TY - JOUR A1 - Kielar, Charlotte A1 - Xin, Yang A1 - Xu, Xiaodan A1 - Zhu, Siqi A1 - Gorin, Nelli A1 - Grundmeier, Guido A1 - Möser, Christin A1 - Smith, David M. A1 - Keller, Adrian T1 - Effect of staple age on DNA origami nanostructure assembly and stability JF - Molecules N2 - DNA origami nanostructures are widely employed in various areas of fundamental and applied research. Due to the tremendous success of the DNA origami technique in the academic field, considerable efforts currently aim at the translation of this technology from a laboratory setting to real-world applications, such as nanoelectronics, drug delivery, and biosensing. While many of these real-world applications rely on an intact DNA origami shape, they often also subject the DNA origami nanostructures to rather harsh and potentially damaging environmental and processing conditions. Furthermore, in the context of DNA origami mass production, the long-term storage of DNA origami nanostructures or their pre-assembled components also becomes an issue of high relevance, especially regarding the possible negative effects on DNA origami structural integrity. Thus, we investigated the effect of staple age on the self-assembly and stability of DNA origami nanostructures using atomic force microscopy. Different harsh processing conditions were simulated by applying different sample preparation protocols. Our results show that staple solutions may be stored at -20 degrees C for several years without impeding DNA origami self-assembly. Depending on DNA origami shape and superstructure, however, staple age may have negative effects on DNA origami stability under harsh treatment conditions. Mass spectrometry analysis of the aged staple mixtures revealed no signs of staple fragmentation. We, therefore, attribute the increased DNA origami sensitivity toward environmental conditions to an accumulation of damaged nucleobases, which undergo weaker base-pairing interactions and thus lead to reduced duplex stability. KW - DNA origami KW - atomic force microscopy KW - stability KW - storage Y1 - 2019 U6 - https://doi.org/10.3390/molecules24142577 SN - 1420-3049 VL - 24 IS - 14 PB - MDPI CY - Basel ER - TY - THES A1 - Möser, Christin T1 - Modular DNA constructs for oligovalent bio-enhancement and functional screening T1 - Modulare DNA-Konstrukte für oligovalente Bio-Verstärkung und funktionelles Screening N2 - Deoxyribonucleic acid (DNA) nanostructures enable the attachment of functional molecules to nearly any unique location on their underlying structure. Due to their single-base-pair structural resolution, several ligands can be spatially arranged and closely controlled according to the geometry of their desired target, resulting in optimized binding and/or signaling interactions. This dissertation covers three main projects. All of them use variations of functionalized DNA nanostructures that act as platform for oligovalent presentation of ligands. The purpose of this work was to evaluate the ability of DNA nanostructures to precisely display different types of functional molecules and to consequently enhance their efficacy according to the concept of multivalency. Moreover, functionalized DNA structures were examined for their suitability in functional screening assays. The developed DNA-based compound ligands were used to target structures in different biological systems. One part of this dissertation attempted to bind pathogens with small modified DNA nanostructures. Pathogens like viruses and bacteria are known for their multivalent attachment to host cells membranes. By blocking their receptors for recognition and/or fusion with their targeted host in an oligovalent manner, the objective was to impede their ability to adhere to and invade cells. For influenza A, only enhanced binding of oligovalent peptide-DNA constructs compared to the monovalent peptide could be observed, whereas in the case of respiratory syncytial virus (RSV), binding as well as blocking of the target receptors led to an increased inhibition of infection in vitro. In the final part, the ability of chimeric DNA-peptide constructs to bind to and activate signaling receptors on the surface of cells was investigated. Specific binding of DNA trimers, conjugated with up to three peptides, to EphA2 receptor expressing cells was evaluated in flow cytometry experiments. Subsequently, their ability to activate these receptors via phosphorylation was assessed. EphA2 phosphorylation was significantly increased by DNA trimers carrying three peptides compared to monovalent peptide. As a result of activation, cells underwent characteristic morphological changes, where they "round up" and retract their periphery. The results obtained in this work comprehensively prove the capability of DNA nanostructures to serve as stable, biocompatible, controllable platforms for the oligovalent presentation of functional ligands. Functionalized DNA nanostructures were used to enhance biological effects and as tool for functional screening of bio-activity. This work demonstrates that modified DNA structures have the potential to improve drug development and to unravel the activation of signaling pathways. N2 - Desoxyribonukleinsäure (DNS, engl. DNA) - Nanostrukturen ermöglichen die Anbringung funktioneller Moleküle an nahezu jede einzigartige Stelle der zugrunde liegenden Struktur. Aufgrund der Basenpaar-Strukturauflösung von DNA können mehrere Moleküle (z.B. Liganden) entsprechend der Geometrie ihres gewünschten Ziels räumlich angeordnet und genau kontrolliert werden, was zu optimierten Bindungs- und/oder Signalwechselwirkungen führt. Diese Dissertation umfasst drei Hauptprojekte. Alle Projekte verwenden Varianten von funktionalisierten DNA-Nanostrukturen, die als Plattform für die oligovalente Präsentation von Liganden dienen. Ziel der vorliegenden Arbeit war es, die Fähigkeit von DNA-Nanostrukturen zur präzisen Positionierung verschiedener Arten von funktionellen Molekülen zu evaluieren und folglich die Wirksamkeit der Moleküle gemäß dem Konzept der Multivalenz zu erhöhen. Außerdem wurde untersucht, wie funktionalisierte DNA-Strukturen in verschiedenen Verfahren zur Erforschung von biologischen Interaktionen eingesetzt werden können. Die entwickelten DNA-basierten Liganden wurden verwendet, um Strukturen auf verschiedenen biologischen Systemen gezielt zu binden. In einem Teil dieser Dissertation wurde versucht, Krankheitserreger mit kleinen modifizierten DNA-Nanostrukturen zu binden. Pathogene, wie Viren und Bakterien, sind für ihre multivalente Anheftung an Wirtszellmembranen bekannt. Durch die oligovalente Blockierung ihrer Rezeptoren für die Erkennung und/oder Fusion mit ihrem Wirt sollte ihre Fähigkeit, sich an Zielzellen anzuheften und in diese einzudringen, beeinträchtigt werden. Bei Influenza A Viren konnte nur eine verstärkte Bindung von oligovalenten Peptid-DNA-Konstrukten im Vergleich zu monovalenten Peptiden beobachtet werden, wohingegen bei Respiratorischen Synzytial-Viren (RSV) sowohl die Bindung als auch die Blockierung der Zielrezeptoren zu einer verstärkten Hemmung der Infektion in vitro führte. Im letzten Teil wurden chimäre DNA-Peptidkonstrukte auf ihre Fähigkeit, an Signalrezeptoren auf der Oberfläche von Zellen zu binden und diese zu aktivieren, getestet. Die spezifische Bindung von mit bis zu drei Peptiden konjugierten DNA-Trimeren an EphA2-Rezeptor-exprimierende Zellen wurde in Durchflusszytometrie-Experimenten untersucht. Anschließend wurde ihre Fähigkeit, diese Rezeptoren durch Phosphorylierung zu aktivieren, beurteilt. Die Phosphorylierung von EphA2 war durch DNA-Trimere, die drei Peptide trugen, im Vergleich zu monovalenten Peptiden signifikant erhöht. Infolge der Aktivierung kommt es zu charakteristischen morphologischen Veränderungen der Zellen, bei denen diese ihre Peripherie "abrunden" und zurückziehen. Die in dieser Arbeit erzielten Ergebnisse beweisen umfassend die Fähigkeit von DNA-Nanostrukturen, als stabile, biokompatible, kontrollierbare Plattformen für die oligovalente Präsentation funktioneller Liganden zu fungieren. Funktionalisierte DNA-Nanostrukturen wurden zur Verstärkung biologischer Effekte und als Werkzeug für das funktionelle Screening von biologischen Interaktionen verwendet. Diese Arbeit zeigt, dass modifizierte DNA-Strukturen das Potenzial haben, die Medikamentenentwicklung zu verbessern und die Aktivierung von Signalwegen zu entschlüsseln. KW - DNA KW - multivalency KW - influenza KW - respiratory syncytial virus KW - nanostructure KW - ephrin KW - DNA KW - Ephrin KW - Influenza KW - Multivalenz KW - Nanostruktur KW - Respiratorisches Synzytial-Virus KW - DNS Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-507289 ER - TY - JOUR A1 - Möser, Christin A1 - Lorenz, Jessica S. A1 - Sajfutdinow, Martin A1 - Smith, David M. T1 - Pinpointed Stimulation of EphA2 Receptors via DNA-Templated Oligovalence JF - International journal of molecular sciences N2 - DNA nanostructures enable the attachment of functional molecules to nearly any unique location on their underlying structure. Due to their single-base-pair structural resolution, several ligands can be spatially arranged and closely controlled according to the geometry of their desired target, resulting in optimized binding and/or signaling interactions. Here, the efficacy of SWL, an ephrin-mimicking peptide that binds specifically to EphrinA2 (EphA2) receptors, increased by presenting up to three of these peptides on small DNA nanostructures in an oligovalent manner. Ephrin signaling pathways play crucial roles in tumor development and progression. Moreover, Eph receptors are potential targets in cancer diagnosis and treatment. Here, the quantitative impact of SWL valency on binding, phosphorylation (key player for activation) and phenotype regulation in EphA2-expressing prostate cancer cells was demonstrated. EphA2 phosphorylation was significantly increased by DNA trimers carrying three SWL peptides compared to monovalent SWL. In comparison to one of EphA2’s natural ligands ephrin-A1, which is known to bind promiscuously to multiple receptors, pinpointed targeting of EphA2 by oligovalent DNA-SWL constructs showed enhanced cell retraction. Overall, we show that DNA scaffolds can increase the potency of weak signaling peptides through oligovalent presentation and serve as potential tools for examination of complex signaling pathways. KW - DNA nanostructure KW - ephrin KW - EphA2 KW - SWL KW - PC-3 cells KW - multivalence Y1 - 2018 U6 - https://doi.org/10.3390/ijms19113482 SN - 1422-0067 VL - 19 IS - 11 PB - MDPI CY - Basel ER - TY - GEN A1 - Möser, Christin A1 - Lorenz, Jessica S. A1 - Sajfutdinow, Martin A1 - Smith, David M. T1 - Pinpointed stimulation of EphA2 receptors via DNA-templated oligovalence T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - DNA nanostructures enable the attachment of functional molecules to nearly any unique location on their underlying structure. Due to their single-base-pair structural resolution, several ligands can be spatially arranged and closely controlled according to the geometry of their desired target, resulting in optimized binding and/or signaling interactions. Here, the efficacy of SWL, an ephrin-mimicking peptide that binds specifically to EphrinA2 (EphA2) receptors, increased by presenting up to three of these peptides on small DNA nanostructures in an oligovalent manner. Ephrin signaling pathways play crucial roles in tumor development and progression. Moreover, Eph receptors are potential targets in cancer diagnosis and treatment. Here, the quantitative impact of SWL valency on binding, phosphorylation (key player for activation) and phenotype regulation in EphA2-expressing prostate cancer cells was demonstrated. EphA2 phosphorylation was significantly increased by DNA trimers carrying three SWL peptides compared to monovalent SWL. In comparison to one of EphA2’s natural ligands ephrin-A1, which is known to bind promiscuously to multiple receptors, pinpointed targeting of EphA2 by oligovalent DNA-SWL constructs showed enhanced cell retraction. Overall, we show that DNA scaffolds can increase the potency of weak signaling peptides through oligovalent presentation and serve as potential tools for examination of complex signaling pathways. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1041 KW - DNA nanostructure KW - ephrin KW - EphA2 KW - SWL KW - PC-3 cells KW - multivalence Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-468828 SN - 1866-8372 IS - 1041 ER -