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Control of RNA Function by Conformational Design

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Control of RNA Function by Conformational Design DISSERTATION / DOCTORAL THESIS Titel der Dissertation /Title of the Doctoral Thesis Control of RNA function by conformational design verfasst von / submitted by Mag. rer. nat. Stefan Badelt angestrebter akademischer Grad / in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) Wien, 2016 / Vienna, 2016 Studienkennzahl lt. Studienblatt / A 794 685 490 degree programme code as it appears on the student record sheet: Dissertationsgebiet lt. Studienblatt / Molekulare Biologie field of study as it appears on the student record sheet: Betreut von / Supervisor: Univ.-Prof. Dipl.-Phys. Dr. Ivo Hofacker Design landscapes >Output Input AGGAACAGUCGCACU ACCCACCUCGACAUC AGCAACA GUAAAUCAAAUUGGA AGGAUCU ACUGAAGCCCUUGGU AGGAUCA CUGGAGUCACCAGGG objective function AGGAACA GGUUUACGUACUACU design Energy landscapes 0.7 0.7 >Input -8.0 Output 3.9 3.7 1.1 2.8 1.2 1.3 2.3 0.8 AGGAACAGUCGCACU 3.5 2.6 2.6 2.8 3.9 2.3 -10.0 3.0 4.2 1.2 0.7 2.3 1.2 4.4 1.3 0.599999 1.3 2.4 1.0 0.8 100 1.5 2.0 1.2 0.8 97 98 99 2.8 1.4 1.0 94 95 93 96 92 2.3 0.7 2.6 0.8 91 1.5 1.3 2.0 2.6 1.6 89 90 1.9 0.8 2.0 86 88 87 2.9 2.3 1.9 1.3 1.3 ACCCACCUCGACAUC 0.6 83 85 84 82 2.6 1.0 0.8 0.8 77 81 79 78 80 0.7 76 1.9 74 75 73 1.5 72 0.7 -12.0 0.8 0.8 70 71 1.1 0.799999 1.2 0.8 69 68 0.8 0.8 0.8 63 65 64 66 67 0.9 1.2 1.2 59 60 58 61 62 1.2 1.4 1.7 1.2 55 54 57 56 3.1 3.0 4.2 52 53 51 49 50 0.7 1.0 45 44 46 47 48 42 43 41 3.5 1.0 38 39 40 1.3 0.6 2.6 1.3 0.8 0.8 0.8 GUAAAUCAAAUUGGA 37 35 36 34 32 33 31 30 28 29 27 1.2 0.8 24 25 26 23 -14.0 22 20 21 2.4 17 18 19 2.2 16 0.8 0.8 ACUGAAGCCCUUGGU 0.8 0.8 15 3.5 14 3.5 13 2.6 2.6 12 free energy 11 10 9 -16.0 0.7 C G C C U C 1.4 C C A U C C C A G A A G 8 CUGGAGUCACCAGGG U C A U C 3.1 A A C A G 3.1 U U C A C G A U A 5 U G 7 6 A U U U U A A U C A U 4 C A U U G U 3 C U G G A C A U G U U C G A U C G A C A C U G G C G C C G U C A U C G C G G G G C G U U A G U A G U U G C G U U U G A A A U A G G A A G U U G -18.0 A G A A C A U A U GGUUUACGUACUACU G C A C U 2 1 modeling design modeling design modeling Reality stefan badelt initial state final state experimental setup final observation Cycling in energy and design landscapes CONTROLOFRNAFUNCTIONBYCONFORMATIONALDESIGN Stefan Badelt: Control of RNA function by conformational design: Cycling in energy and design landscapes A thesis submitted in partial fulfillment of the requirements for the degree of: Ph.D. at the Institut für Theoretische Chemie Fakultät für Chemie Universität Wien © January 2016 ABSTRACT RNAs play an essential role in the life cycle of every cell. RNA is not only the interme- diate between the genetic blueprint (DNA) and the proteins produced, but also per- forms a variety of regulatory tasks. The function of an RNA molecule is determined by its structure, which can be reasonably well predicted following the biophysical rules implemented in the most popular RNA structure prediction programs. I present four projects, two of them in form of a peer-reviewed publication, the other two are unpublished work with preliminary results. The first publication describes how RNAs with catalytic function, ribozymes, can be designed to concatenate multiple copies of themselves (i. e. they self-polymerize), into longer molecules. This is important since the RNA World hypothesis claims that RNA emerged before DNA and proteins. It has, however, been hard to imagine how suffi- ciently long RNA molecules could exist in the RNA world. Our results suggest how pre-biological RNA genomes may have been built up by concatenation of shorter se- quences. The second publication shows conformational switching of RNAs through the inter- action of two copies. Such a conformational self-replication was so far known only from proteins, where it forms the molecular basis of prion diseases such as Creutzfeldt- Jakob. The artificial RNAs designed in this project could help to better understand the mechanism of such diseases, but might also be useful as molecular sensors and amplifiers in biotechnology. A central challenge to both publications mentioned above are RNA-RNA interac- tions. While we have used thermodynamic criteria combined with existing algorithms for intramolecular folding kinetics to design sequences, we are now developing new algorithms to model folding kinetics of interacting RNAs. This problem is much more complicated by the fact that intermolecular base-pairing is concentration dependent. Preliminary results to model the kinetics of small interacting RNAs are promising and will serve as a basis for a separate, peer-reviewed publication. The last major topic addressed in this thesis concerns the synthesis of RNA molecules in cells, i. e. cotranscriptional folding. We first show how small metabolites can be in- cluded into an existing algorithm to model intramolecular folding during transcrip- tion. We have published these results also in the context of a recent book chapter on the computational modeling of riboswitches. However, the approach is limited to short RNA transcripts and we have now developed a faster heuristic to model cotran- scriptional folding for longer molecules. The results presented from this new program, DrTransformer, will also be used in a separate, peer-reviewed publication. v ZUSAMMENFASSUNG RNAs (Ribonukleinsäuren) spielen eine tragende Rolle im Lebenszyklus jeder Zelle. Sie bilden das Bindeglied zwischen dem genetischen Bauplan (DNA) und den da- raus erzeugten Proteinen und übernehmen eine Vielzahl von regulatorischen Auf- gaben. Diese Aufgaben werden großteils von der Molekülstruktur bestimmt, welche wiederum auf Grund experimenteller Daten und der daraus abgeleiteten physikalis- chen Regeln vorhergesagt werden kann. Im Rahmen dieser Dissertation werde ich vier Projekte vorstellen, die sich mit RNA Design und der Faltungskinetik interagieren- der RNAs beschäftigen. Zwei der Projekte sind bereits in öffentlichen Journalen er- schienen, für die beiden anderen werden vorläufige Resultate präsentiert die als Basis für eine separate Publikationen dienen. Die erste Publikation beschäftigt sich mit dem Design von RNAs mit katalytischer Funktion, sogenannten Ribozymen. Das Augenmerk liegt dabei auf Sequenzen die sich selbst prozessieren können, sodass mehrere Kopien des selben Moleküls aneinan- dergehängt werden. Die Resultate zeigen das Potential von RNAs im Ursprung des Lebens, nämlich dass präbiologische RNA-Genome aus kürzeren Sequenzen gebaut werden konnten. In der zweiten Publikation wurden RNAs designed, die die Möglichkeit haben Kopien von sich selbst von einer aktiven Struktur in eine andere umzufalten. Solche Mechanismen sind bisher nur für Proteine beschrieben worden. Die sogenannten Prio- nen sind Auslöser neurologischer Erkrankungen, z.B. von Creutzfeldt-Jakob. Biotech- nologish, können diese RNAs als molekulare Sensoren eingesetzt werden, aber sie kön- nen auch dabei helfen die molekularen Mechanismen von Prionen-Krankheitserregern besser zu verstehen. Eine zentrale Herausforderung der oben genannten Publikationen bestand darin RNA-RNA Interaktionen zu modellieren. Die Konzentrationsabhängikeit der beteiligten Moleküle macht eine genaue Vorhersage der kinetischen Prozesse komplizierter als für intramolekulare Faltung. Die Entwicklung eines Programs zur Modelierung von intermolekluarer Faltunskinetik, ermöglicht detailierte Simulationen von kurzen inter- agierenden RNAs. Das letzte Kapitel betrifft die Synthese von RNA in der Zelle, i. e. co-transkriptionelle Faltung. Anhand eines Beispiels wird beschrieben, wie man den Einfluss kleiner Metabo- liten in die Simulation von co-transcriptioneller Faltung einbeziehen kann. Das Beispiel wurde im Kontext eines Buchkapitels zur Modellierung von Riboswitches verwen- det. Zusätzlich wird hier ein neues Programm präsentiert, DrTransformer, um co- transcriptionelle Faltung auch für längere RNAs mit höherer Genauigkeit vorherzusagen, als es bisher möglich war. vi ACKNOWLEDGMENTS At first I want to thank my supervisor Ivo Hofacker for guiding me through my PhD studies and always being helpful with good advice. He gave me the freedom to follow my own research interests and, at the same time, pointed out different directions when I got stuck. He has changed my perspective on describing biological problems by explaining processes in the context of chemical and physical laws. During my PhD studies Christoph Flamm has been a constant source of inspiration and ideas. A lot of this work builds on his previous publications and he has impressed me with his playful approach to formulate scientific problems as small, exciting chal- lenges, or vice versa. I want to use this possibility to thank my love Anela Tosevska. Our adventures when we are traveling as well as our discussions on lazy days have given me the energy to focus on my work in rough times. Also, she has greatly improved this thesis with critical feedback.
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