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
|
