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Antibody conjugates have taken a great leap forward as tools in basic and applied molecular life sciences that was enabled by the development of chemoselective reactions for the site‐specific modification of proteins. Antibody‐oligonucleotide conjugates combine the antibody's target specificity with the reversible, sequence‐encoded binding properties of oligonucleotides like DNAs or peptide nucleic acids (PNAs), allowing sequential imaging of large numbers of targets in a single specimen. In this report, we use the Tub‐tag® technology in combination with Cu‐catalyzed azide‐alkyne cycloaddition for the site‐specific conjugation of single DNA and PNA strands to an eGFP‐binding nanobody. We show binding of the conjugate to recombinant eGFP and subsequent sequence‐specific annealing of fluorescently labelled imager strands. Furthermore, we reversibly stain eGFP‐tagged proteins in human cells, thus demonstrating the suitability of our conjugation strategy to generate antibody‐oligonucleotides for reversible immunofluorescence imaging., Protein‐oligonucleotide conjugates. We present a novel strategy for the efficient and site‐specific generation of nanobody‐oligonucleotide conjugates in a 1 : 1 stoichiometry by Tub‐tag‐mediated conjugation. We show a proof‐of‐concept that these conjugates can readily be used for reversible staining in confocal fluorescence microscopy.

Superauflosende Mikroskopie ermoglicht optische Bildgebung unterhalb der klassischen Beugungsgrenze von Licht mit bis zu 20-fach verbesserter raumlicher Auflosung. Jedoch ist derzeit das Beobachten mehrerer unterschiedlicher Zielmolekule (“Multiplexing”) schwierig und zeitintensiv in der Durchfuhrung. Hier stellen wir einen einfachen Ansatz fur sequenzielles Multiplexing vor, der auf einem schnellen Austausch von DNA-Sonden basiert. Dies ermoglicht eine effiziente Detektion vieler Zielmolekule mit Superauflosungsmethoden wie (d)STORM, STED und SIM. Wir evaluieren unseren Ansatz mit DNA-Origami-Nanostrukturen, um Markierung, Bildgebung und die Wascheffizienz quantitativ zu testen. Daruber hinaus demonstrieren wir die Anwendbarkeit unserer Methode zur Bildgebung mehrerer Proteine in fixierten Zellen.

Growth cones, the terminal structures of elongating neurites, use extracellular guidance information in order to navigate to appropriate target cells. The directional information of guidance cues is transduced to a cytoplasmic gradient of messenger molecules across the growth cone leading to rearrangements of the cytoskeleton. One messenger molecule regulating growth cone turning is cAMP, which is also known to be sufficient to direct growth cone attraction. Cytoplasmic cAMP gradients have been generated in the present study by photolysing caged cAMP with UV light focused on one side of growth cones of chick sensory neurons. Using this method we show that only specific time patterns of pulsed cAMP release are capable of inducing growth cone turning whereas others, which release the same amount of cAMP, are ineffective. Theoretical calculations show that diverse time patterns produce different intracellular gradients, which were visualized directly in HeLa cells expressing cAMP-sensitive ion channels as a reporter system. Together these data indicate that the spatiotemporal properties of the intracellular gradient are crucial for growth cone turning.