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Sequence-Addressed Assemblies of Trisoligonucleotides Into Nanoscale Motifs and Structural Studies

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Sequence-Addressed Assemblies of Trisoligonucleotides Into Nanoscale Motifs and Structural Studies Sequence-Addressed Assemblies of Trisoligonucleotides into Nanoscale Motifs and Structural Studies Christos Panagiotidis Dissertation zur Erlangung des Grades “Doktor der Naturwissenschaften” an der Fakultät für Chemie und Biochemie der Ruhr-Universität Bochum Bochum/Wuppertal 2016 This dissertation is based on the research work carried out during the period April 2012 to December 2015 at the chair of Organic Chemistry I, Bioorganic Chemistry of the Ruhr- University Bochum, Germany. First referee: Prof. Dr. Günter von Kiedrowski Second referee: Prof. Dr. Frank Schulz Day of submission: 2. Dec. 2015 Day of disputation: 29. Feb. 2016 Herewith I declare that the following work has been carried out independently by myself and all the sources of help and services used during this work have been reported herein. I further declare that I have not submitted this thesis in this or in a similar form to any other university or college. No competing interest is declared. Christos Panagiotidis, December 2015 Danksagungen (Acknowledgements) Ich danke Herrn Prof. Dr. Günter von Kiedrowski für das interessante Forschungsgebiet, die Möglichkeit zur freien Gestaltung und Durchführung der Arbeit, der Diskussionsbereitschaft und interessanten Gesprächen über der Arbeit hinaus. Auch danke ich Herrn Prof. Dr. Frank Schulz für die Übernahme des Koreferates. Herrn Dr. Wolf Matthias Pankau danke ich ebenfalls für die ständige Diskussionsbereitschaft und Herrn Dr. Volker Patzke für das Korrekturlesen des Manuskripts. Herrn Michael „Stoßrohr“ Wüstefeld danke ich für die Durchführung der automatisierten Synthesen, für das offene Ohr und die regen Unterhaltungen. Frau Katja Schulz danke ich für ihre Tätigkeit als chemisch-technische Assistentin, ihre stete Unterstützung beim bioorganischen Praktikum und der freundlichen Atmosphäre. Florian Kaschuba und Carsten Lodwig danke ich für die Behebung technischer Ungereimtheiten. Frau Stephanie Nolte aus der Hollmann-Gruppe danke ich für Einweisung und Nutzung des Bioimagers. Der Bürofee Stefanie Wittmann und ihren zwei Hunden wünsche ich alles Gute im weiteren Leben. Lache immer laut und herzhaft. Meinen Kollegen und Mitstreitern sei herzlichst für die recht entspannte Arbeitsatmosphäre unter Berücksichtigung des allwährenden Wahnsinns gedankt. Wir sind bekloppt und das ist auch gut so. Das wären Daniel Kramer, Nora Leistner, Chris Nielinger, Elena Palmieri, Miriam Patzke, Jana Pyka, Berit Sorge, aber vor allem Markus Rethmeier, der am meisten von meinem Gejammer abbekommen hat und Ilhan Sevim für die gelegentlichen philosophischen Unterhaltungen. Allen Studenten (bzw. das billige Kanonenfutter) aus den Praktika, den Abschlussarbeiten und dem Labor sei gedankt, aber vor allem Sebastian Michalski, Elric Engelage, Holger Rabuske und Ivan Grgic. Bei Herrn Dimitri Kolmanovsky erinnere ich mich gerne an den hilfreichen E-Mail-Austausch speziell am Anfang der Arbeit. Ich danke meiner Familie. “Just a heads up: We're gonna have a superconductor turned up full blast and pointed at you for the duration of this next test. I'll be honest, we're throwing science at the wall here to see what sticks. No idea what it'll do. Probably nothing. Best-case scenario, you might get some superpowers. Worst case, some tumors, which we'll cut out.” Cave Johnson, CEO of Aperture Science, Inc. (Portal 2) Abstract Trisoligonucleotides have been previously established as building blocks in the construction of DNA-based nanostructures, like tetrahedral and dodecahedral scaffolds, possible by a sequence-addressed self-assembly. The resulting care-like shapes are potentially usable as nanoscale containers. Trisoligonucleotides may contain C3h-symmetrical linkers that connect three oligonucleotide arms. One previously established linker was based on 1,3,5-trishydroxypropylbenzene, but required a lengthy and laborious synthesis over up to twelve steps. This is a bottle-neck in subsequent studies with DNA. Six steps of the previously applied linker synthesis pathway have been cut by following a Heck-coupling route, which improved the overall yield. Additionally, a new generation of isocyanurate-based linkers was introduced, which even further shortened preparation to just two or three steps, respectively. Linkers require alkylene chains to allow folding of the single-strands into polyhedral scaffolds. So far studies on the flexibility of methylene- and propylene-chains exist and not for ethylene-chains. Sets of trisoligonucleotides for tetrahedral assemblies were prepared via automated synthesis, purified via preparative polyacrylamide gel electrophoresis and confirmed by MALDI-TOF-MS. Hybridisation products were studied with agarose gel electrophoresis and showed relative stability against enzymatic digestion with mung bean nuclease, which in our hands is a proof for the existence of discrete closed nanoobjects. Likewise, isocyanurate-based trisoligonucleotides shared similar results. An alternative sequence-pattern for trisoligonucleotides was studied, namely, two of three arms sharing an identical oligonucleotide sequence leading to situations below so called maximal instruction. A set of these semi-addressable building blocks were assembled and studied with native agarose gel electrophoresis. New conceivable motifs were accessible expanding the repertoire of trisoligonucleotide-based nanostructures. Tetrahedral scaffolds were intercalated with the anti-cancer drugs dauno- and doxorubicin. UV/VIS spectroscopy and fluorescence quenching experiments were done in comparison to linear oligonucleotides and hint at additional binding pockets close to the vertices of the tetrahedron enhancing the maximally possible binding capacity of duplex DNA. Keywords: structural DNA nanotechnology; C3h-symmetrical linker; isocyanurate; trisoligonucleotide; Sequence- addressability; self-assembly; DNA tetrahedron; nanosynthesis; anthracycline; intercalation; fluorescence quenching Content Content Prologue ........................................................................................................................................... 1 I. Theoretical Background.............................................................................................................. 2 I.1. Nanotechnology ..................................................................................................................... 2 I.2. Deoxyribonucleic acid .......................................................................................................... 3 I.3. DNA Nanotechnology ........................................................................................................... 8 I.4. Chemically unmodified DNA Nanostructures .................................................................. 10 I.5. Chemically modified DNA Nanostructures ....................................................................... 13 I.6. Static DNA Nanostructures for Drug Delivery .................................................................. 21 II. Aim of Work .............................................................................................................................. 23 III. Results and Discussion .......................................................................................................... 25 III.1. Synthesis of C3h-Symmetrical Trislinkers ...................................................................... 25 III.2. Oligonucleotide Synthesis ............................................................................................... 34 III.2.1. Trisoligonucleotide Synthesis Protocol ................................................................... 34 III.2.2. Phosphoramidite Oligonucleotide Synthesis ......................................................... 37 III.2.3. Sequence Design ....................................................................................................... 39 III.2.4. Purification and Quality Control ............................................................................... 43 III.2.5. Long-Term Stability Control ..................................................................................... 47 III.2.6. Solid Supports for Trisoligonucleotide Synthesis .................................................. 48 III.2.7. Fluorous Affinity Purification Studies ..................................................................... 55 III.3. DNA-Tetrahedron Assembly Experiments ................................................................... 68 III.3.1. Hybridization Conditions ............................................................................................ 68 III.3.2. Effect of short Alkylene Chains in T1, T2 and T3 Assembly Studies..................... 70 III.3.3. Assembly and Digestion Experiments of TN .......................................................... 75 III.3.4. Intermixing Assembly Experiments between T1, TN and T3 ................................ 76 III.4. The Concept of UNO-, DOS- and TRE- Sequence Patterns.......................................... 78 Content III.4.1. Introduction ................................................................................................................. 78 III.4.2. Hybridization and Digestion of the TRE-Pattern TY ............................................... 79 III.4.3. Hybridization and Digestion of the DOS-Pattern TD .............................................. 82 III.4.4. Sequence-Addressed Motif Designs via Intermixing Hybridizations .................. 86 III.5. Anthracycline Intercalation ............................................................................................
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