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Nucleic Acid/Inorganic Particle Hybrid Systems from Design to Application Research Collection Doctoral Thesis Nucleic acid/inorganic particle hybrid systems from design to application Author(s): Puddu, Michela Publication Date: 2015 Permanent Link: https://doi.org/10.3929/ethz-a-010609177 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library DISS. ETH No. 23177 NUCLEIC ACID/INORGANIC PARTICLE HYBRID SYSTEMS: FROM DESIGN TO APPLICATION A thesis submitted to attain the degree of DOCTOR OF SCIENCES of ETH ZURICH (Dr. sc. ETH Zurich) presented by MICHELA PUDDU MSc Materials Science born on 02.03.1987 citizen of Italy accepted on the recommendation of Prof. Dr. Wendelin J. Stark, examiner Prof. Dr. János Vörös, co-examiner Dr. Robert N. Grass, co-examiner 2015 2 3 To my parents “Considerate la vostra semenza: fatti non foste a viver come bruti, ma per seguir virtute e canoscenza." Dante Alighieri, Divina Commedia, Inferno, canto XXVI, vv. 18-20. 4 5 Acknowledgments The PhD has been a wonderful and exciting experience, and I want to take a moment to thank the many people who took part in this journey. Foremost, I would like to express my gratitude to Prof. Wendelin Stark for giving me the opportunity to do research and develop a mature scientific approach in a highly stimulating and innovative environment. I am indebted to him for providing me with entrepreneurial spirit and vision that influenced my thinking and career choice. I also thank him for the precious lesson that a balance between research interests and personal pursuits does not keep away success, but it actually brings one step closer to it. My most sincere thanks go to my advisor, Dr. Robert Grass, who oriented and supported me with enthusiasms and promptness through the wonders and frustrations of scientific research. He has been a great inspiration for me, a dedicated teacher and mentor, taking care of both my academic, professional and personal development. It has been a real pleasure to work under his supervision, with heaps of fun and excitement, and the imparted knowledge will be a great help throughout my career and life. I kindly acknowledge Prof. János Vörös for accepting to co-examine my dissertation, concluding together the path started few years ago from his lab, where he firstly welcomed me at ETH for my master thesis project, opening up a range of new opportunities for me. I want to express my gratitude to Prof. Marcy Zenobi-Wong for hosting me in her lab whenever needed, and for the fruitful scientific discussions. Furthermore, I would like to thank Frank Krumeich, for the high quality transmission electron microscopy and energy- dispersive X-ray spectroscopy analysis, and Hanspeter Hächler for the magnetic hysteresis measurements. Many thanks go to Vladimir Zlateski for the great support and the enjoyable time we had together within and outside the lab, in Zurich and around the world. I am grateful to Daniela Paunescu for her contribution both as co-worker and friend. I thank Carlos Mora for the stimulating discussions and exchange of ideas (scientific and non). My most recent work and achievements have benefited from the suggestions and aid of Gediminas Mikutis, who is sincerely acknowledged. I thank Dirk, my office mate, neighbour, eventually trainer, for helping and entertaining me in the lab and outside. I would also like to extend my gratitude to all the other current and former member of this research group for their help and for the cheerful atmosphere: Mirjam, Mario, Elia, Samuel Hess, Philipp, Corinne, Antoine, Lukas, 6 Michael Loepfe, Tino, Christoph Kellenberger, Renzo, Jonas, and Nora. It was fun and pleasant to work and spend time with them all. I want to thank Dr. Neil D. Telling for his support and instructive interaction during my secondment at Keele University. I am grateful to all the “Keele gang” members for their support and for providing a pleasant working atmosphere: Dalibor Soukup, Kaarjel Narayanasamy, Antonella Lisella, and Eva Luther. I thank Rebmann Balder from Freiburg University, with whom I had a productive collaboration and fun time. I would also like to acknowledge my friend Christopher Millan, always available to share ideas and help. My sincere appreciation is extended to my German teacher and friend Vital Lutz, who made the learning of this complicated language a pleasant and entertaining activity. I am most grateful to my beloved parents for their unconditional faith in me and for letting me to be as ambitious as I want (under their watchful eye). The love, help and opportunities they have given me over the years have been essential to me, and their ability to tackle challenges has always been exemplary and inspiring. A special thank goes to my partner Vincent Dabir for his constant patience and support in my work as well in life. This work has been financially supported from the EU-ITN network Mag(net)icFun (PITN- GA-2012-290248), which is kindly acknowledged. 7 Table of contents Acknowledgments 5 Zusammenfassung 11 Summary 13 1 Nucleic acid nanotechnology: the past, the present and the future 15 1.1 The nucleic acid era 16 1.2 The arise of nucleic acid nanotechnology 17 1.3 Nucleic acid interaction with inorganic particles 20 1.4 Nucleic acid as structural elements 22 1.4.1 Hybridization reaction-directed assembly of inorganic particles 22 1.4.2 Nucleic acid-templated fabrication of inorganic particles 23 1.5 Nucleic acid as functional elements 25 1.5.1 Information storage 25 1.5.2 Nucleic acid-delivery systems 27 1.5.3 Sensing 30 2 Magnetically recoverable, thermostable, hydrophobic DNA/silica encapsulates and their application as invisible oil tags 33 2.1 Introduction 34 2.2 Experimental section 36 2.2.1 Particle synthesis 36 2.2.2 Particle characterization 37 2.2.3 DNA recovery 37 2.2.4 qPCR standard curves 38 2.2.5 DNA absolute quantification 38 2.2.6 Thermal stability 39 2.2.7 Sanger sequencing 39 2.3 Results and discussion 39 2.4 Conclusion 49 3 Silica microcapsules for long-term, robust and reliable room temperature RNA preservation 51 3.1 Introduction 52 3.2 Experimental section 54 3.2.1 Handling RNA 54 3.2.2 RNA sources 54 3.2.3 SiO2/RNA microcapsule synthesis 54 3.2.4 Microcapsule characterization 55 8 3.2.5 RNA recovery 55 3.2.6 Gel electrophoresis 55 3.2.7 One-step RT-qPCR 55 3.2.8 RNA absolute quantification 56 3.2.9 Reactive oxygen species treatment 56 3.2.10 RNase treatment 56 3.2.11 Long term RNA stability 56 3.2.12 Capillary electrophoresis 57 3.2.13 Sanger sequencing 57 3.3 Results and discussion 57 3.4 Conclusion 65 4 Magnetically deliverable calcium phosphate nanoparticles for localized gene expression 67 4.1 Introduction 68 4.2 Experimental section 69 4.2.1 Nanoparticle production 69 4.2.2 Nanoparticle characterization 70 4.2.3 Plasmid preparation 70 4.2.4 DNA binding assay 71 4.2.5 Cell culture 71 4.2.6 Transfection of mammalian cells 71 4.2.7 Localization of gene expression 73 4.2.8 Live/dead assay 73 4.2.9 Microscopy 73 4.2.10 Cell counting 73 4.2.11 Statistical analysis 74 4.3 Results and discussion 74 4.4 Conclusion 82 5 Submicrometer-sized thermometer particles exploiting selective nucleic acid stability 83 5.1 Introduction 84 5.2 Experimental section 85 5.2.1 Nucleic acids 85 5.2.2 Iron oxide particle synthesis 86 5.2.3 Encapsulate synthesis 86 5.2.4 Nucleic acid recovery 86 5.2.5 Encapsulate characterization 87 5.2.6 Reactive oxygen species treatment 87 9 5.2.7 qPCR and RT-qPCR analysis 87 5.2.8 Nucleic acid absolute quantification 88 5.2.9 Temperature tests 88 5.2.10 Kinetic studies 88 5.2.11 Calibration curves 89 5.3 Results and discussion 89 5.4 Conclusion 94 6 Conclusion and outlook 95 Appendix 99 A.1 Supporting information to chapter 2 100 A.1.1 Particle density 100 A.1.2 Particle sedimentation velocity 100 A.1.3 Particle aggregation time 101 A.1.4 Independent two-sample t-test 101 A.2 Supporting information to chapter 3 106 A.2.1 Kinetics 106 A.2.2 Simplified procedure for RT-qPCR 107 A.3 Supporting information to chapter 4 110 A.3.1 Particle density 110 A.3.2 Primary particle size 110 A.3.3 Particle size dispersity 110 A.3.4 Number of particles per aggregate 110 A.3.5 Particle long-term storage 111 A.4 Supporting information to chapter 5 115 References 119 10 11 Zusammenfassung Hybridsysteme aus Nukleinsäuren und anorganischen Partikel kombinieren die einzigartigen chemischen und physikalischen Eigenschaften der anorganischen Partikel mit der Hybridisierungs- und Codierungsfähigkeit von Nukleinsäuren. Diese Ausgangsmaterialien sind vielversprechend für die Entwicklung von neuartigen und intelligenten Materialien für biologische und nichtbiologische Anwendungen. Die vorliegende Doktorarbeit setzt sich mit den Fortschritten in der Entwicklung und Anwendung von Nukleinsäure/anorganischen Partikel-Hybriden in verschiedenen Disziplinen auseinander. Kapitel 1 fasst die Auswirkungen der Nukleinsäure-Forschung auf unsere Gesellschaft unter der Betrachtung verschiedener Aspekte zusammen. Es wird ein Überblick über den Wissensstand im Bereich der Manipulation von Nukleinsäuren zur Herstellung von supramolekularer Strukturen aufgezeigt. Zudem wird erklärt wie die Nukleinsäure- Technologie zusammen mit der Partikel-Technologie verwendet werden kann, um die Möglichkeiten der Nukleinsäuren in verschiedenen Bereichen, wie beispielsweise dem "Bottom-up" Aufbau von anorganischen Partikeln, in der Informationsüberlieferung und – speicherung und in der Sensorik, aufzuzeigen.
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