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

总结/摘要 利用光控制蛋白质功能的能力提供了优异的时间和空间分辨率，以精确地测量蛋白质的功能。 这是一项重要的研究，因此对神经科学产生了重大影响。有两个主要障碍 现有的光遗传学方法所强加的：一个是它们不能容易地应用于任何蛋白质， 选择，另一个是在光调制的位点选择中缺乏高特异性和灵活性。这些 这些局限性极大地限制了对神经元过程的研究的范围、精度和深度。到 为了克服这些挑战，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 具有普遍适用性和超特异性。通过基因的扩张 我们将位点特异性地将光可逆非天然氨基酸（Uaas）掺入蛋白质中， 调制一个单一的网站，并建立新的纳米桥能够调节二级结构和域， 以可逆的方式光调节蛋白质活性。与现有方法相比， 蛋白质和结构域，我们的方法仅使用单个Uaa用于光敏感性。因此，我们的方法具有最小的 本发明的方法可以应用于所研究的蛋白质，并且可以一般地应用于任何蛋白质，而不限于蛋白质 类型、功能或细胞定位。此外，与其依赖于蛋白质功能或相互作用来进行光- 与目前的方法一样，我们的方法能够在不知道蛋白质在体内的功能的情况下对蛋白质进行光调节。 提前使用遗传编码的Uaas也使我们的方法与广泛的神经系统兼容。 细胞和模型动物。更重要的是，我们的方法将赋予靶神经元光响应性 蛋白质具有前所未有的分辨率，对所需的亚基、结构域甚至单个残基具有特异性。 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， 精确到极点该项目的成功将为光开关提供一种新的纳米开关平台技术 在体外和体内调节任何神经元蛋白，为新兴的分子生物学奠定了基础。 光学神经生物学，以揭示精细的分子见解以前无法为神经信号。

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.