Photocaged Nanobodies Delivered into Cells for Light Activation of Biological Processes

Abstract

We have recently reported photocaged nanobodies, termed photobodies, that can be light-activated in their antigen binding properties due to the removal of the photolabile protection group. We aimed to establish the delivery and activation of photobodies inside mammalian cells, because intracellular protein manipulation and detection by using antibodies and antibody fragments like nanobodies is highly attractive, however, the spatial and temporal control of their application in cells is extremely limited. AntiGFP photobodies with ortho-nitrobenzyltyrosine in the paratope region were delivered into HeLa cells using the bead-loading technique. Light-activation was carried out with spatial control in single cells to trigger binding to a GFP tag fused to endogenously expressed target proteins. By means of its fused nuclear localization sequence, an activated photobody dragged the GFP fusion protein into the nucleus, the kinetics of which could be monitored using live cell microscopy. These results show the potential of photobodies for intracellular applications.