RT Journal Article SR Electronic T1 Generation of photocaged nanobodies for in vivo applications using genetic code expansion and computationally guided protein engineering JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.04.16.440193 DO 10.1101/2021.04.16.440193 A1 O’Shea, Jack M. A1 Goutou, Angeliki A1 Sethna, Cyrus A1 Wood, Christopher W. A1 Greiss, Sebastian YR 2021 UL http://biorxiv.org/content/early/2021/04/17/2021.04.16.440193.abstract 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.