Study of DNA G-quadruplex structures by Nuclear Magnetic Resonance (NMR) Abdelaziz Kerkour To cite this version: Abdelaziz Kerkour. Study of DNA G-quadruplex structures by Nuclear Magnetic Resonance (NMR). Biomolecules [q-bio.BM]. Université de Bordeaux, 2014. English. NNT : 2014BORD0292. tel- 01281325 HAL Id: tel-01281325 https://tel.archives-ouvertes.fr/tel-01281325 Submitted on 2 Mar 2016 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE PRÉSENTÉE POUR OBTENIR LE GRADE DE DOCTEUR DE L’UNIVERSITÉ DE BORDEAUX ÉCOLE DOCTORALE Sciences de la Vie et de la Santé Spécialité : Interface Chimie-Biologie Présentée par Abdelaziz KERKOUR Study of DNA G-quadruplex structures by Nuclear Magnetic Resonance (NMR) Sous la direction de : Dr. Gilmar SALGADO Soutenue le 15 décembre 2014 Membres du jury Dr. Jean-Jacques Toulmé INSERM U869, Université de Bordeaux Président Dr. Serge Bouaziz CNRS UMR 8015, Université de Paris 5 Rapporteur Dr. Janez Plavec Université de Ljubljana, Slovénie Rapporteur Dr. Patrizia Alberti MNHN, Paris Examinateur Dr. Gilmar Salgado INSERM U869, Université de Bordeaux Directeur de thèse 1 CONTENTS CONTENTS .............................................................................................................................. 1 LIST OF FIGURES ................................................................................................................. 3 SHORTENINGS ....................................................................................................................... 6 ACKNOWLEDGEMENTS ..................................................................................................... 7 Résumé étendu en français ...................................................................................................... 8 A. GENERAL INTRODUCTION ........................................................................................ 12 A.1 DNA structures ......................................................................................................................... 12 A.1.1 DNA components ................................................................................................................ 12 A.1.2 DNA structural polymorphism ............................................................................................ 12 A.1.2.1 Duplex DNA ............................................................................................................................... 12 A.1.2.2 Triplex DNA ............................................................................................................................... 13 A.1.2.3 Guanine quadruplex (G4) structures ........................................................................................... 13 A.2 G4 structures ............................................................................................................................. 16 A.2.1 G-quartets ............................................................................................................................ 16 A.2.2 Structural polymorphism of G4 ........................................................................................... 16 A.2.2.1 Number of strands ....................................................................................................................... 16 A.2.2.2 Orientation of strands .................................................................................................................. 17 A.2.2.3 Conformation of loops ................................................................................................................ 17 A.2.2.4 Nature of cations ......................................................................................................................... 17 A.2.2.5 Intra-molecular G4 motifs .......................................................................................................... 18 A.2.2.6 Bulges in G4 ............................................................................................................................... 18 A.3 Biological functions of G4 ........................................................................................................ 20 A.3.1 G4 in silico evidence ........................................................................................................... 20 A.3.2 Examples of G4 localization in genomes ............................................................................ 21 A.3.2.1 Promoters of genes ...................................................................................................................... 21 A.3.2.2 Minisatellites .............................................................................................................................. 22 A.3.2.3 Immunoglobulin class switching regions .................................................................................... 23 A.3.2.4 RNA quadruplex ......................................................................................................................... 24 A.3.2.5 Telomeres .................................................................................................................................... 24 A.3.2.5.1 G4 structures of human telomeric DNA .............................................................................. 25 A.4 G4 ligands .................................................................................................................................. 29 A.4.1 π-π stacking interaction with G-quartets.............................................................................. 30 A.4.1.1 In situ protonated ligands ............................................................................................................ 30 A.4.1.2 Aromatic N-methylated ligands .................................................................................................. 32 A.4.1.3 Neutral and negatively-charged macrocyclic ligands .................................................................. 34 A.4.1.4 Metallo-organic ligands............................................................................................................... 34 A.4.2 Ligands of grooves and loops .............................................................................................. 35 A.5 Goals of the doctoral research ................................................................................................. 36 A.5.1 Binding studies of 2,4,6-triarylpyridine family ligand with Human Telomeric Repeat G4 36 A.5.2 Characterization of a tetra-molecular G4 model d[TG4T]4 and its interaction with specific G4 ligands inside cells ................................................................................................................... 36 A.5.3 NMR study of KRAS G4 ........................................................................................ 37 1 Part 1 ......................................................................................................................................... 2 B. Interaction of 2,4,6-triarylpyridine family ligand with the Human Telomeric Repeat G-quadruplex motif ................................................................................................................ 38 B.1 Introduction............................................................................................................................... 38 B.2 Materials and Methods ............................................................................................................. 40 B.2.1 Biophysical characterization of G4 ...................................................................................... 40 B.2.1.1 Synthesis and purification ........................................................................................................... 40 B.2.1.2 Ligand preparation ..................................................................................................................... 41 B.2.1.3 CD and CD melting ..................................................................................................................... 41 B.2.2 NMR spectroscopy .............................................................................................................. 42 B.2.3 Assignment and structure calculation .................................................................................. 43 B.3 Results and discussions ............................................................................................................. 45 B.3.1Characterization of 22AG G-quadruplex formation by CD and NMR spectroscopy ........... 45 B.3.2 Characterization of TAP compound .................................................................................... 48 B.3.3 TAP binds to and stabilizes 22AG ....................................................................................... 50 B.3.4 NMR analysis of the TAP-22AG complex and binding site determination ........................ 53 B.3.5 Assignment of resonances belonging to the bound 22AG ................................................... 56 B.3.6