Optogenetics for all: A general method for optical control of protein activity

描述（由申请人提供）：光控制遗传定义的蛋白质活性，即光遗传调节蛋白质，一直是生物学的梦想。如果我们能够开发出一种可推广的方法来光学控制感兴趣的蛋白质的活性，它可能会深刻地改变生物实验，并赋予基于基因和细胞的治疗新的能力。例如，蛋白质协调的细胞功能，如生存、凋亡、分化、迁移或连接（在神经系统中），可以在体内控制，以微米级的分辨率研究生物体生理学。治疗性植入的细胞同样可以通过光来控制，以精确地将治疗集中在所需的解剖位置。还有无数其他可能的应用。鉴于光学蛋白调控的这些优势，人们已经花费了大量的努力来适应已知的光响应信号域来调节哺乳动物蛋白。然而，现有的策略需要广泛的筛选来产生光反应蛋白，或者依靠蛋白质的重新定位来间接调节活性。因此，这些方法仅用于控制少数蛋白质。因此，需要一种方法来制造感兴趣的光调节蛋白质

DESCRIPTION (provided by applicant): Optical control of genetically defined protein activities, i.e. optogenetic regulation of proteins, has long been a dream in biology. If we could develop a generalizable method to optically control activities of proteins of interest, it could profoundly transform biological experimentation and impart new capabilities to gene- and cell-based therapies. For instance, the cellular functions that proteins coordinate, such as survival, apoptosis, differentiation, migration or connectivity (in the nervous system) could be controlled in vivo to study organismal physiology with micron-level resolution. Therapeutically implanted cells could be similarly controlled by light to precisely focus treatment at desired anatomical locations. There are countless other possible applications. Given these advantages of optical protein regulation, considerable efforts have been expended to adapt known light-responsive signaling domains to regulate mammalian proteins. However, existing strategies require extensive screening to create light-responsive proteins, or rely on protein relocalization to indirectly regulate activity. As a result, these methods have been used to control only a few proteins. Thus there exists a need for a method to create light-regulated proteins of interest that is generalizable. We have recently discovered a new class of light-mediated protein-protein interaction, and translated this discovery into a generalizable method for controlling protein activities with light. We hypothesized that fluorescent proteins (FPs) could undergo light-dependent conformational changes that drive changes in oligomerization state. Indeed, we found that tetrameric and dimeric variants of the reversibly photoswitching green FP Dronpa undergo dissociation as they are switched from bright to dark states by cyan light. We then discovered that fusing Dronpa domains at both ends of an enzymatic domain of interest cages it in the dark but allows uncaging upon illumina