Wachowius F.   Mechanistic characterization of RNA-ligating deoxyribozymes by chemical and biophysical methods and spin-labeled RNA for EPR spectroscopy: Diss. (Gottingen, 2012). - ОГЛАВЛЕНИЕ / CONTENTS

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ОбложкаWachowius F. Mechanistic characterization of RNA-ligating deoxyribozymes by chemical and biophysical methods and spin-labeled RNA for EPR spectroscopy: Diss. for the award of the degree Doctor rerum naturalium (Dr. rer. nat.) of the Georg-August-Univ. Gottingen / F.Wachowius. - Gottingen: Sierke Verlag, 2012. - XIX,194 S.: ill. - Bibliogr.: S. 180-194. - ISBN 978-3-86844-429-2
 

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Оглавление / Contents
 
1    General introduction ....................................... 1
1.1  RNA as catalyst ............................................ 1
1.2  RNA as gene regulation element ............................. 6
1.3  Artificial ribozymes and catalytic DNA ..................... 8
1.4  Thesis outline ............................................ 10

2    Functional characterization of deoxyribozymes ............. 11
2.1  Introduction .............................................. 11
     2.1.1  In vitro selection as tool to identify
            deoxyribozymes ..................................... 11
     2.1.2  RNA-cleaving deoxyribozymes ........................ 13
     2.1.3  RNA-ligating deoxyribozymes ........................ 17
            2.1.3.1  Formation of native 3'-5' RNA linkages .... 17
            2.1.3.2  Formation of 2',5'-branched and lariat
                     RNA ....................................... 18
            2.1.3.3  The 7S11 deoxyribozyme .................... 20
            2.1.3.4  Applications of RNA-ligating
                     deoxyribozymes ............................ 22
     2.1.4  Deoxyribozymes with other catalytic properties ..... 24
     2.1.5  Combinatorial methods for analyzing structure and
            function of nucleic acids .......................... 25
     2.1.6  Solid-phase RNA synthesis using phosphoramidite
            building blocks .................................... 26
2.2  Objectives ................................................ 30
2.3  Results and discussion .................................... 32
     2.3.1  Combinatorial mutation interference analysis
            (CoMA) ............................................. 32
            2.3.1.1  Method development ........................ 32
            2.3.1.2  CoMA of 9DB1 .............................. 36
            2.3.1.3  CoMA of 10-23 ............................. 40
     2.3.2  Nucleotide analogue interference mapping of DNA
            (dNAIM) ............................................ 43
            2.3.2.1  Synthesis of modified ribonucleotides
                     for dNAIM ................................. 44
            2.3.2.2  Synthesis of individual nucleoside
                     building blocks ........................... 46
            2.3.2.3  dNAIM method development .................. 49
            2.3.2.4  dNAIM of the DNA AMP aptamer .............. 51
            2.3.2.5  dNAIM of 9DB1 ............................. 53
     2.3.3  Functional characterization of the 7S11
            deoxyribozyme ...................................... 58
            2.3.3.1  CoMA of 7S11 .............................. 58
            2.3.3.2  dNAIM of 7S11 ............................. 60
            2.3.3.3  dNAIM with different branch-site
                     nucleotides ............................... 63
            2.3.3.4  Substrate requirements for 7S11-
                     catalyzed ligation ........................ 68
            2.3.3.5  Phosphorothioate interference and metal
                     ion rescue at the branch-site ............. 74
            2.3.3.6  Leaving group analysis of 7S11, 10DM24
                     and 9FQ4 .................................. 78
2.4  Conclusion and Outlook .................................... 86

3    Spin-labeling of RNA for EPR spectroscopy ................. 88
3.1  Introduction .............................................. 88
     3.1.1  EPR spectroscopy ................................... 88
     3.1.2  Spin-labeled RNA for application in EPR
            spectroscopy ....................................... 91
            3.1.2.1  Spin labeling of the nucleobase ........... 92
            3.1.2.2  Spin labeling of the sugar phosphate
                     backbone .................................. 93
            3.1.2.3  Recent DNA spin-labels and future
                     perspectives for RNA labeling ............. 93
3.2  Objective ................................................. 95
3.3  Results and discussion .................................... 97
     3.3.1  Post-synthetic spin-labeling of RNA ................ 97
            3.3.1.1  The convertible nucleoside approach ....... 97
            3.3.1.2  Synthesis and characterization of TEMPO-
                     labeled RNA ............................... 98
            3.3.1.3  UV melting analysis and CD spectroscopy
                     of spin-labeled RNA ...................... 102
            3.3.1.4  Distance measurement of spin labeled
                     RNA by pulsed EPR ........................ 104
     3.3.2  Çm as a rigid spin probe for RNA .................. 110
            3.3.2.1  Design and synthesis of spin-labeled
                     phosphoramidite Çm ....................... 111
            3.3.2.2  Synthesis and purification of spin-
                     labeled RNA .............................. 111
            3.3.2.3  Characterization of Çm-labeled RNA by
                     UV melting and CD spectroscopy ........... 112
            3.3.2.4  Analysis of different RNA secondary
                     structures by CW-EPR ..................... 117
            3.3.2.5  Distance measurements by PELDOR .......... 121
3.4  Conclusion and Outlook ................................... 123

4    Material and Methods ..................................... 125
4.1  Synthesis of Nucleoside Building Blocks .................. 125
     4.1.1  Standard methods and reagents ..................... 125
     4.1.2  Synthesis of 1-Methylguanosine (m1G) .............. 126
     4.1.3  Synthesis of N2-Methylguanosine (m2G) ............. 129
     4.1.4  Synthesis of N2, N2-Dimethylguanosine (m22G) ...... 132
     4.1.5  Synthesis of 2-aminopurine (AP) ................... 135
     4.1.6  Synthesis of N6, N6-dimethyladenosine (m62A) ...... 139
4.2  Methods .................................................. 142
     4.2.1  RNA/DNA solid-phase synthesis and deprotection .... 142
            4.2.1.1  Phosphoramidites ......................... 142
            4.2.1.2  Solid-phase synthesis .................... 143
            4.2.1.3  Deprotection of oligonucleotides ......... 147
            4.2.1.4  RNA spin-labeling by the convertible
                     nucleoside approach ...................... 148
            4.2.1.5  Nucleobase modifications by the
                     convertible nucleoside approach .......... 148
     4.2.2  Oligonucleotides .................................. 149
     4.2.3  Purification of oligonucleotides .................. 154
            4.2.3.1  Analysis and purification of
                     oligonucleotides by HPLC ................. 154
            4.2.3.2  Oligonucleotide purification by PAGE ..... 155
     4.2.4  Electrophoretic mobility shift assay .............. 156
     4.2.5  Synthesis of 5'-activated oligonucleotide
            substrates ........................................ 157
            4.2.5.1  In vitro transcription of 5'-
                     triphosphorylated RNA .................... 157
            4.2.5.2  Chemical synthesis of triphosphorylated
                     DNA ...................................... 157
            4.2.5.3  Enzymatic synthesis of AppDNA ............ 159
            4.2.5.4  Chemical synthesis of AppRNA or AppDNA ... 160
     4.2.6  Enzymatic modification of oligonucleotides ........ 161
            4.2.6.1  5'-phosphorylation of synthetic
                     oligonucleotides ......................... 161
            4.2.6.2  5'-radioactive labeling of
                     oligonucleotides ......................... 162
            4.2.6.3  3'-radioactive labeling of
                     oligonucleotides ......................... 162
            4.2.6.4  Ligation of deoxyribozyme libraries
                     to R-RNA by T4RNA ligase ................. 162
     4.2.7  DNA-catalyzed reactions and separation of CoMA
            and dNAIM pools ................................... 163
     4.2.8  dNAIM of the DNA AMP aptamer ...................... 163
     4.2.9  Alkaline hydrolysis of DNA libraries and
            analysis of interference pattern .................. 165
     4.2.10 Kinetic assays of deoxyribozyme-catalyzed
            ligation .......................................... 167
     4.2.11 Quantification of oligonucleotides by UV
            absorbance ........................................ 168
     4.2.12 UV melting curves ................................. 169
     4.2.13 CD Spectroscopy ................................... 171
     4.2.14 EPR measurements and data analysis ................ 172
            4.2.14.1 Continuous-wave EPR spectroscopy
                     measurements ............................. 172
            4.2.14.2 Pulsed EPR spectroscopy measurements ..... 172
4.3  Instruments and Special Materials ........................ 174

5    References ............................................... 180


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