Nukleinsäuren und Peptide erlauben es, Kontrolle über molekulare Prozesse auszuüben. In dieser Arbeit werden strukturgebende Elemente wie Coiled-Coil-Peptide oder PNA∙DNA-Strukturen genutzt, um Rezeptoren auf lebenden Zellen zu markieren und in ihrem Verhalten zu modulieren, oder cytosolische Proteine in ihrem Bindungsverhalten zu steuern. Im ersten Konzept wird die Interaktion des Coiled-Coil-Paars K3/E3 genutzt, um eine Transferreaktion, in welcher eine PNA-Sequenz vom K3-Donor auf den E3-Akzeptor übertragen wird, zu induzieren. Durch die Fusion des Akzeptorpeptids mit einem Rezeptor werden kovalente PNA-Rezeptorkonjugate auf der Oberfläche lebender Zellen geschaffen. Die Reaktion zwischen Thiol und Thioester erlaubt dabei einen schnellen Transfer. So wurden Rezeptoren aus der Familie der GPCR sowie der EGFR mit einem PNA-Strang versehen und durch fluoreszente PNA oder DNA selektiv markiert. Zusätzlich wurden verzweigte DNA-Architekturen mit mehreren Fluorophoren genutzt, um die Helligkeit der Markierung quantitativ zu erhöhen. Die PNA-EGFR-Konjugate wurden durch eine zwei Rezeptoren verbrückende Cy3-DNA adressiert und so zeitgleich markiert und dimerisiert. Dadurch wurde die Rezeptoraktivität gesteigert, was über Western Blot-, Immunofluoreszenz- und Fluoreszenzmikroskopieanalyse belegt wurde. In weiteren Ansätzen wurden Coiled-Coil-Systeme genutzt, um i) parallel zwei verschiedene Akzeptorpeptide mit verschiedenen Fluorophoren zu markieren und ii) Coiled-Coil-Peptide schaltbar zu machen. Durch die asymmetrische Verlängerung von K3/E3-Paaren mit Coiled-Coil-Sequenzen kann die Interaktion der Peptide an und aus geschaltet werden. Dies wurde sowohl in einem Fluoreszenzassay als auch in einer direkten Anwendung an der Syk-Kinase demonstriert. Die Liganden der Kinase wurden an den schaltbaren Peptiden angebracht und so die Affinität zur Syk-Kinase kontrolliert. / Nucleic acids and peptides can be used to obtain control over molecular processes within living cells. In this work, structural elements as coiled-coil peptides or PNA∙DNA-structures were used to label and modulate receptor behavior on living cells and to control ligand binding of cytosolic proteins. For the first concept the K3/E3-coiled-coil peptide pair was used to establish a proximity-guided, covalent transfer of a PNA strand from a K3-donor peptide onto the complementary E3-acceptor peptide. By fusion of the acceptor peptide to a receptor, PNA-receptor-conjugates were generated selectively on living cells. The native chemical ligation type of reaction allowed a fast PNA-transfer within minutes. Receptors from the family of GPCRs and the EGFR were tagged with a PNA-sequence and subsequently labeled by the addition of a fluorescent DNA or PNA. By recruiting branched DNA architectures which were decorated with several fluorophores, the total brightness of the labeling was increased quantitatively. A twice complementary Cy3-DNA was used to simultaneously label and dimerize the EGFR. Thereby, an artificially induced increase in receptor activity could be achieved, which was shown in Western Blot and immunofluorescence analysis as well as in fluorescence microscopy. In two other approaches coiled-coil peptides were used to i) label two different acceptor peptides simultaneously with two different dyes and ii) introduce coiled-coil peptides as part of a dynamic switchable system. Using an asymmetric coiled-coil elongation on the K3/E3 pair the interaction of both can be turned on and off. This was demonstrated in a fluorescence assay and applied to the Syk kinase, were Syk ligands were attached to the switchable peptides. Those ligands were changed from a bi- to a monovalent presentation status and thus the affinity of the Syk kinase towards its ligands can be controlled.

Um die Funktion von Proteinen in ihrer natürlichen Umgebung zu verstehen, ist es unerlässlich ihre Lokalisation und Bewegung im lebenden System durch z.B Fluoreszenz-markierung sichtbar zu machen. Eine ideale Markierungsmethode zeichnet sich dadurch aus, dass sie das Zielprotein (protein of interest, POI) selektiv und in kurzer Zeit mit einer maßgeschneiderten Reportergruppe ausstattet, ohne die Proteinfunktion und -lokalisation zu beeinflussen. Dabei ist die Größe der Erkennungssequenz von großer Bedeutung. In dieser Arbeit wird die Entwicklung einer Markierungsstrategie beschrieben, bei der die Ausbildung eines parallelen Coiled-Coil-Motivs den Transfer einer Reportergruppe auslöst. Untersucht wurde dabei die Übertragung eines Sulfonat-gebundenen Fluorophors auf ein Cystein in der Erkennungssequenz durch nukleophile Substitution. Ebenfalls untersucht wurde der Transfer verschiedener Thioester-verknüpfter Reporter auf ein N-terminales Cystein der Erkennungssequenz durch eine Acyltransferreaktion. Beide Strategien zeichnen sich durch eine hohe Selektivität und einen geringen Massenzuwachs am Zielprotein aus. Der Acyltransfer mit Arylthioestern zeigte zudem eine bemerkenswerte Reaktivität und erlaubte eine Markierung innerhalb weniger Minuten Reaktionszeit. Die Vielfältigkeit dieser Methode wurde anhand der Fluoreszenzmarkierung von sieben verschiedenen G-Protein gekoppelten Membranrezeptoren auf der Oberfläche lebender Zellen demonstriert. Die markierten Rezeptoren blieben dabei funktional und konnten ihren entsprechenden Liganden mit hoher Affinität binden. / In order to understand the function of proteins in their native environment, it is crucial to visualize their localization and trafficking in living cells by means of e.g. fluorescence labeling. An ideal labeling method adds a custom reporter group to the protein of interest (POI) in a selective and fast manner without disturbing the POIs function and localization. Hence the size of the recognition sequence is of major concern. This work describes the development of a labeling strategy in which the formation of a parallel coiled coil motif triggers the transfer of a reporter group. The transfer of a sulfonate-linked fluorescence dye onto the cysteine-modified recognition sequence via a nucleophilic substitution reaction was tested. Also the transfer of various thioester-linked reporters onto the N-terminal cysteine of the recognition sequence via an acyl transfer reaction was investigated. Both strategies are characterized by a high selectivity and low mass increase at the target protein. The acyl transfer with aryl thioesters also showed a remarkable reactivity and allowed labeling reactions to proceed within minutes. The versatility of this method was demonstrated by applying it to the labeling of seven different G-protein coupled membrane receptors on the surface of living cells. The labeled receptors remained functional and were able to bind their respective ligand with high affinity.

Genome sequencing and annotation projects define the complete sets of RNA and protein components for living systems. They also present the challenge to generate functional information for thousands of previously uncharacterized genes. Protein tagging with fluorescent or affinity tags provides a generic way to describe protein expression and localization patterns and protein-protein interactions. The genome wide application of this approach in Saccharomyces cerevisiae has resulted in a comprehensive picture of the core proteome of a simple, well-studied model system. Extending these studies to more complex, multicellular model organisms, would allow us to place protein function onto a 4 dimensional space-time map, and will improve our understanding of the complex processes of development and differentiation. This will require efficient protein tagging methods and new high performance tags. Here we present a generic protein tagging approach for the model nematode Caenorhabditis elegans. The method is based on recombination mediated DNA engineering of genomic BAC clones into tagged transgenes for integrative transformation. C.elegans offers unique advantages for function discovery through protein tagging: compact and a well annotated genome, combined with a simple and well-understood anatomy and pattern of development. However, the methods for protein tagging in C.elegans have so far been inefficient and largely dependent on artificial cDNA based constructs, which can lack important regulatory elements. In contrast, our approach combines the advantages of authentic regulation with a new application of recombineering, which is simple, fast and efficient. For the first time we apply liquid culture cloning for multiple recombineering steps. This is particularly important when high throughput applications are considered, as it offers significant advantages in scale up and automation. We show that the BAC derived transgenes can be used for stable, integrative transformation in C. elegans. We show that the tagged transgene can take over the function of its endogenous counterpart. Using florescent reporter, we reproduce known and document new expression patterns. The second part of the thesis describes a project that we undertook to develop improved double affinity cassettes for protein purification. We evaluated the performance of 5 new double tag combinations in vitro and in mammalian culture cells. All of the new cassettes performed well and present a valuable tool for protein interaction studies in higher model systems.

Genome sequencing and annotation projects define the complete sets of RNA and protein components for living systems. They also present the challenge to generate functional information for thousands of previously uncharacterized genes. Protein tagging with fluorescent or affinity tags provides a generic way to describe protein expression and localization patterns and protein-protein interactions. The genome wide application of this approach in Saccharomyces cerevisiae has resulted in a comprehensive picture of the core proteome of a simple, well-studied model system. Extending these studies to more complex, multicellular model organisms, would allow us to place protein function onto a 4 dimensional space-time map, and will improve our understanding of the complex processes of development and differentiation. This will require efficient protein tagging methods and new high performance tags. Here we present a generic protein tagging approach for the model nematode Caenorhabditis elegans. The method is based on recombination mediated DNA engineering of genomic BAC clones into tagged transgenes for integrative transformation. C.elegans offers unique advantages for function discovery through protein tagging: compact and a well annotated genome, combined with a simple and well-understood anatomy and pattern of development. However, the methods for protein tagging in C.elegans have so far been inefficient and largely dependent on artificial cDNA based constructs, which can lack important regulatory elements. In contrast, our approach combines the advantages of authentic regulation with a new application of recombineering, which is simple, fast and efficient. For the first time we apply liquid culture cloning for multiple recombineering steps. This is particularly important when high throughput applications are considered, as it offers significant advantages in scale up and automation. We show that the BAC derived transgenes can be used for stable, integrative transformation in C. elegans. We show that the tagged transgene can take over the function of its endogenous counterpart. Using florescent reporter, we reproduce known and document new expression patterns. The second part of the thesis describes a project that we undertook to develop improved double affinity cassettes for protein purification. We evaluated the performance of 5 new double tag combinations in vitro and in mammalian culture cells. All of the new cassettes performed well and present a valuable tool for protein interaction studies in higher model systems.

Die Durchflusszytometrie und Fluoreszenzmikroskopie sind zentrale Techniken zur Analyse von Zellen, Geweben und Organen. Besonders in der Immunologie werden sie zur Identifizierung und Charakterisierung von Biomolekülen mittels fluoreszenzmarkierter Antikörper verwendet. Fluoreszenzmarker müssen je nach Anwendung hohe Helligkeit, geringe Größe und minimierte Löschung des Signals aufweisen. Stark markierte Konstrukte leiden jedoch oft unter Fluoreszenzlöschung oder großen Molekularmassen. Diese Arbeit untersucht verzweigtes Polyethylenglykol (PEG) als Träger für Fluorophore. PEG-Ketten wurden als räumliche Trennmittel identifiziert und an Aminodextran gekoppelt, wodurch hochgradig multimerisierte Fluorophor-PEG-Dextran-Zwischenprodukte entstanden. Diese Konjugate, gekoppelt mit Antikörpern, zeigen hohe Fluoreszenzintensität und wurden bei der Detektion von CAR SUP-T1-Zellen erfolgreich eingesetzt. PEG-basierte Reagenzien durchdringen jedoch oft die Zellmembran nicht, was für intrazelluläre Ziele und größere Gewebe wichtig ist. Sequentielle Multiplex-Analysen sind durch unvollständige Spaltung und Restsignale problematisch. Deshalb wurden synthetische Peptide als Rückgrat für die Fluorophor-Multimerisierung untersucht. Diese Konstrukte, verbunden mit Nanokörpern, zeigten erhöhte Helligkeit und Gewebepenetration in der Lichtblattmikroskopie von Mausorganen. Zudem wurde ein dualer Entfernungsmechanismus in die REAdyelease-Technologie integriert. Basierend auf Oligonukleotiden, Disulfiden oder Peptiden in Kombination mit Aminodextran konnte eine schnellere Signalreduktion ermöglicht werden. Dies wurde in der Konfokalmikroskopie an einer Pankreastumorzelllinie demonstriert. / Flow cytometry and fluorescence microscopy are crucial for analyzing cells and tissues, especially in immunology, where immunofluorescence is used for identifying, visualizing, and characterizing biomolecules with fluorescently labeled antibodies. These labels must meet various requirements: high brightness, small size, and the ability to be rendered non-fluorescent. However, highly labeled constructs often suffer from fluorescence self-quenching or high molecular masses, limiting their effectiveness. This work demonstrates that branched polyethylene glycol (PEG) serves as an efficient fluorophore multimerization platform for protein labeling. I explored factors critical for preventing fluorophore self-quenching in multi-fluorophore systems. Fluorescent PEGs were multimerized on an amino-dextran scaffold, generating highly multimerized fluorophore-PEG-dextran intermediates. When conjugated to antibodies, these intermediates allowed bright labeling of biomarkers on cells and tissues and were successfully used in detecting CAR SUP-T1 cells. Despite their strengths, PEG-based reagents often lack deep tissue penetration, essential for intracellular targets and 3D organ imaging. To enhance tissue penetration, I designed small peptide-based backbones for fluorophore multimerization. These constructs, coupled with nanobodies, produced homogeneous fluorescent conjugates that quickly penetrated mouse organs and enabled bright staining in light-sheet microscopy. The final part of the thesis focuses on synthesizing labels for cyclic immunofluorescence. I addressed the issue of incomplete label removal by creating erasable conjugates with two release sites. Fluorescent conjugates based on oligonucleotides, disulfides, or peptides combined with amino-dextran can be rapidly erased from labeled epitopes using a dual-release approach. This method was demonstrated in confocal microscopy and used for iterative imaging of biomarkers on a sample of a pancreatic tumor cell line.