find molecules that can bind and stabilize G-quadruplexes, the second one is to find molecules capable of inhibiting interaction between G-quadruplexes and effectors, with ligand that can bind to Gquadruplexes or to effectors. In this work, we focused on KRAS G-quadruplexes and especially the sequence called KRAS32R looking for molecules that can be used as candidates for cancers therapies. We were identified good stabilizers of KRAS32R and inhibitors of interaction with UP1, a hnRNP A1 derivative known to unfold KRAS G-quadruplexes (21R and 32R). Previous studies have identified ligands against KRAS G-quadruplexes, however, there is no structural information concerning this interaction and only few information concerning KRAS Gquadruplexes structure alone. In order to give new insights in the field of KRAS Gquadruplexes, we determined the major scaffold adopted in KRAS32R and we studied the interaction between this G-quadruplex and ligands by also assessing the interaction with UP1.

I. Determination of KRAS32R structure G-quadruplex

In this work, we successfully provided new structural insights by determining two structures of KRAS32R G-quadruplexes at physiological temperature. Indeed, we rapidly saw and confirmed by NMR that KRAS32R is polymorphic adopting several different conformations.

After performing several mutations to understand the G-quadruplexes formation, we proposed a model with two major conformations in equilibrium along with uncharacterized minor conformations (<5%). These conformations, named KRAS32R G9T and KRAS32R G25T, are the two structures we determined. Before using mutated sequences, we tried to isolate one conformation in the wild type sequence by influencing the equilibrium. We tried to change the experiment conditions (temperature, pH or ionic strength). In addition, we performed ligand and protein binding interactions to see if we could induce the formation of a preferred conformation. Unfortunately, I was not able to shift the equilibrium far enough by playing with the experimental conditions. A future work to be done to better understand the exchange between conformers of 32R and characterize a possible intermediate could be to perform temperature-induced folding and unfolding events using 2D 1 H-1 H NOESY type spectra. By looking at the appearance and disappearance of peaks, we could in principle decipher the transition between both states. But those experiments would require to analyze the extremely complex 32R WT in addition to G9T and G25T at different conditions. It would require a steady access to a high field an instrument > 900 MHz. Another possible issue has also to be addressed. In our model, we proposed the two major structures that also coexist with minor confirmations. I identified a sliding in the last tract of guanines in G25T conformation that could lead to the existence of one of these minor conformations. The implication of guanines in minor confirmations is not clear as we cannot observe any other base involved in KRAS32R G-quadruplexes except the ones we already observed and characterized in the G4 core and triad. This observation means that the base exchange leading to minor conformations could be too slow to be observed in our experiments. Thanks to these new insights we have now structural information that could be used to study interaction of both ligands and transcription factors such as the truncated version of hnRNP A1 (UP1). We also studied the structure of KRAS22RT G-quadruplex which corresponds to the major scaffold adopted by KRAS21R. This sequence is a part of KRAS32R and has been shown, as 32R, to have an effect on transcription when it is deleted and it is able to interact with hnRNP A1. In a previous work, KRAS22RT structure has been determined at 20°C and we then studied this structure at 37°C. In addition to the help in the more heavily studies about KRAS32R. The shorter G4 structure of KRAS could be used as structural base to study the effect of ligands and UP1. In this sequence of studies, I also think that now it will be possible to further extend the KRAS sequence and try to understand the G-quadruplex structures that could be formed in longer sequences (5' AGGGCGGTGTGGGAAGAGGGAAGAGGGGGAGGCAGCGAGCGCCG 3') called KRAS44R, which is also known to form G-quadruplexes. When compared to KRAS32R, the additional residues, contain five additional guanines but not in a tract meaning that they will not be implicated in the G-quadruplex formation, and five additional cytosines which could be implicated in the formation of a duplex segment. Thus duplex stem could further stabilize the G-quadruplex formation and influence the equilibrium between G9T and G25T species. Finally, another point that need to be discussed is related to the comparison between the structures I determined in vitro and how these results could be expanded in vivo or ex vivo conditions. A major difference is the staggering amount of possible partners and soft interactions that could emerge in complex media that can be found in cells. The G4 structure and G4 folding could be modulated by proteins such as transcription factors, histones, zincfingers, polymerase and so on. Despite the fact that they could have an effect on G-quadruplex formation and structure, their quantity will also lead to an increased viscosity in the cell which is far from our NMR conditions. One way to simulate this condition is to use polymer such as PEG or Ficoll to increase viscosity in our sample and looked at the effect on our G-quadruplex structure. So, one important aspect that must be investigated is the effect of high viscosity environment in the G4 equilibrium. A similar approach was studied by the group of Prof. N.

Sugimoto (Konan University Japan). They studied the effect of (poly)ethylene glycols (PEG)

with different lengths on G-quadruplex formation. And determined G4 structures in presence of different cosolutes [290] . They found that the G4 core remained unchanged in presence of PEG but they observed several changes especially in loops. Indeed, their results showed that PEGs are capable of interacting with G-quadruplexes mainly due to dehydration. Moreover.

Heddi and colleagues determined the structure of a tetrameric G-quadruplex in presence of PEG [291] . They showed that this G4, which was known as polymorphic, could formed a monomorphic structure in crowed solution. It is therefore reasonable to consider that viscosity may affect our equilibria between G9T and G25T.

Structure determination of KRAS32R conformers, as well as KRAS22RT G-quadruplex at physiological temperature brought new structural features that could be used in interaction study with ligands or proteins. Indeed, we could specifically identify moieties implicated in interaction with both ligands and proteins, which could allow for example the design of better ligands or competitors that ultimately would interfere with G4 metabolism in the cell.

II. UP1 interaction with KRAS G-quadruplexes

In this work, we tried to assess the interaction that takes place between KRAS G-quadruplexes and the hnRNP A1 derivative UP1. As demonstrated by the group of L. Xodo (Univ. Udine -Italy), UP1 is capable of interacting and unfolding KRAS21R and 32R G4s. We decided to test the activity of UP1 on the KRAS G4 forming sequences KRAS22RT, 32R and both G9T and G25T conformers. We showed that UP1 could interact with each of the four sequences to form a molecular complex of different oligomerization states. Nevertheless, it is also capable of interacting with a mutated sequence which cannot form a G4 structure. These results were not so surprising considering that UP1 RRM domains are not very selective in terms of DNA binding sequence. In collaboration with Anne Bourdoncle and Samir Amrane, we performed ITC experiments and showed that UP1 had very similar binding affinities for all four G4

sequences and an unfolded control-sequence. We also found that two proteins, or four RRM domains, are involved in each G4-UP1 complex. In addition, HSQC experiments showed that the interaction with G9T and G25T G4s and isotopically labelled UP1 resulted in very similar results in terms of binding. We were not able to decipher exactly which nucleotides interact with UP1, but in terms of protein surface the results were very similar. Further experiments need to be performed with a non-forming G-quadruplex sequence such as the one we used in ITC. Concerning the unfolding role of UP1, our preliminary studies did not confirm the hypothesis according to which UP1 unfolds G4 structures. However, we only used one equivalent of UP1, and it seems that two proteins may be required to form G4-UP1 complex.

Even with one equivalent of UP1 we saw that G-quadruplexes started to be partially unfolded which could support the unfolding role of UP1. However, these results have to be carefully taken because our sample may aggregate. These experiments need to be repeated with an increased amount of UP1, different incubation times and amounts of K + salt in order to decrease re-folding process of the G4 once unfolded by UP1. Although we had only proven that the UP1 is capable of interacting with KRAS G-quadruplexes, it is enough to start looking for ligands which can inhibit this interaction, which is one of the main objectives of my research work.

III. Ligands binding studies with KRAS G-quadruplexes

My research time was also focused on the screening of ligands with high affinity for the KRAS G4 sequences. We first focused on 22RT since we completed the structure calculations on the smaller G4 from KRAS and we then started our investigations on 32R WT, G9T and G25T sequences. In the initial screens for ligands against 22RT, we tested a pool of compounds representing a large spectra of chemical families. Compounds were selected from FRET melting results and the best compounds in terms of ΔTm were subjected to different biophysical experiments in order to assess not only the G4 stabilization, but also the capacity to protect G4 from the complementary strand, the cytotoxicity and cellular localization. We also investigated peak dispersion in 1D and 2D NMR spectra to continue characterize the structure of a drug-G4 complex. Some biophysical methods were not well suited for ligand binding studies due to many reason: among those methods SPR and ITC were the most deceiving. Nevertheless, I identified candidates showing good results in several of the tests, which allow to proceed to structural studies. For example, C8, an acridine derivative, was able to stabilize both 22RT and 32R (with 40°C ΔTm at 2 equivalents for 22RT and 20°C ΔTm at 5 equivalents for G25T for example), was also good to protect the G4 against duplex hybridization, and was also capable of entering the nucleus and killing KRAS-driven addicted cancer cells. Unfortunately, it exhibited a high degree of cytotoxicity in non-malignant cells.

TriPropil, from (Naphthalene DiImide) NDI, showed very good results in stabilization and cellular penetration (being localized in the cell nucleus), but not a good capacity to protect 22RT G4 from complementary strand hybridization. It was also found to be cytotoxic in normal cells. Regarding the NMR titrations results, both ligands showed a decent peak separation that would render them capable for further structural studies in order to determine the complex with 22RT G4. One of the best ligands that showed an excellent profile for NMR structural studies was a Ni-Salphen derivative (AG). This ligand only showed average results in stabilization and protection from complementary strand hybridization and it was not efficient in entering the nucleus, leading to poor cytotoxicity properties for both normal and cancer cells. However, we started to study this ligand to identify chemical moieties involved in the G4 interaction. Based on these results, we could modify the ligand to remove the useless parts for the interaction and include chemical moieties that would for example, improve passage through the membrane and localization to the nucleus. The same experiments need to be repeated with the longer KRAS sequences. This global strategy needs to be used for other ligands such as C8 and TriPropil to increase their selectivity against KRAS G-quadruplexes. This is the first point I want to discuss. Indeed, most of the actual studies are focused on inspecting ligands for their stabilization and cytotoxicity capacity, and modified them depending on the results obtained. These modifications comprise often the addition of aliphatic arms or the modification of the metal cation but without structure-based strategy. Within all studies that have been performed with ligands against G-quadruplexes (KRAS or not), several good candidates have been identified. It is necessary to performed structural studies with the best identified candidates to improve their properties or design new derivatives to be close to be the "perfect" ligand. This approach "rational structure-based drug design" is well stablished in other segments of drug development such as in the case of kinases where several block buster drugs were design such as Imatinib or the antiviral Zanamivir. There are still too many studies performed so far looking for good ligands against G4s structures focused primarily in stabilization studies obtained from screening large libraries with a big "fishing net". The results

have not been good at all and I assume that it is not the proper way to tackle the problem. I understand that this strategy is also the result of lack of proper numbers and quality of structures of G4s. Fortunately there is also some excellent research that uses ligands in original and relevant ways such as using a mixture of several molecules in order to maximize the effect against tumors. The main idea is to select several good candidates and improve their effects thanks to structural studies to maximize their potential. I think that in the future we will be find very good G4 ligands that will have the potential to be included in a "drug-cocktail" in order to improve the synergy between drugs and maximize the chance of tumor-growth inhibition.

IV. Global conclusion

We provided novel structural insights concerning the organization of G-quadruplexes in the NHE region of KRAS. I solved the structure of three G-quadruplexes by NMR methods, including a small and two large G4s that are in equilibrium under in vitro conditions. The structure determination of 32R was considered as a huge challenge for years among our peers.

We also used the previous structure of KRAS22RT at 20°C to translate this structure at physiological temperature also. Any of these three structures have the potential to be used for rational drug design. Now we have a target we could focus on to develop ligands with increased affinity for KRAS G4s. I believe this is a major step for studying the complexes between these G-quadruplexes and ligands that have been identified as good candidates in order to extract information concerning the implication of the different moieties, and improve ligand selectivity. We started to perform studies in order to find good ligands and obtain some structural information with a complex formed between KRAS22RT and ligands (AG, C8). The next steps of this work will be to continue looking to the complexes formed with KRAS Gquadruplexes and good ligands at atomic level and improve the salphen and the acridine derivatives. For KRAS32, more ligands need to be tested especially with NMR titrations to identify good candidates for further NMR structural studies. This work can be used in drug design applied to KRAS G-quadruplexes and can help to solve the major societal problem represented by pancreatic, lung or colorectal cancers. These cancers are among the deadliest cancers in the world and this work could be an additional step to fight their cancer growth and tumor spreading. Résumé : L'oncogène KRAS code pour une protéine GTPasique hautement mutée qui agit comme un « interrupteur » entre des états actifs et inactifs, un mécanisme important dans les processus comme la réplication ou la prolifération cellulaire. Quand ils sont dérégulés, ces processus sont à l'origine des cancers. Les mutations de KRAS sont particulièrement impliquées dans les cancers des poumons (30%), colorectaux (44%) et pancréatiques (97%). Malgré le fait que ces mutations soient bien connues, aucune molécule ne cible KRAS car toutes les stratégies actuelles ne sont pas assez efficaces pour les thérapies contre le cancer. C'est pourquoi de nouvelles stratégies ont émergé il y a quelques années visant directement la région promotrice de KRAS et plus précisément des structures appelées Gquadruplexes (G4). Même si le phénomène n'est pas encore parfaitement compris, de nombreux exemples dans la littérature montre que ces structures peuvent se former in vitro et dans les conditions cellulaires. Il a été montré que les G4 formés dans la région promotrice de KRAS peuvent lier des facteurs de transcription et perturber le processus en agissant comme un bloc lorsque l'enzyme vient lire la séquence. La stabilisation ou la destruction des G4, en utilisant de petits ligands chimiques par exemple, pourrait devenir une nouvelle voie de thérapie. Ce travail se concentre sur une séquence de 32 résidus (KRAS32R) qui peut former un G4 et correspond également au domaine minimal d'interaction de certains facteurs de transcription comme MAZ ou hnRNP A1. Cette dernière est capable de lier les G4 de KRAS32R et de les défaire favorisant ainsi la transcription de KRAS. Ce projet vise à comprendre la formation du G4 de KRAS32R au niveau atomique ainsi que son interaction avec de petites molécules organiques qui pourraient agir sur la transcription

Mots clés : G-quadruplexe, RMN, Ligands, Cancer, KRAS Title: NMR structural studies of G-quadruplexes nucleic acids from KRAS and their interaction with ligands.

Abstract: KRAS gene codes for a highly mutated GTPase protein acting as a « switch » between an active and an inactive state, a mechanism found to be important in biological processes such as cell replication and proliferation. When misregulated, these processes are found to be at the origin many types of cancer. KRAS mutations are particularly implicated in lung (30%), colorectal (44%) and pancreatic (97%) cancers. Despite the fact that those mutations are well known, KRAS is still an undruggable target because all the actual strategies (RAS activator inhibitors, membrane association inhibitors, and so on) are not efficient enough as cancer therapies. That is why new strategies have emerged recently, such as directly targeting the KRAS promoter region and especially some specific structures called G-quadruplexes (G4). Although we do not understand well the phenomena, there are plenty of evidence in the literature that these structures can assemble both in vitro and in cellular conditions. It was shown that G4 within KRAS promoter region can bind transcription related proteins and disturb transcription process acting as a block mechanism when transcription machinery is reading the genetic sequence. Stabilization of these structures, using small chemical ligands for example, could become a new area of therapy. In my thesis work, I am focused on a 32 nucleotide sequence (KRAS32R) which can form G4 and also corresponds to the minimal interaction domain of transcription proteins such as MAZ or hnRNP1. This last protein is capable of binding to KRAS32R G-quadruplexes and possibly unfolding it, favoring the transcription of KRAS. This project aims to understand the folding of this KRAS32R G-quadruplex at atomic level and its interaction with small organic molecules that would have some effect on transcription process.

Figure 1 .

 1 Figure 1. Chemical structure of a nucleotide with the different bases implicated in its composition. .............................................................................................................................

Figure 2 .

 2 Figure 2. Simple view of Chargaff's rule ..................................................................................

Figure 3 .

 3 Figure 3. Structure of the double-helical structure of DNA discovered by Watson and Crick

Figure 4 .

 4 Figure 4. Differences between Watson-Crick base and Hoogsteen base pairing ....................

Figure 5 .

 5 Figure 5. Schematic view of a stem loop structure ..................................................................

Figure 6 .

 6 Figure 6. Schematic view of the formation of a H-type pseudoknot with the crystal structure of a viral RNA pseudoknot ........................................................................................................

Figure 7 .

 7 Figure 7. Schematic view of a kissing complex with the crystal structure of an TAR-R06 RNA kissing complex.........................................................................................................................

Figure 8 .

 8 Figure 8. (A) Formation of a triplex from a duplex and an external TFO; and in (B) Representation of the different possible motifs ......................................................................

Figure 9 .

 9 Figure 9. C-C base pairing with the protonated cytosine and schematic representation of the i-motif intercalation with TCCT as a simple model ..................................................................

Figure 10 .

 10 Figure 10. Examples of i-motifs with four, two and one strand ..............................................

Figure 11 .

 11 Figure 11. Representation of the G-quartet with the four guanines linked in a Hoogsteen hydrogen bonds network .........................................................................................................

Figure 12 .

 12 Figure 12. Examples of different G-quadruplexes depending on the number of implicated strands, their orientation and their connectivity .....................................................................

Figure 13 .

 13 Figure 13. Representation of anti and syn conformations for guanosine ...............................

Figure 14 .

 14 Figure 14. Several examples of telomeric G-quadruplex structures .......................................

Figure 15 .

 15 Figure 15. Hypothesizes about the putative role of G-quadruplexes during transcription in the promoter region .................................................................................................................

Figure 16 .

 16 Figure 16. G-quadruplex structures from several oncogene promoter regions .....................

Figure 17 .

 17 Figure 17. Principle of Antisense Oligonucleotides in therapeutics ........................................

Figure 18 .

 18 Figure 18. Strategy using siRNA leading to the degradation of messenger RNA (mRNA) .......

Figure 19 .

 19 Figure 19. CRISPR-Cas9 technology principle leading to DNA editing .....................................

Figure 20 .

 20 Figure 20. Schematic view of the putative role of triplex formation in transcription .............

Figure 21 .

 21 Figure 21. Examples of the most studied ligands for G-quadruplexes stabilization ...............

Figure 22 .

 22 Figure 22. Examples of the most studied ligands designed to block interaction between Gquadruplexes and associated proteins ....................................................................................

Figure 23 .

 23 Figure 23. Kras signaling activation in EGF pathway ................................................................

Figure 24 .

 24 Figure 24. Overview of strategies in KRAS-driven cancers ......................................................

Figure 25 .

 25 Figure 25. General principle of synthetic lethality in KRAS-driven cancers .............................

Figure 26 .

 26 Figure 26. Identification of the three main G-quadruplex forming sequences within KRAS promoter region .......................................................................................................................

Figure 27 .

 27 Figure 27. The ten lowest structures deposited on PDB under code 6T51 with a table containing restrains ..................................................................................................................

Figure 28 .

 28 Figure 28. Comparison between KRAS22RT G-quadruplex at 20°C and 37°C .........................

Figure 29 .

 29 Figure 29. KRAS32R wild type relaxation measurements at 37°C in 1X buffer .......................

Figure 30 .

 30 Figure 30. Different NMR methods that can be used for dynamics studies............................

Figure 31 .

 31 Figure 31. Schematic view of our model for the formation of KRAS32R G-quadruplex with G9T, G25T and the sliding phenomenon in equilibrium with other minor conformation ....

Figure 32 .

 32 Figure 32. Concept of Drug Design against KRAS22RT G-quadruplex ...................................

Figure 33 .

 33 Figure 33. Schematic view of hnRNP A1 and UP1 proteins ...................................................

Figure 34 .

 34 Figure 34. FRET melting experiments with a panel of ligands against KRAS22RT G-quadruplex ................................................................................................................................................

Figure 35 .

 35 Figure 35. CD titrations of the best ligands identified in FRET melting experiments against 22RT G-quadruplex .................................................................................................................

Figure 36 .

 36 Figure 36. Different profiles we can observe in NMR titrations with ligands to determine the type of exchange we encounter .............................................................................................

Figure 37 .

 37 Figure 37. NMR titrations performed with the best stabilizers .............................................

Figure 38 .

 38 Figure 38. NMR competitions results (without ligand) ..........................................................

Figure 39 .

 39 Figure 39. NMR competitions results for AG and TriPropil with NMR spectra and the corresponding fit ....................................................................................................................

Figure 40 .

 40 Figure 40. NMR competitions results for C8 an acridine orange derivative ligand ...............

Figure 41 .

 41 Figure 41. NMR competitions results for all ligands tested in this study ..............................

Figure 42 .

 42 Figure 42. Relative cell viability of HeLa cancer cells (first line) and NDHF cells (second line) measured by the MTT assay after 72 h incubation with ligands AG and TriPropil ................

Figure 43 .

 43 Figure 43. Relative cell viability of the different cancer cell line measured by the MTT assay after 72 h incubation with ligands at different concentrations .............................................

Figure 44 .

 44 Figure 44. Confocal microscopy images of the subcellular localization of compounds AG and TriPropil in HeLa cells .............................................................................................................

Figure 45 .

 45 Figure 45. Imino peaks assignment of KRAS22RT in presence of AG ....................................

Figure 46 .

 46 Figure 46. Structure of AG ligand with all protons and the corresponding peaks assigned in 1D 1 H NMR spectrum .............................................................................................................

Figure 47 .

 47 Figure 47. Interactions between AG ligand and KRAS22RT G-quadruplex observed in 1H-1H NOESY spectrum .....................................................................................................................

Figure 48 .

 48 Figure 48. FRET melting experiments with a panel of ligands against 32R G-quadruplexes using wild type, G9T and G25T ...............................................................................................

Figure 49 .

 49 Figure 49. CD melting experiments with C8 ligand against 32R G-quadruplexes using wild type, G9T and G25T sequences ..............................................................................................

Figure 50 .

 50 Figure 50. NMR titrations performed with the best stabilizers .............................................

Figure 51 .

 51 Figure 51. Native gel experiments to assess KRAS G-quadruplexes and UP1 by using different sequences ...............................................................................................................................

Figure 52 .

 52 Figure 52. Thermodynamics parameters obtained from Isothermal Titration Calorimetry bonding studies with UP1 protein ..........................................................................................

Figure 53 .

 53 Figure 53. Superimposition of 15 N-1 H NMR HSQC spectra of UP1 showing each residue with NH bond of the backbone, measured alone and with increasing amount of KRAS32 G9T Gquadruplex .............................................................................................................................

Figure 54 .

 54 Figure 54. Superimposition of 15N-1H NMR HSQC spectra of UP1 showing each residue with NH bond of the backbone, measured alone and with increasing amount of KRAS32 G25T Gquadruplex .............................................................................................................................

Figure 55 .

 55 Figure 55. Plotted chemical shifts with their corresponding residue with a schematic view of UP1 structure .........................................................................................................................

Figure 56 .

 56 Figure 56. UP1 structure with the two RRM domains with the most shifted residues in NMR ................................................................................................................................................

Figure 57 .

 57 Figure 57. 32R G9T and G25T G-quadruplexes imino region after the addition of 1 equivalent of UP1 at different time periods. ...........................................................................................

Figure 58 .

 58 Figure 58. Native gel experiments to study the role of PhenDC3 and C8 ligands in UP1 interaction ..............................................................................................................................

Figure 59 .

 59 Figure 59. CD signal of KRAS32R WT, G9T and G25T upon the addition of 2 equivalents of UP1 and then PhenDC3 ..........................................................................................................

Figure A1 .

 A1 Figure A1. Chemical structures of the ligands used for KRAS G-quadruplexes interaction with their corresponding chemical families ...................................................................................

Figure A2 .

 A2 Figure A2. FRET melting experiments with a panel of ligands against DNA duplex ..............

Figure A3 .

 A3 Figure A3. All CD titrations with the tested ligands against KRAS22RT parallel G-quadruplex performed at 37°C ..................................................................................................................

Figure A4 .

 A4 Figure A4. All NMR titrations with the tested ligands with KRAS22RT parallel G-quadruplex performed at 37°C with increased addition of ligands ..........................................................

Figure A5 .

 A5 Figure A5. All NMR competitions with the other tested ligands with KRAS22RT parallel Gquadruplex performed at 37°C in function of time ...............................................................

Figure A6 .

 A6 Figure A6. Spectra superimposition of AG alone (blue) and KRAS22RT in presence of 2 equivalents AG (pink) .............................................................................................................

  Abbreviations

Figure 1 .

 1 Figure 1. Chemical structure of a nucleotide with the different bases implicated in its composition.

Figure 2 .Figure 3 .

 23 Figure 2. Simple view of Chargaff's rule

Figure 4 .Figure 5 .

 45 Figure 4. Differences between Watson-Crick base and Hoogsteen base pairing

Figure 6 .

 6 Figure 6. Schematic view of the formation of a H-type pseudoknot with the crystal structure of a viral RNA pseudoknot (code PDB: 1L2X)

Figure 7 .

 7 Figure 7. Schematic view of a kissing complex with the crystal structure of an TAR-R06 RNA kissing complex (code PDB:2JLT)

Figure 8 .

 8 Figure 8. (A) Formation of a triplex from a duplex and an external TFO; and in (B) Representation of the different possible motifs depending on the base coming from TFO (purine motif if A or G; pyrimidine motif if T or C). Watson and Crick (red) and Hoogsteen (blue) base pairings are represented

Figure 9 .

 9 Figure 9. C-C base pairing with the protonated cytosine and schematic representation of the i-motif intercalation with TCCT as a simple model

Figure 10 .

 10 Figure 10. Examples of i-motifs with four, two and one strand. Tetramolecular i-motif is the NMR structure of 5' AACCCC 3' (PDB: 1YBL); bimolecular i-motif is the NMR structure of the dimer 5' CCTCACTCC 3' (PDB: 2AWV) and intramolecular i-motif corresponds to the NMR structure of 5' CCTTTCCTTTACCTTTCC 3'

Figure 11 .

 11 Figure 11. Representation of the G-quartet with the four guanines linked in a Hoogsteen hydrogen bonds network (red)

Figure 12 .

 12 Figure 12. Examples of different G-quadruplexes depending on the number of implicated strands, their orientation and their connectivity (loops)

Figure 13 .

 13 Figure 13. Representation of anti and syn conformations for guanosine (purines). For pyrimidines N9 and C4 are respectively N1 and C2

Figure 14 .

 14 Figure 14. Several examples of telomeric G-quadruplex structures PDB codes are (from left to right and from up to bottom): 143D, 1KF1, 2KKA, 2HY9, 3IBK and 5MBR.

Figure 15 .

 15 Figure 15. Hypothesizes about the putative role of G-quadruplexes during transcription in the promoter region

Figure 16 .

 16 Figure 16. G-quadruplex structures from several oncogene promoter regions PDB codes are (from left to right and from up to bottom): 1XAV, 2M27, 2F8U, 2KZD, 6AC7 and 2O3M.

Figure 17 .

 17 Figure 17. Principle of Antisense Oligonucleotides in therapeutics Modified from Nat Struct Mot Biol, Judy Lieberman, 2018

Figure 18 .

 18 Figure 18. Strategy using siRNA leading to the degradation of messenger RNA (mRNA) Modified from Nat Struct Mot Biol, Judy Lieberman, 2018

Figure 19 .

 19 Figure 19. CRISPR-Cas9 technology principle leading to DNA editing Modified from Nat Struct Mot Biol, Judy Lieberman, 2018

Figure 20 .

 20 Figure 20. Schematic view of the putative role of triplex formation in transcription

Figure 21 .

 21 Figure 21. Examples of the most studied ligands for G-quadruplexes stabilization which belong to different chemical families (porphyrin, acridine, fluoroquinolone…)From Molecules, Zhi-YinSun et al., 2019

Figure 22 .

 22 Figure 22. Examples of the most studied ligands designed to block interaction between Gquadruplexes and associated proteinsFrom Molecules, Zhi-YinSun et al., 2019

Figure 23 .

 23 Figure 23. Kras signaling activation in EGF pathway From Genes & Diseases, Kyle Knickelbein and Lin Zhang, 2015

Figure 24 .

 24 Figure 24. Overview of strategies in KRAS-driven cancers From Trends in Cancer, Meagan B. Ryan et al., 2015

Figure 25 .

 25 Figure 25. General principle of synthetic lethality in KRAS-driven cancers From Genes & Diseases, Kyle Knickelbein and Lin Zhang, 2015

Figure 26 .

 26 Figure 26. Identification of the three main G-quadruplex forming sequences within KRAS promoter regionModified from BBA,Morgan et al., 2016

Figure 27 .

 27 Figure 27. The ten lowest structures deposited on PDB under code 6T51 with a table

Figure 28 .

 28 Figure 28. Comparison between KRAS22RT G-quadruplex at 20°C and 37°C with views from the top and from the side.

Figure 29 .

 29 Figure 29. KRAS32R wild type relaxation measurements at 37°C in 1X buffer. Longitudinal (T1), transverse (T2) relaxations and imino 15 N NOE effect were plotted for each residue of KRAS32R G-quadruplex.

Figure 30 .

 30 Figure 30. Different NMR methods that can be used for dynamics studies with the corresponding time scales and structural changes that can be observed in each experiment. From 10 -12 to 10 3 seconds time scale, NMR provide a panel of tools to study several mechanisms.From Gabriel Ortega, Miquel Pons, Oscar Millet, Advances in Protein Chemistry and Structural Biology, 2013

  and most plausible structures of the 32R sequence in the NHE region of KRAS. The 3D structures could represent important new targets for the development of new drugs targeting the organization of the G-quadruplex in the NHE region and interfere with regulation by proteins such as MAZ or hnRNPA1 transcription factors.

Figure 31 .

 31 Figure 31. Schematic view of our model for the formation of KRAS32R G-quadruplex with G9T, G25T and the sliding phenomenon in equilibrium with other minor conformation

Figure 32 .

 32 Figure 32. Concept of Drug Design against KRAS22RT G-quadruplex by studying the interaction with several ligands from different families and determining parameters such as, pharmacophoric-important chemical moieties, stabilization, overall binding mode or the KD.

Figure 33 .

 33 Figure 33. Schematic view of hnRNP A1 and UP1 proteins with two RRM domains and the glycine rich domain containing RGG box in hnRNP A1 whereas UP1 only contains the two RRM domains

FRET

  melting experiments have been used to screen ligands by studying their capacity to stabilize KRAS G4s. In order to perform the experiment, we need to use labelled sequences with two different fluorophores that are compatible for emission and quenching phenomenon. Quenching is happening when the emission spectrum of one of the fluorophores is overlapping the absorption spectrum of the second fluorophore leading to extinction of the second one. The condition for quenching is that the two fluorophores have to be close in space. Based on this concept, we can use our G4 forming sequences with fluorescein (FAM) as a 5' label and tetramethylrhodamine (TAMRA) as a 3' label observing FAM fluorescence. When the G-quadruplex is formed we cannot observe any FAM fluorescence and the by increasing temperature, we will recover fluorescence because of G4 denaturation which leads to an increased distance between fluorophores. All FRET melting experiments were done with Statagene Mx3005P device. Buffer contains 10 mM LiCaCo, 10 mM KCl et 90 mM LiCl à pH 7,2 for KRAS22RT experiments and we used 1X buffer for 32R experiments. Each well contains 0.2 µM of 22RT sequence or duplex sequence. Ligands are tested at different two concentrations 0.4 and 1µM corresponding to 2 and 5 ligand equivalence conditions. Negative control contains water instead of ligand and we used PhenDC3 ligand as a positive control knowing its stabilizer effects on G-quadruplexes. Each well is duplicated in each plate and experiments were repeated three times per ligand. In the case of testing ligands that are capable of absorbing the same wavelengths as our fluorophores, we would test their stabilization effect by Circular Dichroism melting experiments.

  These experiments have been done in collaboration with Dr. Carla Cruz and her PhD student Josué Carvalho at the University of Beira Interior in Portugal and with David Santamaria and his colleague Benjamin Drogat at European Institute of Chemistry and Biology.

Figure 34 .

 34 Figure 34. FRET melting experiments with a panel of ligands against KRAS22RT Gquadruplex. All ligands have been tested at 2 and 5 equivalent of ligand. ΔTM have been obtained by subtracting TM of negative control in TM of each well.

Figure 35 .

 35 Figure 35. CD titrations of the best ligands identified in FRET melting experiments against 22RT G-quadruplex. We added increasing amount of ligand up to 5 equivalents

Figure 36 .

 36 Figure 36. Different profiles we can observe in NMR titrations with ligands to determine the type of exchange we encounter From BBA, I.R. Kleckner and M.P. Foster, 2011

Figure 38 .

 38 Figure 38.

Figure 38 .

 38 Figure 38. NMR competitions results (without ligand) with the disappearance of imino peaks corresponding to G4 between 10.5 and 12 ppm, and the appearance of peaks corresponding to the duplex formation between 12.5 and 14 ppm.

Figure 40 .Figure 41 .Figure 42 .

 404142 Figure 40. NMR competitions results for C8 an acridine orange derivative ligand tested in a previous study

Figure 43 .

 43 Figure 43. Relative cell viability of the different cancer cell line measured by the MTT assay after 72 h incubation with ligands at different concentrations. Results with different cells lines are plotted in graph and in a table with summarized values

Figure 44 .

 44 Figure 44. Confocal microscopy images of the subcellular localization of compounds AG and TriPropil in HeLa cells after 3 h incubation at 10 and 1 μM concentration, respectively. Cell nucleus was counterstained with Hoechst 33342 (blue)

Figure 45 .

 45 Figure 45. Imino peaks assignment of KRAS22RT in presence of AG using 1 H-15 N 1D HMQC -filtered NMR spectra of samples containing ≈5% of 15 N-enriched isotope

Figure 46 .

 46 Figure 46. Structure of AG ligand with all protons and the corresponding peaks assigned in 1D 1 H NMR spectrum

Figure 47 .

 47 Figure 47. Interactions between AG ligand and KRAS22RT G-quadruplex observed in 1H-1H NOESY spectrum implicating protons from ligand imidazole group and several residues from KRAS22RT

Figure 48 .

 48 Figure 48. FRET melting experiments with a panel of ligands against 32R G-quadruplexes using wild type, G9T and G25T. All ligands have been tested at 2 and 5 molar equivalents of each ligand. ΔTM have been obtained by subtracting the TM of a negative control from the TM of each well

Figure 49 .

 49 Figure 49. CD melting experiments with C8 ligand against 32R G-quadruplexes using wild type, G9T and G25T sequences. The table on the right summarizes the variation of TM in presence of ligand.

Figure 50 .

 50 Figure 50. NMR titrations performed with the best stabilizers with increasing amount of ligands up to the point we started observing aggregate formation in the NMR tube.

Figure 51 .

 51 Figure 51. Native gel experiments to assess KRAS G-quadruplexes and UP1 by using different sequences: KRAS 22RT, 32RWT, 32RG9T, 32RG25T and 32RMut in presence of 1 and 2 equivalents of ligand. A ladder has been added containing several polythymine sequences T9, T15, T30, T45, T60 and T90 respectively corresponding to 2.7, 4.5, 9.0, 13.6, 18.2 and 27.3 kDa.

7 Figure 52 .

 752 Figure 52. Thermodynamics parameters obtained from Isothermal Titration Calorimetry bonding studies with UP1 protein as target and WT, G9T, G25T and G9T G25T sequence as ligands at 37°C with the corresponding curves

Figure 54 .

 54 Figure 54. Superimposition of 15N-1H NMR HSQC spectra of UP1 showing each residue with NH bond of the backbone, measured alone and with increasing amount of KRAS32 G25T G-quadruplex

Figure 55 .

 55 Figure 55. Plotted chemical shifts with their corresponding residue with a schematic view of UP1 structure to identify regions implicated in the interaction with KRAS32R G9T and

Figure 56 .

 56 Figure 56. UP1 structure with the two RRM domains with the most shifted residues in NMR colored (red for strong shifts and orange for medium shifts)

Figure 57 .

 57 Figure 57. 32R G9T and G25T G-quadruplexes imino region after the addition of 1 equivalent of UP1 at different time periods.

Figure 58 .

 58 Figure 58. Native gel experiments to study the role of PhenDC3 and C8 ligands in UP1 interaction. We used KRAS 22RT, 32RWT, 32RG9T, 32RG25T and 32RMut and added 2 equivalents of each UP1 first and the ligand after. A ladder has been added containing several polythymine sequences T9, T15, T30, T45, T60 and T90

Article 1 :

 1 Figure A1. Chemical structures of the ligands used for KRAS G-quadruplexes interaction with their corresponding chemical families

Figure A3 .

 A3 Figure A3. All CD titrations with the tested ligands against KRAS22RT parallel G-quadruplex performed at 37°C

  the most mutated isoform present in 90% of pancreatic cancers, 35-40% of colorectal cancers and 15-20% of lung cancers. As a comparison, mutations in HRAS and NRAS in colorectal cancers are around 5%. The mutated isoform is also present in other cancer types but with less frequency such as cervical or liver cancer. Perkins and colleagues showed that the most frequent mutations in KRAS gene occur at codons 12,13 and 61 and with less frequency at codons63, 117, 119 and 146

  aliquots of titrant with titrant injections made at 400 s intervals. The integrated heat data were corrected for the heat of dilution and blank effects and the corrected data fit for a binding model by nonlinear regression. The binding isotherms were sigmoidal and well fit with the standard one-site binding model incorporated into the Microcal Origin VP-ITC software.

	II.8. NMR experiments
	NMR spectra were recorded on in house Bruker Avance NEO 700 and 800 MHz and 400 MHz
	(Bruker Avance III). In addition, some spectra were collected at CEITEC (Central European
	Institute of Technology, Masaryk University, Brno) of in 850 and 950 MHz spectrometers
	equipped with cryogenically cooled probes (850 and 950 MHz). Experiments were usually
	performed at 37 °C for routine experiments, otherwise temperatures from 4 °C up to 60°C
	were used for different experiments such as NMR melting profiles. For solution NMR, standard
	3 mm NMR tubes were used. The samples were prepared in 1X buffer with 10% D2O. Most of
	the

[START_REF] Dahm | Friedrich Miescher and the discovery of DNA[END_REF]

H 1D spectra were recorded using an echo pulse sequence, which selectively removes resonance due to water without affecting other resonances including those which are in fast exchange with water. Resonance assignments were made using 5%

15 N,

References

Nevertheless, this result needs to be carefully taken since it very difficult to correctly integrate the duplex signal. In the acridine orange paper (Article 2 in Annexes) we identified the C8 ligand as a very good stabilizer with promising cytotoxicity results and an interesting cellular colocalization. The NMR competition assays with 22RT are depicted in Figure 40. We performed titrations using isotopically labelled protein and followed with 2D 1 H-15 N HSQC NMR spectroscopy allowing us to follow the evolution of each protein residue upon the addition of DNA. We could extract chemical shift deviations (Δδ / ppm) for the amide group for the most affected amino acids. We measured HSQC spectrum without G4 sequences and

VII. Conclusions

In this part of the study, we tested a panel of different ligands from several chemical families in order to find good candidates for G-quadruplexes stabilization and prevent UP1 interaction through competition. Among all the ligands we tested, there is no ligand with desired properties. Several of them gave good results in term of thermal stabilization, protection from the complementary strand or UP1 protein, cytotoxicity or cellular uptake. We have tested several interesting ligands, among those PhenDC3 which can efficiently stabilize KRAS Gquadruplex, gives a good IC50 in KRAS-driven cancer cells and can inhibit UP1 interaction.

Another one was TriPropil revealed to be a good stabilizer but it was too toxic. C8 is probably our best ligands regarding its stabilization effect and its capacity to protect KRAS G4s against complementary strand. Unfortunately, it seems that it cannot avoid UP1 interaction, nevertheless it has good cytotoxicity results with good IC50 but not enough specific for cancer cells. Although AG is not the best ligand, we decided to performed advanced structural studies with 22RT because it was the best candidate to this end. We wanted to obtain structural information of the interaction to know which part of the ligand is important for the interaction. We obtained a list of cross-peaks giving distance restrains that we could use in molecular dynamics between 22RT and AG, and finally determine the structure of the complex. For 32R G-quadruplexes, the best candidates for these studies would be G9T conformer with PhenDC3 looking at the spectrum we obtained in NMR titrations. Concerning