Nanobodies - Little helpers unravelling intracellular signaling

The identification of diagnostic and therapeutic targets requires a comprehensive understanding of cellular processes, for which advanced technologies in biomedical research are needed. The emergence of nanobodies (Nbs) derived from antibody fragments of camelid heavy chain-only antibodies as intracellular research tools offers new possibilities to study and modulate target antigens in living cells. Here we summarize this rapidly changing field, beginning with a brief introduction of Nbs, followed by an overview of how target-specific Nbs can be generated, and introduce the selection of intrabodies as research tools. Intrabodies, by definition, are intracellular functional Nbs that target ectopic or endogenous intracellular antigens within living cells. Such binders can be applied in various formats, e.g. as chromobodies for live cell microscopy or as biosensors to decipher complex intracellular signaling pathways. In addition, protein knockouts can be achieved by target-specific Nbs, while modulating Nbs have the potential as future therapeutics. The development of fine-tunable and switchable Nb-based systems that simultaneously provide spatial and temporal control has recently taken the application of these binders to the next level.