Nano-switches for optogenetic control of neuronal proteins with ultra-specificity

摘要/摘要 用光控制蛋白质功能的能力为精确的 现场调查，因此对神经科学产生了重大影响。有两个主要的障碍 由现有的光遗传方法施加的：一种是它们不能很容易地应用于任何蛋白质上 二是光调制的地点选择缺乏高度的特异性和灵活性。这些 局限性显著地限制了对神经元突起的研究的范围、精度和深度。至 克服这些挑战，我们在这里提出了一种用于神经元蛋白质光学控制的纳米开关技术 在其原生环境中，具有普遍适用性和超强的特异性。通过基因的扩展 代码，我们将定点地将光可逆的非天然氨基酸(UAA)整合到蛋白质中，以 调制单个位置，并建立能够调制二级结构和结构域的新型纳米桥，因此 以可逆的方式光调节蛋白质的活性。与现有方法相比，使用大型 对于蛋白质和结构域，我们的方法只使用单一的UAA来检测光敏性。因此，我们的方法具有最小的 对正在研究的蛋白质的扰动，可以普遍应用于任何蛋白质，不受蛋白质的限制 类型、功能或细胞定位。此外，与其依靠蛋白质的功能或相互作用来拍照- 与目前的方法一样，我们的方法能够在不知道蛋白质功能的情况下对蛋白质进行光调制 前进。使用遗传编码的尿酸也使我们的方法能够与广泛的神经 细胞和模型动物。更重要的是，我们的方法将赋予目标神经元光响应性。 具有前所未有的分辨率的蛋白质，专用于所需的亚基、结构域，甚至单个残基。 这种无与伦比的特异性将为研究神经元过程提供巨大的新机会。 精确定位。该项目的成功将为光通信领域提供一种新型的纳米开关平台技术 在体外和体内对任何神经元蛋白的调节，为新出现的分子奠定了基础 光神经生物学，以揭示神经信号以前无法获得的精细分子洞察力。

Summary/Abstract The ability to control protein function with light provides excellent temporal and spatial resolution for precise investigation in situ, and thus is having significant impact on neuroscience. There are two major barriers imposed by existing optogenetic methods: one being that they cannot be readily applied on any protein of choice, and the other being lack of high specificity and flexibility in site selection for photo-modulation. These limitations significantly restrain the scope, precision, and depth of investigations on neuronal processes. To overcome these challenges, we propose here a nano-switch technology for optical control of neuronal proteins in their native settings with general applicability and ultra-specificity. Through the expansion of the genetic code, we will site-specifically incorporate photo-reversible unnatural amino acids (Uaas) into proteins to modulate a single site, and to build novel nano-bridges able to modulate secondary structures and domains, so as to photo-regulate protein activities in a reversible manner. Compared with existing methods using large proteins and domains, our method uses only a single Uaa for light sensitivity. Our method thus has minimal perturbation to proteins under study, and can be generally applied to any protein without limitations to protein type, function, or cellular localization. In addition, rather than relying on protein function or interaction for photo- regulation as in current methods, our method is able to photo-modulate a protein without knowing its function in advance. Using genetically encoded Uaas also enables our method compatible with a broad range of neural cells and model animals. More importantly, our method will confer photo-responsiveness on target neuronal proteins with unprecedented resolution that is specific for desired subunits, domains, and even single residues. This unparalleled specificity will open vast new opportunities for investigation of neuronal processes with pinpoint accuracy. The success of this project will afford a novel nano-switch platform technology for optical modulation of any neuronal protein both in vitro and in vivo, laying a foundation for the emerging molecular opto-neurobiology to uncover fine molecular insights previously inaccessible for neural signaling.