RNA Synthetic Biology using the Hammerhead Ribozyme: Engineering of Artificial Genetic Switches Dissertation submitted for the Doctor of Natural Sciences (Dr. rer. nat.) presented by Benedikt Klauser at the Faculty of Natural Sciences Department of Chemistry Date of the oral examination: 16 March 2015 First referee: Prof. Dr. Jörg S. Hartig Second referee: Prof. Dr. Valentin Wittmann Konstanzer Online-Publikations-System (KOPS) URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-0-287233 This thesis was conducted from 2010 to 2014 in the group of Prof. Dr. Jörg S. Hartig (Chair of Biopolymer Chemistry) at the University of Konstanz. It was supported by scholarships of the Research Training Group 1331 (12/2010 - 02/2011), of the Chemical Industry Fund of the Chemical Industry Association (VCI; 03/2011 - 02/2013) and of the Collaborative Research Center 969 (03/2013 - 09/2014). Parts of this thesis are published in: Klauser, B., Atanasov, J., Siewert, L. K., and Hartig, J. S. (2014) Ribozyme-based aminoglycoside switches of gene expression engineered by genetic selection in S. cerevisiae, ACS synthetic biology. Klauser, B., and Hartig, J. S. (2013) An engineered small RNA-mediated genetic switch based on a ribozyme expression platform, Nucleic acids research 41, 5542- 5552. Publications not integrated in this thesis: Klauser, B., Rehm; C., Hartig, J.S. (2015) Engineering of ribozyme-based aminoglycoside switches of gene expression by in vivo genetic selection in Saccharomyces cerevisiae, Methods Enzymology 550, 301-20. Rehm; C., Klauser, B.,Hartig, J. S. (2015) Screening of genetic aptazyme switches for conditional gene expression using a mammalian cellular system, Methods in Molecular Biology, in press. Saragliadis, A., Klauser, B., and Hartig, J. S. (2012) In vivo screening of ligand- dependent hammerhead ribozymes, Methods in molecular biology 848, 455-463. Klauser, B.*, Saragliadis, A.*, Ausländer, S., Wieland, M., Berthold, M. R., and Hartig, J. S. (2012) Post-transcriptional Boolean computation by combining aptazymes controlling mRNA translation initiation and tRNA activation, Molecular BioSystems 8, 2242-2248. Wieland, M., Benz, A., Klauser, B., and Hartig, J. S. (2009) Artificial ribozyme switches containing natural riboswitch aptamer domains, Angewandte Chemie 48, 2715-2718. * authors contributed equally Danksagung Als Erstes möchte ich mich ganz besonders bei meinem Doktorvater Prof. Dr. Jörg S. Hartig bedanken. Vielen Dank für die Unterstützung meiner wissenschaftlichen Arbeit und das in mich gesetzte Vertrauen. Ich hatte immer die Möglichkeit, meine eigenen Ideen zu verwirklichen. Dank deiner verständnisvollen Art, deiner vielen Ratschläge und deiner ausgezeichneten Betreuung werde ich mich immer an die schöne Zeit in deiner Arbeitsgruppe zurückerinnern. Deine Begeisterung und dein Engagement für die Wissenschaft haben mich inspiriert und motiviert. Ich danke Prof. Dr. Valentin Wittman für die Übernahme des Zweitgutachtens. Mein weiterer Dank gilt dem Prüfungsvorsitzenden Prof. Dr. Christof R. Hauck und Prof. Dr. Martin Scheffner als Mitglieder meines Dissertations-Komitees. Eure Anmerkungen, Anregungen und Unterstützung waren mir immer von großer Hilfe. Weiterhin möchte ich bei meinen Kooperationspartnern Dr. Dietmar Funck und Dr. martin Gamerdinger sowie Dr. Daniel Summerer für die wissenschaftlichen Ratschläge danken. Meine Promotion wäre ohne die Anwesenheit vieler weiterer Mitarbeiter und Freunde nur halb so schön gewesen. Und ohne Euch wäre meine Promotion in dieser Form nicht möglich geworden: Ich bedanke mich bei allen ehemaligen und derzeitigen Mitgliedern der AG Hartig für die große Unterstützung, Hilfsbereitschaft und ausgezeichnete Arbeitsatmosphäre. Vor allem möchte ich mich bei Charlotte Rehm für die schöne und unterhaltsame Zeit in unserem gemeinsamen Labor bedanken. Vielen Dank auch Isabelle Holder und Mark Hauser für eure seit dem ersten Semester andauernde Unterstützung und Astrid Joachimi für deine große Hilfsbereitschaft. Vielen Dank euch allen für die wissenschaftlichen Diskussionen und die gemeinsame schöne Zeit auch außerhalb des Labors. Außerdem bedanke ich mich bei allen Doktoranden des RTG 1331 und allen, die mich (nicht nur wissenschaftlich) unterstützt haben und mein Studium zu einer für mich unvergesslichen Zeit gemacht haben. Mein größter Dank geht an meine Familie, inbesondere an meine Eltern. Ihr wart während meines Studiums und meiner Promotion immer für mich da und habt mir immer den notwendigen Rückhalt gegeben, meine Ziele zu erreichen. Vielen Dank Euch allen! Table of Contents 1. Introduction 1 1.1 Basics of gene expression 1 1.1.1 Reading and translating the genetic code 1 1.1.2 Characteristics of prokaryotic gene expression 4 1.1.3 Characteristics of eukaryotic gene expression 5 1.2 Natural regulation of gene expression 5 1.2.1 Gene regulation by transcription factors 6 1.2.2 RNA as regulatory molecule 7 1.3 RNA synthetic biology 13 1.3.1 Small RNA-mediated regulation of gene expression 14 1.3.2 Artificial riboswitches 15 1.3.3 Artificial riboswitches based on the hammerhead ribozyme motif 17 1.3.4 Generation and application of artificial riboswitches 21 2. Aim of this Work 22 3. Results and Discussion 23 3.1 Rational design of synthetic HHR-based genetic switches that sense small RNAs in bacteria 23 3.1.1 General considerations 23 3.1.2 Construction of an expression system 25 3.1.3 Rational design of TR-HHR 25 3.1.4 Mutational and structural characterization of the taRNA-TR-HHR interaction 29 3.1.5 Alterations within the secondary structure of the taRNA impede riboregulatory function 32 3.1.6 Conclusion 35 3.2 Genetic selection of ribozyme-based aminoglycoside switches in S. cerevisiae 37 3.2.1 General considerations 37 3.2.2 Impact of the 5‟-leader sequence on GAL4 expression 39 3.2.3 Regulation of GAL4 expression with 3‟-UTR hammerhead ribozymes 40 3.2.4 In vivo selection of theophylline-dependent type I HHRs 42 3.2.5 Development of neomycin-dependent hammerhead ribozymes 46 3.2.6 Conclusion 53 3.3 Application of ribozyme-based genetic switches in Arabidopsis thaliana and Caenorhabditis elegans - cross-kingdom transferability of HHR motifs 55 3.3.1 General considerations 55 3.3.2 Aptazymes as artificial riboswitches in Arabidopsis thaliana 55 3.3.3 Aptazymes as artificial riboswitches in Caenorhabditis elegans 60 3.3.4 Investigation of the cross-kindom transferability of natural hammerhead ribozyme motifs by a comparative in vivo analysis 62 5. Summary and Outlook 71 Zusammenfassung und Ausblick 75 6. Materials 79 6.1 Chemicals 79 6.2 Nucleotides 79 6.3 Enzymes 79 6.4 Standards and Kits 80 6.5 Organisms 80 6.6 Consumables 81 6.7 Equipment 81 6.8 Media and buffer 82 7. Methods 84 7.1 General methods 84 7.1.1 Extraction of DNA from biological samples 84 7.1.2 Purification of DNA by agarose gel electrophoresis 84 7.1.3 Quantification of DNA 84 7.1.4 Polymerase chain reaction 84 7.1.5 Endonucleolytic digestion of DNA for molecular cloning procedures 85 7.1.6 Dephosphorylation of DNA 85 7.1.7 Ligation of DNA 86 7.1.8 Genetic manipulation of model organisms 86 7.1.9 Sequence analysis 87 7.2 Supplementary methods: Results and Discussion 3.1 87 7.2.1 Plasmid construction 87 7.2.2 E. coli strain and growth conditions 87 7.2.3 Gene expression and quantification 88 7.2.4 Quantification of RNA-levels 88 7.3 Supplementary methods: Results and Discussion 3.2 89 7.3.1 Reagents 89 7.3.2 Cell culture and growth conditions 89 7.3.3 Plasmid construction 89 7.3.4 Construction and screening of aptazyme libraries 89 7.3.5 Reporter gene assays in S. cerevisiae 90 7.3.6 Mfold analysis 90 7.4 Supplementary methods: Results and Discussion 3.3 91 7.4.1 Plasmid construction for gene expression in A. thaliana 91 7.4.2 Plasmid construction for gene expression in C. elegans 91 7.4.3 Plasmid construction for gene expression in E. coli 91 7.4.4 Plasmid construction for gene expression in S. cerevisiae 92 7.4.5 Plasmid construction for gene expression in mammalian cells 92 7.4.6 Mammalian cell culture 92 7.4.7 Transfection of mammalian cells 92 7.4.8 Gene expression and quantification in S. cerevisiae 92 7.4.9 Gene expression and quantification in E. coli 93 8. References 94 9. Appendix 104 9.1 DNA Sequences: Results and Discussion 3.1 104 9.2 DNA sequences: Results and Discussion 3.2 106 9.3 DNA sequences: Results and Discussion 3.3 107 9.4 Plasmids 111 9.5 Plasmid maps 115 10. Record of contributions 116 11. Abbreviations 117 1. Introduction Synthetic biology is a rapidly emerging field that seeks to engineer biological systems to perform beneficial functions (1). Promised technological advances include the generation of tailor-made organisms for basic research, for therapeutical applications, for environmental issues, and for manufacturing chemicals, pharmaceuticals and biofuels. Synthetic biology is an engineering-driven discipline aiming for the application of robust and predictable genetic technologies to rationally design genetic networks by modifying and reassembling well characterized genetic devices (1). The functional complexity of artificial networks emerges from the interplay of individual genetic switches for tuning gene expression in response to a defined stimulus. A prerequisite for realizing the many goals of synthetic biology is the development of universal concepts that ease the generation and optimization of genetic switches to rapidly respond to environmental and cellular signals. Artificial RNA-based switches can be applied for precisely regulating gene expression at various stages without the need for protein factors (2; 3). For the development of novel concepts for artificial gene regulation it is important to have a thorough understanding of the general mechanism that rule natural gene expression and its regulation. 1.1 Basics of gene expression Cellular systems have evolved an intricate molecular machinery to maintain cellular homeostasis, to diversify, to process environmental signals and to self-replicate.