Super-specific DNA methylation by a DNA methyltransferase coupled with a triple helix-forming oligonucleotide Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation vorgelegt von Maria Maluszynska-Hoffman aus Poznań, Polen Berichter: Universitätsprofessor Dr. Elmar Weinhold Universitätsprofessor Dr. Markus Albrecht Tag der mündlichen Prüfung: 13.11.2008 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. This work has been carried out at the Institute of Organic Chemistry, RWTH Aachen University under the supervision of Prof. Dr. Elmar Weinhold from September 2005 to September 2008. I would like to thank Prof. Dr. Elmar Weinhold for giving me the great opportunity to come to Aachen and work in his research group. I am grateful for the very interesting and challenging project assignment, as well as for his guidance and encouragement throughout my study. I would like to thank Dr. Reinhold Wasserkort for his work on bisulfite sequencing. Dr. Marianne G. Rots, Dr. Marcel Ruiters and Ieneke van der Gun for carrying out the in vivo experiments and for their hospitality during my stay in Groningen. I would like to thank all participants of the IMEDGEN project for the stimulating partnership. I am very grateful to the whole research group, for their help, friendship and the very pleasant working atmosphere as well as for the priceless discussions and suggestions concerning science, chemistry and my own work. Among all, I would especially like to acknowledge Matthias Bahr for his most valuable help and cooperation. To my family. TABLE OF CONTENTS TABLE OF CONTENTS 1 INTRODUCTION.............................................................................................................1 1.1 The structure of double-stranded DNA ................................... ...... ...... ...... ...... ...... .. 1 1.2 DNA methylation ............................................................................................ ...... ...... 2 1.2.1 DNA methyltransferases......................................................................................2 1.2.2 DNA methylation in prokaryotes........................................................................7 1.2.3 DNA methylation in eukaryotes..........................................................................7 1.2.4 DNA methylation pattern.....................................................................................8 1.2.5 DNA methylation and human disease..................................................................9 1.2.6 DNA MTase from Spiroplasma sp. strain MQ1 (M.SssI)..................................10 1.3 Super-specific DNA methylation.............................................................................12 1.3.1 Triple helix DNA...............................................................................................12 1.3.2 Polyamides.........................................................................................................18 1.3.3 DNA-binding domains.......................................................................................19 1.4 DNA-protein conjugation methods.........................................................................20 1.4.1 Heterobifunctional crosslinking.........................................................................21 1.4.2 Coupling DNA to the N- or C-terminus of recombinant proteins ....................22 1.5 Objectives..................................................................................................................26 2 RESULTS AND DISCUSSION......................................................................................28 2.1 Bifunctional crosslinking ........................................................................................28 2.1.1 Conjugation of M.SssI(C141S) with TFO.........................................................28 2.1.2 Conjugation of M.SssI with ODN.....................................................................31 2.2 Expressed protein ligation.......................................................................................37 2.2.1 Construction of pBAM3-M.SssI(Q147L)..........................................................37 2.2.2 Synthesis of Cys-PEG27-NH-C6-TFO..............................................................39 2.2.3 Effect of linker length on expressed protein ligation.........................................42 i TABLE OF CONTENTS 2.3 Super-specific DNA methyltransferase..................................................................43 2.3.1 Design of the TFO.............................................................................................44 2.3.2 In vitro methylation assay on Litcon54.............................................................45 2.3.3 Bisulfite DNA sequencing.................................................................................47 2.3.4 Thermal denaturation studies ............................................................................51 2.3.5 Fluorescence resonance energy transfer...........................................................52 2.3.6 Kinetics and thermodynamics of triple helix formation....................................53 2.3.7 Protein-DNA interactions..................................................................................55 2.3.8 Cell delivery ....................................................................................................... 59 3 SUMMARY AND OUTLOOK......................................................................................62 3.1 Summary...................................................................................................................62 3.2 Outlook......................................................................................................................65 4 EXPERIMENTAL..........................................................................................................67 4.1 Materials...................................................................................................................67 4.1.1 Chemicals..........................................................................................................67 4.1.2 Enzymes.............................................................................................................68 4.1.3 DNA and plasmids............................................................................................68 4.1.4 Escherichia coli strains......................................................................................70 4.1.5 Instruments........................................................................................................70 4.2 Methods.....................................................................................................................72 4.2.1 DNA sequencing ...............................................................................................72 4.2.2 HPLC.................................................................................................................72 4.2.3 FPLC..................................................................................................................73 4.2.4 ESI-Q-TOF .......................................................................................................74 4.2.5 UV-Vis absorption spectroscopy........................................................................74 4.2.6 Fluorescence spectroscopy................................................................................74 4.3 Molecular biology methods.....................................................................................74 4.3.1 Agarose gel electrophoresis...............................................................................74 ii TABLE OF CONTENTS 4.3.2 Gel filtration.......................................................................................................75 4.3.3 SDS-PAGE.........................................................................................................75 4.3.4 Determination of protein concentration.............................................................76 4.3.5 Determination of DNA concentration................................................................76 4.3.6 Amplification and isolation of plasmid DNA....................................................76 4.3.7 Preparation of electrocompetent E. coli cells....................................................76 4.3.8 Transformation by electroporation....................................................................77 4.3.9 Construction of Litcon54...................................................................................77 4.3.10 Construction of pBAM3-M.SssI(Q147L)........................................................77 4.4 Protein purification and activity assays.................................................................78 4.4.1 Purification of M.SssI, M.SssI(C141S) and M.SssI(Q147L)............................78 4.4.2 Purification of M.SssI(Q147L)-thioester...........................................................79 4.4.3 In vitro activity assay on λ DNA.......................................................................80 4.5 Bifunctional crosslinker synthesis...........................................................................81 4.5.1 Synthesis of Fmoc-NH-PEG27-NHS................................................................81 4.6 DNA modifications...................................................................................................82