2-aminopurine and pyrenylethyne-modified uridine as fluorescent probes for the investigation of nucleic acids and their interactions [Elektronische Ressource] / Manuela Johanna Rist

Manu Alma

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2003
• Nombre de lectures: 40
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Institut für Organische Chemie und Biochemie
der Technischen Universität München



2-Aminopurine and Pyrenylethyne-Modified Uridine as
Fluorescent Probes for the Investigation of Nucleic Acids and
Their Interactions



Manuela Johanna Rist



Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität
München zur Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr. J. Buchner
Prüfer der Dissertation: 1. f. H. Kessler
2. Adjunct Assistant Professor J. P. Marino, PhD,
University System of Maryland/USA
3. Univ.-Prof. Dr. M.-E. Michel-Beyerle, i. R.


Die Dissertation wurde am 22.01.2003 bei der Technischen Universität München eingereicht
und durch die Fakultät für Chemie am 28.02.2003 angenommen.































dedicated to my parents and Burkhard
Thanks

The work for this PhD thesis has been carried out from October 1999 to December 2001 at
the Center for Advanced Research in Biotechnology in Rockville, Maryland, in the group of
Prof. Dr. J. P. Marino, and from January 2002 to January 2003 in the group of Prof. Dr. H.
Kessler at the “Institut für Organische Chemie und Biochemie der Technischen Universität
München” under the supervision of Dr. Hans-Achim Wagenknecht.

First and foremost, I would like to thank Prof. Dr. John P. Marino for making it possible for
me to spend two years of my time as a PhD student in his group, enjoying excellent facilities
and resources. I was so fortunate as to reap the benefits of his superb supervision and
guidance but at the same time having the freedom I needed to perform my work.
My special thanks go to Prof. Dr. Horst Kessler for taking me up in his group so
spontaneously. In Munich I enjoyed a hearty welcome and excellent working conditions.
Also, I would like to thank Dr. Hans-Achim Wagenknecht for his preeminent supervision
during my time in his group and for always being available for questions and discussions.
Prof. Dr. Hannelore Daniel I thank very much for supporting my extravagant idea of spending
most of my PhD time in the US.

At CARB I would like to thank
• Dr. Karen A. Lacourciere for introducing me into the secrets of fluorescence
spectroscopy and biochemical lab work in real life,
• my lab colleagues Dr. Burkhard Luy, Nanda Mahashetty, Dr. Eric S. Dejong and Dr. M.
Rita Mihailescu for a warm and hearty lab atmosphere and many interesting
discussions,
• Prof. Dr. James T. Stivers for stimulating discussions and help with the interpretation of
sometimes confusing fluorescence data,
• Dr. Fenhong Song for the synthesis of many DNA templates and labeled RNA samples,
• Nadik Abdulaev for his help with the HPLC,
• Pat Alexander, Mike Tangrea, Kathryn Fisher, Prof. Phil Bryan and Prof. John Orban
for countless lunchtime discussions and insights into the American way of seeing
things,
• Mariela Urrutia, Dr. Paloma Fernandez-Varela, Dr. Pia Bonander and Dr. Nicklas
Bonander for the oasis in the 'other world' of CARB, • Tom Belcher, Tim Bennett, Grace Bromfield, Cindy Denisac, Darwin Diaz, Jonathan
Dill, Tom Dimsdale, Al Ledford, Davin Mackesy, Michelle Stanton and Frank Wallis
for the assistance with all the ‘little’ things,
• and all the other wonderful people at CARB who made my stay there so interesting and
unforgettable.

I would also like to thank
• Prof. Lynne Regan (Yale University) for providing us with purified Rom protein, and
• Dr. T. E. Shrader (Albert Einstein College of Medicine) for providing us with plasmid
pT7-911Q

In Munich I would like to thank
• my lab colleagues Nicole Amann, Alessandro Righi, Elke Mayer, Stefan Mitschke,
Peter Kaden, Clemens Wagner and Linda Valis for the great lab atmosphere, many
stimulating and also entertaining discussions,
• Nicole especially for making the new start in a new lab much easier and for ceaselessly
trying to turn me into a 'real' chemist,
• Georgette Thumshirn for the help with the HPLC,
• Claudia Dahmen for the occasional lift home,
• Herrn Helmut Krause for the prompt measurements of many MALDI-TOF mass
spectra,
• Dr. Torsten Fiebig and his coworkers for the time-resolved transient absorption
measurements,
• Dr. Rainer Häßner and Alexander Frenzel for help with all kinds of computer problems,
• Dr. Burkhard Luy and Dr. Hans-Achim Wagenknecht for the critical reading of this
manuscript,
• Evelyn Bruckmaier and Marianne Machule for secretarial assistance
and all mentioned and unmentioned co-workers for the excellent working atmosphere and for
being not just colleagues but also friends.

My very special thanks go to Dr. Burkhard Luy who helped me regain the fascination for
science and research whenever I had doubts about my work, and without whom this work
would not have been possible in this way.
Index i
______________________________________________________________________________________________________
INDEX I
ABBREVIATIONS V
1 INTRODUCTION AND MOTIVATION 1
2 BACKGROUND 6
2.1 Fluorescence 6
2.1.1 General Introduction to Fluorescence 6
2.1.2 Investigations of Nucleic Acid Interactions Using Fluorescence Spectroscopy 14
2.1.3 Fluorescent and Chemical Properties of the Nucleotide Base Analogs Used in
this Thesis 15
2.2 Time-Resolved Transient Absorption Spectroscopy 21
2.3 Antisense RNA Systems 22
2.4 Energy and Charge Transfer in DNA 24
2.4.1 Excited State Energy Transfer 24
2.4.2 Charge Transfer 24
2.4.2.1 Mechanisms of Hole Transfer 26
2.4.2.1.1 Superexchange Mechanism 26
2.4.2.1.2 Hopping Mechanism 28
2.4.2.2 Experimental Techniques for Charge Transfer Detection 28
2.4.2.2.1 Oxidative Hole Transfer 29
2.4.2.2.2 Reductive Electron Transfer 32
2.4.2.2.3 Biotechnological Applications of Electron Transfer in DNA 37
3 QUANTITATIVE ANALYSIS OF AN ANTISENSE RNA LOOP-LOOP
COMPLEX FROM THE COLE1 PLASMID REPLICATION SYSTEM 39
3.1 Biochemical Background 39
3.2 Sample Preparation and Characterization 41
3.2.1 RNA Oligonucleotide Preparation 41
3.2.2 Samples Used 43 ii Index
______________________________________________________________________________________________________
3.2.3 Native Gel Electrophoresis and UV Melt 43
3.3 Fluorescence Experiments 46
2+3.3.1 Kinetics of Kiss Formation with Mg 46
3.3.2 Proposed Model: a Two-Step Mechanism for RNA Kissing 53
3.3.3 Metal Ion Requirement for the RNA Loop-Loop Complex Formation. 55
3.3.4 Rom Binding Kinetics 60
3.4 Summary 62
4 INVESTIGATION OF THE DIMERIZATION AND STRUCTURAL
ISOMERIZATION OF THE DIINITIATION SITE (DIS) STEM-
LOOP FROM HIV-1 64
4.1 Introduction 64
4.2 Preliminary Experiments 69
4.2.1 Homodimers 69
4.2.2 Heterodimers 71
4.3 Fluorescence Experiments with Second Set of Heterodimers 75
4.3.1 Sample Design 75
4.3.2 Results 76
4.3.2.1 DIS Kissing Dimer Kinetics 76
4.3.2.2 DIS Conversion 80
4.3.2.3 NCp7 Mechanism 83
4.3.2.4 Junction Mutants 88
4.3.2.5 Proposed Model for DIS Conversion 91
4.4 Summary 95
5 EXCIMER FORMATION IN DNA STUDIED WITH 2-AP FLUORESCENT
MARKERS 97
5.1 Introduction 97
5.2 Sample Design and Control Experiments 97 Index iii
______________________________________________________________________________________________________
5.3 Results and Discussion 100
5.3.1 Steady-State Fluorescence Spectroscopy 100
5.3.2 Time-Resolved Transient Absorption Spectroscopy 105
5.4 Conclusion 107
6 STUDIES OF CHARGE TRANSFER IN 2-AMINOPURINE CONTAINING
DINUCLEOTIDES 108
6.1 Introduction 108
6.2 Sample Design and Control Experiments 108
6.3 Results and Discussion 110
7 REDUCTIVE ELECTRON TRANSFER STUDIED WITH 5-(1-
PYRENYLETHYNYL)-2' DEOXYURIDINE (PY-≡-DU) 116
7.1 Introduction 116
7.2 Sample Preparation and Control Experiments 117
7.3 Results 121
8 SUMMARY 126
9 MATERIALS AND METHODS 128
9.1 Materials 128
9.2 Frequently Used Buffers 128
9.3 General Work Procedures 128
9.3.1 Denaturing Polyacrylamide Gel Electrophoresis (PAGE) 128
9.3.2 Native Polyacrylamide Gel Electrophoresis 129
9.3.3 High-Performance Liquid Chromatography (HPLC) 130
9.3.4 Lyophilization 130
9.3.5 Annealing of RNA or DNA Duplexes
9.3.6 Snap Cooling 130 iv Index
______________________________________________________________________________________________________
9.3.7 MALDI-TOF Mass Spectroscopy 130
9.3.8 UV/Vis Spectroscopy 130
9.3.9 UV Melts 131
9.3.10 Circular Dichroism Spectroscopy (CD)
9.4 Protein Preparations 132
9.4.1 T7 Polymerase 132
9.4.2 Nucleocapsid Protein (NCp7)
9.5 Oligonucleotide Preparations 134
9.5.1 RNA Transcripti