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Pdf [Accessed August 19, 2013] THÈSE En vue de l'obtention du DOCTORAT DE L’UNIVERSITÉ DE TOULOUSE Délivré par l’Université Toulouse 3 Paul Sabatier (UT3 Paul Sabatier) Discipline ou spécialité Biologie Structurale et Fonctionelle Présentée et soutenue par Olivier MARTINEZ Le 18 décembre 2013 Titre : Aptamères ADN : du Cell-SELEX à l’imagerie JURY François COUDERC, Professeur, IMRCP, Toulouse Président Carmelo DIPRIMO, Chargé de Recherche, IECB, Bordeaux Rapporteur Frédéric DUCONGE, Chargé de Recherche, CEA, Orsay Rapporteur Jean-Louis MARTY, Professeur, Images, Perpignan Examinateur Vincent ECOCHARD, Chargé de Recherche, IPBS, Toulouse Directeur de thèse Laurent PAQUEREAU, Professeur, IPBS, Toulouse Directeur de thèse Ecole doctorale : Biologie-Santé, Biotechnologies Unité de recherche : Institut de Pharmacologie et de Biologie Structurale, CNRS, UMR5089 SOMMAIRE 3 Table des matières Introduction ................................................................................................................. 9 1 Cancer Epithélial Ovarien .................................................................................. 10 1.1 Marqueur tumoral CA 125 .......................................................................... 13 1.2 Traitement anti-angiogénique ..................................................................... 16 2 Les Aptamères .................................................................................................. 17 3 Les Avantages des Aptamères .......................................................................... 18 4 Le SELEX .......................................................................................................... 21 5 Les Librairies ..................................................................................................... 27 6 Les Molécules Cibles ......................................................................................... 32 7 Processus de Sélection ..................................................................................... 38 7.1 Liaison à la cible ......................................................................................... 38 7.2 Partitionnement .......................................................................................... 39 7.3 Amplification ............................................................................................... 41 7.4 Génération de la sous-librairie d’ADN simple-brin. ..................................... 42 7.4.1 PCR asymétrique : ................................................................................ 44 7.4.2 Single-Primer Limited Amplification (SPLA) : ........................................ 44 7.4.3 Brins de longueur inégale : ................................................................... 44 7.4.4 Digestions par des exonucléases : ....................................................... 45 7.4.5 Séparation Biotine-Streptavidine : ......................................................... 46 7.4.6 Comparaison des méthodes : ............................................................... 46 8 Suivi de l’Evolution du SELEX ........................................................................... 48 8.1 Dosage de fluorescence : ........................................................................... 48 8.2 Chromatographie liquide de haute-performance en condition dénaturante (dHPLC) : .............................................................................................................. 49 8.3 Analyse des températures de renaturation ................................................. 49 4 SOMMMAIRE 8.4 Analyse du polymorphisme par la longueur de fragments de restriction (RFLP) : ................................................................................................................ 49 9 Identification des Séquences ............................................................................. 50 9.1 Clonage – séquençage .............................................................................. 50 9.2 Séquençage haut-débit Illumina ................................................................. 51 10 Analyse des Populations de Séquences ........................................................... 54 10.1 Approche classique .................................................................................... 54 10.2 Approche haut-débit ................................................................................... 55 10.2.1 Identification par le taux d’enrichissement : ....................................... 55 10.2.2 Classification par polymorphisme en clusters : .................................. 55 10.2.3 Classification structurale en clusters : ................................................ 56 10.2.4 Identification acyclique d’aptamères : ................................................ 56 10.2.5 Identification par analyse des k-mer : ................................................ 57 11 Caractérisation et Optimisation des Aptamères ................................................ 58 11.1 Caractérisation : ......................................................................................... 58 11.2 Optimisation des aptamères identifiés : ...................................................... 60 11.2.1 Etudes de tronquage : ....................................................................... 60 11.2.2 Modifications chimiques pour applications ......................................... 61 12 Les Aptamères comme Outils Thérapeutiques et Diagnostiques ...................... 62 12.1 La thérapie ................................................................................................. 62 12.2 Diagnostic in vitro ....................................................................................... 64 12.3 Diagnostic par imagerie .............................................................................. 65 13 Objectifs ............................................................................................................ 83 Résultats ................................................................................................................... 85 1 Problématique de Purification du Simple Brin à partir des Banques ADN ......... 86 1.1 « α,β-D-constrained nucleic acids are strong terminators of thermo-stable DNA polymerases in polymerase chain reaction », article publié dans le journal PLoSOne en 2011. ............................................................................................... 86 SOMMAIRE 5 1.2 Données non publiées sur la contrainte induite par 2 modifications terminatrices de polymérisation espacées dans la matrice ................................... 97 2 SELEX IL-8 ...................................................................................................... 100 2.1 Non-SELEX par électrophorèse capillaire ................................................ 101 2.1.1 Sélection ............................................................................................. 101 2.1.2 Clonage .............................................................................................. 103 2.1.3 Tests d’affinités ................................................................................... 105 2.2 Filter-Binding SELEX contre l’IL-8 ............................................................ 106 2.2.1 Sélection ............................................................................................. 106 2.2.2 Interaction de la sous-librairie du cycle 6 avec la cible IL-8 ................ 107 2.2.3 Séquençage haut débit ....................................................................... 107 2.3 Comparaison des séquences obtenues par les deux approches CE-SELEX et Filter-Binding SELEX ...................................................................................... 109 3 Sélection d’Aptamères Ciblant la Mucine16 : .................................................. 109 3.1 Test d’expression de la Mucine16 sur les lignées cellulaires NIH:OVCAR3 et SK-OV-3 .............................................................................................................. 110 3.2 Création d’une lignée cellulaire NIH:OVCAR3 Down Mucine16 (Muc16Sh- GFP) ................................................................................................................. 111 3.2.1 Contrôle du taux d’expression ............................................................. 112 3.2.2 Dilution limite ....................................................................................... 112 3.2.3 Utilisations des cellules NIH:OVCAR3 Down Muc16 .......................... 113 3.3 Sélection .................................................................................................. 114 3.3.1 La librairie : ......................................................................................... 114 3.3.2 Purification des ADNsb sur colonne streptavidine : ............................ 115 3.3.3 Conditions de stringence utilisées en SELEX ..................................... 117 3.3.4 Suivi du Cell-SELEX : ......................................................................... 118 3.4 Clonage .................................................................................................... 124 3.5 Séquençage haut-débit ............................................................................ 128 6 SOMMMAIRE 3.5.1 Occurrence moyenne des séquences ................................................. 128 3.5.2 Taille des régions dégénérées ............................................................ 128 3.5.3 Recherche visuelle
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