Modified oligonucleotides [Elektronische Ressource] : investigations and applications of the Diels-Alderase ribozyme / presented by Florentine Wahl

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Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Diplom-Chemikerin Florentine Wahl born in Freiburg i. Br., Germany rdoral-examination: March 3 2011 Modified Oligonucleotides: Investigations and Applications of the Diels-Alderase Ribozyme Referees: Prof. Dr. Andres Jäschke Prof. Dr. Stefan Wölfl Acknowledgements First and foremost I want to thank my supervisor Prof. Andres Jäschke, for giving me the opportunity to work in his research group, as well as for his constant support, encouragement and guidance during my thesis. I am thankful to Professor Wölfl for accepting to review and co-examine this thesis. Furthermore I want to thank Prof. Fricker and Prof. Haefeli to who agreed to take part in my oral exam. I owe sincere and earnest thankfulness to Dr. Roberto Fiammengo for mentoring me and for his support and friendship throughout all these years. My thanks also go to Viola Funk and Karin Weiß, who supported me in any possible way. The personal of the IPMB, Heiko Rudy for everything he ever did help me with, Tobias Timmermann for acquiring NMR spectra and his support with computer problems, Besarta Ljaic and Sandra Suhm for their synthetic expertise, Steffi Dräger for her help.

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Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences












presented by

Diplom-Chemikerin Florentine Wahl
born in Freiburg i. Br., Germany
rdoral-examination: March 3 2011






Modified Oligonucleotides:
Investigations and Applications of the
Diels-Alderase Ribozyme















Referees: Prof. Dr. Andres Jäschke
Prof. Dr. Stefan Wölfl
Acknowledgements

First and foremost I want to thank my supervisor Prof. Andres Jäschke, for giving me the
opportunity to work in his research group, as well as for his constant support, encouragement
and guidance during my thesis.

I am thankful to Professor Wölfl for accepting to review and co-examine this thesis. Furthermore
I want to thank Prof. Fricker and Prof. Haefeli to who agreed to take part in my oral exam.

I owe sincere and earnest thankfulness to Dr. Roberto Fiammengo for mentoring me and for
his support and friendship throughout all these years.

My thanks also go to Viola Funk and Karin Weiß, who supported me in any possible way.
The personal of the IPMB, Heiko Rudy for everything he ever did help me with, Tobias
Timmermann for acquiring NMR spectra and his support with computer problems, Besarta
Ljaic and Sandra Suhm for their synthetic expertise, Steffi Dräger for her help.
Big thanks also go to my interns Michael Wrede, Helen Hagen, Michael Morgen and
Michaela Stach for their excellent work.

Special thanks to Benjamin Strauss, Marie Winz, Dr. Roberto Fiammengo, Anna Wiesmayer,
Dr. Alexander Niert and André Krause for proof-reading the manuscript.
Furthermore I want to thank my friends Dr. Bernd Sterling, Dr. Dave Smith and Martina
Mechler for their invaluable support and especially Dr. Thomas Schultz for believing in me
and make me believe in myself.

I am obliged to many of my colleagues - the present and past members of the Jäschke lab as
well as the Helm group, who all helped me, contributed to this thesis and always created a
good working atmosphere. Anna, for her help in the lab, as well with my terrace and the
breaks spent together. Benjamin, for invaluable help during the last couple of months. Marie,
for her advice. Sandeep, for great discussions and his support with my project. Armine and
Martin, for introducing me into the word of biology. Matthias for his support. Ayan, for the
good discussions. Juliane, André, Alex, Steffi K., Ankita, Surjendu, Hana, Steffi P., Marco,
Salifu, Markus P., Felix, Madeleine, Michaela, Markus H., Mihaela … I am truly indebted and thankful to all my friends, who supported me during these years and
helped me to get through the ups and downs of this thesis. Terje for the good times spent
together.

My parents and my sister have always provided strong and consistent support. Without them I
wouldn’t be where I am now and I will forever be grateful for that.







For my family Abstract Florentine Wahl, Diplom-Chemikerin

Modified Oligonucleotides: Investigations and Applications of the Diels-Alderase
Ribozyme.

st1 Referee: Prof. Dr. Andres Jäschke
nd2 Referee: Prof. Dr. Stefan Wölfl

The hypothesis of the origin of life was fundamentally changed by the discovery of catalytic
RNA. The fascinating fact that RNA can function as a catalyst in the formation of carbon-
carbon bonds, thereby creating enantiomeric centers and thus potential building blocks for
life-sustaining structures, is of great importance to those who wish to understand biocatalytic
processes. Ribozymes are generally identified by in vitro selection. Such an artificial
ribozyme was utilized for the work presented in this dissertation; specifically, the Diels-
Alderase ribozyme was the focus of the investigation. This ribozyme catalyzes a Diels-Alder
reaction between anthracene and maleimide. The work presented herein revolved around
modifications of oligonucleotides on different levels. Modification of RNA with organic
moieties is indispensable for effective in vitro selection of such ribozymes.
This thesis consists of three parts. In the first, new pathways were established for the synthesis
of modified guanosine monophosphates (GMPs). The ability of the enzyme T7 RNA
polymerase to incorporate these modified GMPs during transcription priming coined the term
initiator nucleotides. The syntheses of a substituted anthracene and aldehyde-modified r nucleotides connected with a flexible polyethylene glycol linker were realized in just
three synthetic steps. The incorporation of the initiator nucleotides into short RNA transcripts
was investigated, as well as their incorporation into an RNA library with a randomized region
eligible for in vitro selection. Excellent incorporation rates up to 77% were obtained for the
transcription into the RNA library. The reactivity of thereby modified RNA molecules was
subsequently studied in chemical reactions. Whereas the anthracene derivative could be
reacted in a Diels-Alder reaction, the aldehyde led to full conversion in the reaction with
hydrazides.
In the second part of this thesis, the effect of divalent metal ions on the folding and stability of
the Diels-Alderase ribozyme was studied in UV thermal denaturation experiments. Low
2+concentrations of Mg cations led to secondary structure stabilization of up to 85°C. Thermal
denaturation studies of the wild-type ribozyme and comparison to mutants complement other
ongoing investigations of the complex interplay of non-standard base pairs and triples in the
ribozyme structure. While the wild-type ribozyme exhibited a significant stabilization in the
2+presence of Mg cations, a comparable stabilization of the mutants could not be observed. In
accordance with the findings in other studies, the best interpretation of this effect on stability
was that the mutants, despite their ability to form a pseudoknot structure, could not undergo
further compaction.
In the third part of this thesis, the Diels-Alderase ribozyme, which causes a remarkable
acceleration of the Diels-Alder reaction rate, was successfully used as a catalyst for
fluorescently labeling oligonucleotides in cis and in trans. Investigations of the substrate
specificity of the ribozyme revealed that only fluorescent maleimide dyes with five carbon
atoms next to the dye were accepted as substrates. This distinct feature was utilized as a
bioorthogonal labeling strategy for the Diels-Alder bioconjugation of anthracene-modified
oligonucleotides with equimolar proportions of fluorescent dyes. A new dual orthogonal
labeling strategy was achieved by using an iodoacetamide dye for the thiol modification and a
maleimide dye for the anthracene modification within the same DNA strand. For the
anthracene modification, the Diels-Alderase ribozyme was employed as a catalyst.