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Site-Selective Characterization of the Dynamics of the Nucleic Acid Site-selective characterization of the dynamics of the nucleic acid chaperone properties of HIV-1 nucleocapsid protein using innovative fluorescent probes Marianna Sholokh To cite this version: Marianna Sholokh. Site-selective characterization of the dynamics of the nucleic acid chaperone prop- erties of HIV-1 nucleocapsid protein using innovative fluorescent probes. Biophysics. Université de Strasbourg, 2016. English. NNT : 2016STRAJ031. tel-01561541 HAL Id: tel-01561541 https://tel.archives-ouvertes.fr/tel-01561541 Submitted on 13 Jul 2017 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. UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANT É UMR CNRS 7312 Laboratoire de Biophotonique et Pharmacologie THÈSE présentée par : Marianna SHOLOKH soutenue le : 12 juillet 2016 pour obtenir le grade de : Docteur de l’université de Strasbourg Discipline/ Spécialité : Sciences du vivant/Biophysique Caractérisation site -sélective de la dynamique des propriétés chaperonnes de la protéine de la nucléocapside de VIH - 1 vis-à-vis de ses cibles nucléiques, à l’aid e de sondes fluorescentes innovantes THÈSE dirigée par : M. MELY Yves Professeur , université de Strasbourg Mme ZAPOROZHETS Olga Professeur, université nationale Taras Chevtch enko de Kyiv RAPPORTEURS : Mme TISNÉ Carine Docteur, université Paris Descartes M. DEPREZ Eric Docteur, école normale supérieure Cachan AUTRES MEMBRES DU JURY : Mme TEULADE-FICHOU Marie -Paule Docteur, institut Curie M. RUFF Marc Docteur, université de Strasbourg UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE DES SCIENCES DE LA VIE ET DE LA SANTÉ UMR CNRS 7312 Laboratory of Biophotonics and Pharmacology THESIS presented by : Marianna SHOLOKH on : 12 July 2016 to obtain the degree of : Doctorate of University of Strasbourg Discipline/ Specialization : Life sciences/Biophysics Site-selective characterization of the dynamics of the nucleic acid chaperone properties of HIV-1 nucleocapsid protein using innovative fluorescent probes THESIS directed by : M. MELY Yves Professeur, université de Strasbourg Mme ZAPOROZHETS Olga Professeur, université nationale Taras Chevtchenko de Kyiv RAPPORTEURS : Mme TISNÉ Carine Docteur, université Paris Descartes M. DEPREZ Eric Docteur, école normale supérieure Cachan AUTRES MEMBRES DU JURY : Mme TEULADE-FICHOU Marie-Paule Docteur, institut Curie M. RUFF Marc Docteur, université de Strasbourg Table of contents Acknowledgements ................................................................................................................................. 4 List of abbreviations ................................................................................................................................ 6 Chapter 1. Bibliographical review ................................................................................................ 7 Part 1. Overview on the human immunodeficiency virus type 1 (HIV-1) ...................................... 8 1.1.1. Discovery of HIV-1 ..................................................................................................... 8 1.1.2. Structure of the HIV-1 viral particle............................................................................ 9 1.1.3. Genetic organization of the HIV-1 genome .............................................................. 10 1.1.4. Virus replication cycle ............................................................................................... 18 1.1.5. Antiretroviral therapies .............................................................................................. 22 Part 2. Nucleocapsid protein as a potential target for anti-HIV therapy ............................... 24 1.2.1. Structural characteristics of the nucleocapsid protein (NC) ...................................... 24 1.2.2. Binding of NC to nucleic acids ................................................................................. 27 1.2.3. Chaperone properties of NC ...................................................................................... 30 1.2.4. Role of NC in HIV-1 reverse transcription ............................................................... 32 1.2.5. Role of NC in HIV-1 assembly ................................................................................. 39 Part 3. Fluorescent amino acid analogs as markers for peptide-nucleic acid interactions .... 44 1.3.1. Introduction to fluorescence ...................................................................................... 44 1.3.2. Fluorescent probes ..................................................................................................... 46 1.3.3. Fluorescence approaches to study protein – nucleic acid interactions ...................... 50 1.3.4. Natural fluorescent amino acids ................................................................................ 53 1.3.5. Environment-sensitive probes ................................................................................... 54 1.3.6. Ratiometric environment-sensitive probes based on 3-hydroxychromones .............. 59 1.3.7. Applications of 3HC probes and 3HC-based fluorescent amino acid analogs for monitoring biomolecular interactions....................................................................................... 65 Part 4. Fluorescent nucleic acid analogs: diversity, properties and applications .................. 70 1.4.1. Introduction to nucleic acids ..................................................................................... 70 1.4.2. Emissive nucleoside analogs and their classification ................................................ 73 1.4.3. Applications of fluorescent nucleoside analogs ........................................................ 83 Research objectives ............................................................................................................................... 86 Chapter 2. Materials and Methods .............................................................................................. 89 Part 1. Materials .................................................................................................................... 90 2.1.1. Synthesis of fluorescent amino acid and nucleoside analogs .................................... 90 2.1.2. Peptide synthesis ....................................................................................................... 91 2.1.3. Preparation of Zn-bound NC(11-55) peptides ........................................................... 94 2.1.4. Peptide activity tests .................................................................................................. 94 2.1.5. Oligonucleotide sequences used in this work ............................................................ 95 2.1.6. Synthesis of the labeled 3HCnt-()PBS DNA and dthG7()PBS DNA .................... 96 2 2.1.7. Preparation of the double-stranded ODNs................................................................. 97 2.1.8. Lipids ......................................................................................................................... 97 2.1.9. Experiments with free nucleosides ............................................................................ 99 2.1.10. Preparation of M3HFaa in a polystyrene film ....................................................... 99 Part 2. Physical measurements and procedures .................................................................... 99 2.2.1. UV/visible absorption and steady-state fluorescence measurements ........................ 99 2.2.2. Thermal denaturation experiments .......................................................................... 101 2.2.3. Competition experiments ......................................................................................... 102 2.2.4. Quenching experiments ........................................................................................... 102 2.2.5. Time-resolved fluorescence spectroscopic measurements ...................................... 103 2.2.6. Time-resolved fluorescence anisotropy measurements ........................................... 105 2.2.7. Fluorescence lifetime imaging microscopy (FLIM) ................................................ 106 2.2.8. Kinetic measurements ............................................................................................. 108 2.2.9. Cell cultures ............................................................................................................. 112 2.2.10. Microinjection ..................................................................................................... 113 2.2.11. Deconvolution of the spectra ............................................................................... 113 Chapter 3. Results and Discussion ............................................................................................ 115 Part 1. Interaction of NC with its molecular partners using fluorescent amino acid analogs based on 3-hydroxychromones ..................................................................................................
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