PT - JOURNAL ARTICLE AU - O’Shea, Jack M. AU - Goutou, Angeliki AU - Sethna, Cyrus AU - Wood, Christopher W. AU - Greiss, Sebastian TI - Generation of photocaged nanobodies for <em>in vivo</em> applications using genetic code expansion and computationally guided protein engineering AID - 10.1101/2021.04.16.440193 DP - 2021 Jan 01 TA - bioRxiv PG - 2021.04.16.440193 4099 - http://biorxiv.org/content/early/2021/04/17/2021.04.16.440193.short 4100 - http://biorxiv.org/content/early/2021/04/17/2021.04.16.440193.full AB - Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo-activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular-dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo-activation, binding is restored. We use this approach to generate improved photocaged variants of two anti-GFP nanobodies. These variants exhibit photo-activatable binding triggered by illumination with 365nm light. We demonstrate that the photocaged nanobodies we have created are highly robust and function in a complex cellular environment. We apply them to control subcellular protein localisation in the nematode worm C. elegans. Our approach provides a rare example of computationally designed proteins being directly applied in living animals and demonstrates the importance of accounting for in vivo effects on protein-protein interactions.Competing Interest StatementThe authors have declared no competing interest.