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DNA and Peptide Aptamer Selection for Diagnostic Applications

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DNA and Peptide Aptamer Selection for Diagnostic Applications DNA and Peptide Aptamer Selection for Diagnostic Applications vorgelegt von Diplom-Ingenieurin Janine Michel aus Berlin Von der Fakultät III – Prozesswissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades Doktorin der Ingenieurwissenschaften -Dr.-Ing.- genehmigte Dissertation Promotionsausschuss: Vorsitzender: Prof. Dr. Leif-A. Garbe Berichter: Prof. Dr. Jens Kurreck Berichter: PD Dr. Andreas Nitsche Tag der wissenschaftlichen Aussprache: 27.09.2013 Berlin 2013 D83 To Olaf and my loving family, especially to grandpa Bernd. I miss you! I Acknowledgments This work would have been impossible to complete without the help of many persons, including colleagues, family, and friends. Since there are so many of them I cannot acknowledge every single contribution by name, but I would like to thank everyone who helped me through this demanding and challenging but interesting time, regardless of the type of support. Above all, I would like to thank Andreas Nitsche for giving me the opportunity to do my PhD project and for the continuous support. Many thanks go to my dear colleagues Lilija Miller and Daniel Stern who helped me especially in the beginning of this project. Thank you for introducing me to the basics of phage display and aptamers and for the fruitful and valuable scientific discussions and for frequent encouragement. I am grateful to Lilija Miller who helped me with phage display selections and subsequent peptide characterizations. Further, I would like to thank all members of the ZBS1 group for the friendly atmosphere and the company during lunch. A considerable contribution was made by students I supervised during my PhD project. Carolin Ulbricht, Daniel John and Alina Sobiech contributed to the “DNA aptamer selection and characterization project” during their bachelor’s thesis, internships, and master’s thesis, respectively. Guido Vogt contributed to the “peptide aptamer selection and characterization project” during his internship and his subsequent bachelor’s thesis. Wojtek Dabrowski contributed to the same project by programming the “Library Insert Finder” software, making the analysis of the numerous phage clones a lot easier. Furthermore, Wojtek Dabrowski performed the analysis of the NGS data and provided me with a workable amount of DNA sequences. In this context I would like to thank Aleksandar Radonić for introducing me to NGS library preparation and for the sequencing of these libraries. The staff of the sequencing lab, namely Julia Tesch, Julia Hinzmann, Silvia Muschter, Marlies Panzer, and Angelina Targosz, provided me with numerous DNA sequences. Thank you for this, I know it has been a lot of work. Many thanks go to Jörg Döllinger who performed the mass spectrometric analysis, to Janett Piesker who provided me with these wonderful EM pictures, and to Kazimierz Madela for the great help with the confocal laser scanning microscope pictures. Sincere thanks go to Jeffrey Drimmer and Ursula Erikli for copy-editing and continuously improving my English language skills. I am most grateful to my parents and my sister for always believing in me and supporting me in whatever I do. Thank you so much! Last but not least, I would like to thank my partner Olaf for the continuous support and always having an ear and the right words for me in times of despair. II Declaration of Authorship I certify that the work presented here is, to the best of my knowledge and belief, original and the result of my own investigations, except as acknowledged. The present work has not been submitted, either in part or in its entirety, for a degree at this or any other University. Berlin, Janine Michel III Abstract The availability of stable and reproducible detection methods is an important factor in the reliable detection and identification of common clinical infections, newly emerging pathogens, and bio-threat agents. Classical detection methods are based on the detection of nucleic acids or proteins, using monoclonal and polyclonal antibodies as detection molecules. While nucleic acid-based detection methods depend on the detection of the genetic material of pathogens, antibodies have some considerable limitations, including tedious and expensive production, limited durability, and stability. To circumvent the many limitations of antibody-based detection systems, there is an increasing interest in employing alternative recognition molecules for the detection and identification of pathogens. Such alternative molecules can be represented by aptamers. Aptamers are short synthetic molecules that can comprise of nucleic or amino acids. They are selected in vitro from huge libraries of molecules containing up to several billion randomly created sequences that can bind with high affinity and specificity to literally any target. Screenings of random synthetic DNA- and bacteriophage-based libraries were utilized in the present study to evaluate the applicability of synthetic nucleic acid and peptide aptamers for poxvirus detection. First, a suitable protocol for the selection of DNA aptamers using SELEX (systematic evolution of ligands by exponential enrichment) was established, and the amplification step was optimized using the Taguchi approach. Together with different random DNA libraries against native vaccinia virus particles, SELEX resulted in the identification of 6 oligonucleotide sequences, using the classical cloning and sequencing approach. The massively parallel sequencing of enriched DNA libraries resulted in identification of further 24 oligonucleotide sequences. These oligonucleotide sequences were characterized to determine affinities, specificities, and cross-reactivities. Of these 24 clones, 15 oligonucleotide sequences bound specifically to vaccinia virus particles and were characterized further. These 15 aptamers could be utilized for the detection of different poxvirus species in a sandwich assay in combination with a monoclonal anti-vaccinia antibody. Additionally, peptide aptamers for the specific detection of poxvirus particles were selected. For this, the combinatorial phage display methodology was used. Affinity selections of random peptide phage display libraries resulted in 17 recurring peptides, indicating the enrichment of specific vaccinia virus-binding phage clones. After characterization of these 17 phage clones, five peptide sequences were synthesized and characterized. One phage- derived synthetic peptide (VV1) was able to bind specifically to vaccinia virus particles with the poxviral surface protein D8 as the interaction partner of VV1. The functionality of VV1 as capture molecule in combination with a polyclonal anti-vaccinia antibody could be shown. Furthermore, VV1 was successfully applied for the detection of different poxvirus species. At last, the successful combination of the selected and characterized SELEX-derived DNA- and phage display-derived peptide aptamers for the detection of vaccinia viruses in an aptamer-based sandwich assay could be shown. IV Zusammenfassung Ein wichtiger Faktor für die zuverlässige Detektion und Identifizierung verbreiteter klinischer Infektionen, neuartiger Pathogene und Bioterror-Agenzien ist die Verfügbarkeit stabiler und reproduzierbarer Detektionsmethoden. Klassische Detektionsmethoden basieren auf dem Nachweis von Nukleinsäuren oder Proteinen unter Verwendung monoklonaler und polyklonaler Antikörper. Während Nukleinsäure-basierte Detektionsmethoden darauf angewiesen sind, das genetische Material von Pathogenen nachzuweisen, weisen Antikörper erhebliche Nachteile auf. Diese Nachteile umfassen deren aufwändige und teure Produktion und eingeschränkte Stabilitäten. Auf Grund der vielfältigen Limitationen Antikörper-basierter Detektionsmethoden gibt es ein gesteigertes Interesse an der Verwendung alternativer Detektionsmoleküle. Aptamere können solch alternative Moleküle darstellen. Aptamere sind kurze, synthetische Moleküle, die aus Nuklein- oder Aminosäuren bestehen und mit hoher Affinität und Spezifität an buchstäblich jedes Zielmolekül binden können. Sie werden in vitro aus riesigen Bibliotheken selektiert, die mehrere Milliarden randomisierte Sequenzen enthalten können. Hier wurden randomisierte DNA- und Bakteriophagen-basierte Bibliotheken verwendet, um die Anwendbarkeit synthetischer Nukleinsäure- und Peptidaptamere für die Detektion von Pockenviren zu evaluieren. Zunächst wurde ein Protokoll für die Selektion von DNA- Aptameren unter Verwendung der SELEX (systematic evolution of ligands by exponential enrichment) Technologie etabliert und der Amplifikationsschritt mit Hilfe der Taguchi- Methode optimiert. Die Selektion gegen native Vaccinia-Virus-Partikel, unter Anwendung des klassischen Klonierungs- und Sequenzierungsansatzes, resultierte in der Identifizierung von 6 Oligonukleotiden. Die Hochdurchsatzsequenzierung angereicherter DNA-Bibliotheken führte zur Identifizierung 24 weiterer Oligonukleotide. Diese Oligonukleotide wurden hinsichtlich ihrer Affinitäten, Spezifitäten und Kreuzreaktivitäten charakterisiert. Von den 24 Oligonukleotiden banden 15 spezifisch an Vaccinia-Virus-Partikel, wurden weiter charakterisiert und konnten erfolgreich für die Detektion verschiedener Pockenviren in Kombination mit einem monoklonalen anti-Vaccinia-Antikörper eingesetzt werden. Zusätzlich zu den DNA-Aptameren über SELEX wurden Peptidaptamere als Pockenvirus- spezifische Detektionsmoleküle über Phagen-Display selektiert. Die Affinitätsselektion randomisierter Peptid-Phagen-Display-Bibliotheken resultierte in 17 wiederkehrenden Peptiden. Nach
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