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.

蛋白质为生命各个阶段的许多生物过程提供必要的功能调节；然而，需要专门调节生命系统和工程生物材料中蛋白质活性的新技术，以更好地探究基本细胞信号传导并指导生物命运的高级决策。在这里，我们建立了一种可推广的策略，通过完全时空控制快速且不可逆地激活蛋白质功能。通过开发基因编码和光激活的 SpyLigation (LASL)，在短暂暴露于细胞相容性光下（通常为几分钟）后，生物活性蛋白可以从非功能性分裂片段对中稳定地重新组装。采用易于使用的光刻处理技术来指定光连接发生的时间、地点和程度，我们展示了在溶液、生物材料和活体哺乳动物细胞中使用 LASL 对 UnaG、NanoLuc 和 Cre 重组酶的精确蛋白质激活，以及对蛋白质亚细胞定位的光学控制。展望未来，我们预计这些基于光点击的光遗传学方法将在探测和指导时间和三维空间中复杂的细胞命运方面发挥巨大的作用。