Spatiotemporal functional assembly of split protein pairs through a light-activated SpyLigation
Spatiotemporal functional assembly of split protein pairs through a light-activated SpyLigation
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DOI：
10.1038/s41557-023-01152-x
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发表时间：
2023-04-17
期刊：
影响因子：
21.8
通讯作者：
DeForest, Cole A.
中科院分区：
文献类型：
--
作者：
Ruskowitz, Emily R.;Munoz-Robles, Brizzia G.;DeForest, Cole A.
蛋白质在所有生命尺度上提供许多生物过程的基本功能调节;然而，需要新技术来特异性地调节生命系统内和工程生物材料中的蛋白质活性，以更好地询问基本细胞信号传导并指导生物命运的高级决策。在这里，我们建立了一个可推广的策略，以快速和不可逆地激活蛋白质的功能与完整的时空控制。通过开发遗传编码和光激活的SpyLigation（LASL），生物活性蛋白可以在短暂暴露于细胞相容性光（通常为几分钟）后从非功能性分裂片段对稳定地重新组装。采用易于访问的生物处理技术，以指定何时，何地和多少光连接发生，我们证明了精确的蛋白质激活UnaG，NanoLuc和Cre重组酶使用LASL在溶液中，生物材料和活的哺乳动物细胞，以及光学控制蛋白质亚细胞定位。展望未来，我们预计这些基于photoclick的光遗传学方法将在时间和三维空间中探测和指导复杂的细胞命运方面具有巨大的实用性。
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.