Oligodeoxynucleotide-polypeptide block copolymers [Elektronische Ressource] / Sabine Flügel

Bine

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2010
• Nombre de lectures: 43
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Oligodeoxynucleotide-Polypeptide
Block Copolymers




Dissertation
zur Erlangung des Grades
„Doktor der Naturwissenschaften“
im Promotionsfach Chemie

am Fachbereich Chemie, Pharmazie und Geowissenschaften
der Johannes Gutenberg-Universität in Mainz.

Sabine Flügel
geboren in Fulda

Mainz 2010
 
Die vorliegende Arbeit wurde im Zeitraum von Januar 2007 bis März 2010 am Institut für
Physikalische Chemie der Johannes Gutenberg-Universität Mainz angefertigt.














Dekan:
1. Berichterstatter:
2. Berichterstatter:
Tag der mündlichen Prüfung: 16. April 2010


 

To my family



















Don’t curse the darkness
-light a candle.
Chinese proverb

  

Contents
Contents
 
1 Motivation ...................................................................................................................................... 7
2 Background ................................................................................................................................... 11
2.1 DNA ........................................................................................................................................ 11
2.1.1 Structure ...................................................................................................................... 11
2.1.2 Chemical Synthesis .................................................................................................... 14
2.1.3 DNA four-arm junctions ........................................................................................... 17
2.1.4 Junction Design ........................................................................................................... 18
2.1.5 Junction Geometry ..................................................................................................... 19
2.1.6 Controlled self-assembly of DNA ............................................................................ 20
2.2 Elastin-like Polypeptides (ELPs) ........................................................................................ 22
2.2.1 Elastin ........................................................................................................................... 22
2.2.2 Genetic Engineering of ELPs .................................................................................... 23
2.2.3 Thermosensitivity of ELPs 26
2.2.4 Applications of ELPs .................................................................................................. 27
2.3 Bioconjugation ....................................................................................................................... 29
2.3.1 Conjugation by activated ester chemistry ............................................................... 29
2.3.2 Conjugation by thiol addition .................................................................................. 30
2.3.3 Oligonucleotide conjugates ....................................................................................... 31
2.4 Characterization Methods ................................................................................................... 33
2.4.1 Gel Electrophoresis .................................................................................................... 33
2.4.2 UV/Vis Absorption Spectroscopy ........................................................................... 36
2.4.3 High-performance liquid chromatography (HPLC) ............................................. 38
2.4.4 Dynamic Light Scattering .......................................................................................... 40
3 DNA four-arm junctions ............................................................................................................ 43
3.1 Thermal Stability of the Seeman junction J1 ..................................................................... 43
3.2 Four-arm DNA junction K4 with 23bp per arm ............................................................... 47
3.2.1 Effect of dangling ends on the hybridization efficiency ....................................... 49
3.2.2 Rehybridization efficiency ........................................................................................ 50
3.2.3 Effect of hybridization procedure ............................................................................ 51
nd3.3 2 Generation junction K5 .................................................................................................. 53
3.4 3rd Generation junction K6 ................................................................................................. 55
3.5 Summary ................................................................................................................................ 56

2 Contents

4 Hydrophobic cysteine functionalized ELPs ........................................................................... 57
4.1 Genetic engineering ............................................................................................................. 57
4.2 Polypeptide Expression ....................................................................................................... 61
4.3 UV cloud point measurements ........................................................................................... 64
4.4 Dynamic light scattering (DLS) .......................................................................................... 67
4.5 Summary................................................................................................................................ 70
5 ODN-ELP Diblocks .................................................................................................................... 71
5.1 Thiol Addition to methacrylate modified ODN .............................................................. 71
5.2 Modification of amino oligonucleotides by heterobifunctional linkers ....................... 74
5.2.1 Hydrolysis kinetics of crosslinkers with NHS ester functionality ...................... 75
5.2.2 Modification of amino ODNs by BMPS and sulfo-SMCC.................................... 77
5.2.3 ODNs by SMCC ................................................................. 78
5.2.4 HPLC analysis of the SMCC modification reaction .............................................. 80
5.3 Synthesis of ODN conjugates by thiol addition ............................................................... 83
5.3.1 Addition of thiol-PEG to maleimide modified ODNs ......................................... 83
5.3.2 thiol functionalized ODNs to maleimide modified ODNs ............. 84
5.3.3 Addition of cysteine containing hexapeptide to maleimide ODNs .................... 85
5.3.4 ELPs to maleimide modified ODNs .................................................. 87
5.3.5 Translocation of ODN-ELP diblock copolymers ................................................... 91
5.4 Synthesis of ODN conjugates by Activated Ester Chemistry ........................................ 94
5.4.1 Protection of the cysteine moiety ............................................................................. 95
5.4.2 ODN-ELP diblock copolymers ................................................................................. 96
5.4.3 Purification of ODN-ELP diblock copolymers ....................................................... 99
5.4.4 ODN-ELP diblock copolymer library .................................................................... 102
5.4.5 DLS characterization of ODN-ELP diblock copolymers .................................... 103
5.5 Self Assembly of ODN-ELP diblock copolymers ........................................................... 107
5.6 Summary.............................................................................................................................. 109
6 ODN-Peptide-ODN Triblocks ................................................................................................ 111
6.1 ODN-Hexapeptide-ODN conjugates .............................................................................. 111
6.2 Self assembly of ODN-di-hexapeptide-ODN triblocks ................................................. 114
6.3 Synthesis ODN-ELP-ODN triblock copolymers ............................................................ 117
6.4 Isolation of ODN23-ELP-ODN23 triblock copolymers by adsorption onto
gold nano spheres .............................................................................................................. 122
6.5 Summary.......... 124
7 Conclusions and Outlook ........................................................................................................ 125

3 Contents
8 Materials and Methods ............................................................................................................ 129
8.1 DNA four-arm junctions .................................................................................................... 129
8.1.1 Junction sequences ................................................................................................... 129
8.1.2 Gel extraction protocol ....................