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

用于超特异性神经元蛋白光遗传学控制的纳米开关

基本信息

批准号：
9379982
负责人：
Lei Wang
金额：
$ 175.18万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-01 至 2020-09-30
项目状态：
已结题

项目摘要

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.

摘要/摘要用光控制蛋白质功能的能力提供了出色的时间和空间分辨率，以实现精确的原位研究，因此对神经科学产生重大影响。有两个主要障碍现有光遗传学方法所强加的：其中之一是它们不能轻易应用于任何蛋白质的选择，另一个是光调制位点选择缺乏高度特异性和灵活性。这些局限性极大地限制了神经元过程研究的范围、精度和深度。到克服这些挑战，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特异性。通过基因的扩展代码中，我们将位点特异性地将光可逆非天然氨基酸（Uaas）整合到蛋白质中调节单个位点，并构建能够调节二级结构和域的新型纳米桥，因此以可逆的方式光调节蛋白质活性。与现有方法相比，使用大蛋白质和结构域，我们的方法仅使用单个 Uaa 来实现光敏感性。因此我们的方法具有最小的对所研究蛋白质的扰动，通常可应用于任何蛋白质，不限于蛋白质类型、功能或细胞定位。此外，光不是依赖于蛋白质功能或相互作用，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其进行光调节进步。使用基因编码的 Uaas 还使我们的方法能够与广泛的神经网络兼容细胞和模型动物。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开辟巨大的新机会精确度。该项目的成功将为光学提供一种新颖的纳米开关平台技术在体外和体内调节任何神经元蛋白质，为新兴分子奠定了基础光神经生物学揭示以前神经信号传导无法获得的精细分子见解。