Site-specific functionalization of nanobodies: From labeling to cellular uptake

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The long-term goal of this collaborative project is to develop and validate novel methods to visualize and manipulate biochemical processes within living cells with fluorescently labeled proteins functionalized for cellular uptake. For the latter, we will utilize cyclic cell penetrating peptides and engineer them for intracellular cleavage and release of the cargo proteins in the cytosol and nucleus of the cell. For intracellular binding of different target structures we will use nanobodies specific against GFP, the DNA replication and repair core component PCNA and the HIV viral capsid protein. Different strategies for site-specific modification of proteins including intein expression, incorporation of artificial amino acids by amber suppression, novel chemoenzymatic modification strategies and bioorthogonal reactions will be employed to introduce functional modules like fluorescent labels and artificial cyclic cell penetrating peptides. Orthogonal functionalization strategies will initially be tested with the GFP binding nanobodies. We will validate the binding of the modified versus unmodified nanobodies in vitro and in vivo to their respective intracellular targets. We will monitor and quantify the uptake of modified nanobodies by live cell confocal microscopy. Physiological effects on target structures including the disruption of protein interactions will be quantified by automated image analysis. This proposed collaboration, combines expertise in nanobody engineering, bioorthogonal chemistry and protein semi-synthesis, cellular uptake mechanisms, microscopy and cell biology. Consequently, this joint effort should lead to the development of entirely new research tools to detect, study and manipulate antigens in living cells.