Spatiotemporal functional assembly of split protein pairs through a light-activated SpyLigation

Proteins provide essential functional regulation of many bioprocesses across all scales of life; however, new techniques to specifically modulate protein activity within living systems and in engineered biomaterials are needed to better interrogate fundamental cell signalling and guide advanced decisions of biological fate. Here we establish a generalizable strategy to rapidly and irreversibly activate protein function with full spatiotemporal control. Through the development of a genetically encoded and light-activated SpyLigation (LASL), bioactive proteins can be stably reassembled from non-functional split fragment pairs following brief exposure (typically minutes) to cytocompatible light. Employing readily accessible photolithographic processing techniques to specify when, where and how much photoligation occurs, we demonstrate precise protein activation of UnaG, NanoLuc and Cre recombinase using LASL in solution, biomaterials and living mammalian cells, as well as optical control over protein subcellular localization. Looking forward, we expect that these photoclick-based optogenetic approaches will find tremendous utility in probing and directing complex cellular fates in both time and three-dimensional space. Techniques to specifically modulate protein activity are needed to interrogate spatial effects in cellular processes. A genetically encoded method for site-specific protein–protein conjugation based on a photoclick chemical reaction has now been developed. This method permits rapid and irreversible reassembly of bioactive proteins from non-functional split fragment pairs with full spatiotemporal control in solution, biomaterials and living mammalian cells.