中文摘要：红光控制的蛋白质二聚体系统可以增加组织渗透深度，是临床应用如细胞治疗和基因治疗急需的工具。然而，现有红光诱导的二聚系统在哺乳动物中的应用受到了限制，原因在于其两个组分：通常需要哺乳动物外源生色团的光敏蛋白(或光感受器)；和通常具有复杂结构和非理想结合特性的天然感光结合蛋白，。本文介绍了一种高效、可推广的方法（COMBINES-LID），以制备高度特异、可逆的光诱导异源二聚系统，无需依赖任何现有的光受体结合剂。该方法涉及组合纳米体库的两步结合剂筛选(噬菌体展示和酵母双杂交)，以获得选择性结合光激活形式的光开关蛋白或结构域，而非暗形式的结合剂。构建基于纳米体的红光诱导二聚（nanoReD）系统提，该系统包括使用哺乳动物内源性生色团、胆绿素和光形成特定纳米体的截短细菌光敏色素感觉模块。nanoReD系统具有低暗活性和高诱导特异性的生化特性，并进一步证明了其能可逆控制小鼠蛋白质易位和基因表达激活。总的来说，COMBINES-LID为创造用于生物过程光学操作的基因编码驱动器提供了新的机会。
外文摘要：Protein dimerization systems controlled by red light with increased tissue penetration depth are a highly needed tool for clinical applications such as cell and gene therapies. However, mammalian applications of existing red light-induced dimerization systems are hampered by limitations of their two components: a photosensory protein (or photoreceptor) which often requires a mammalian exogenous chromophore and a naturally occurring photoreceptor binding protein typically having a complex structure and nonideal binding properties. Here, we introduce an efficient, generalizable method (COMBINES-LID) for creating highly specific, reversible light-induced heterodimerization systems independent of any existing binders to a photoreceptor. It involves a two-step binder screen (phage display and yeast two-hybrid) of a combinatorial nanobody library to obtain binders that selectively engage a light-activated form of a photoswitchable protein or domain not the dark form. Proof-of-principle was provided by engineering nanobody-based, red light-induced dimerization (nanoReD) systems comprising a truncated bacterial phytochrome sensory module using a mammalian endogenous chromophore, biliverdin, and light-form specific nanobodies. Selected nanoReD systems were biochemically characterized, exhibiting low dark activity and high induction specificity, and further demonstrated for the reversible control of protein translocation and activation of gene expression in mice. Overall, COMBINES-LID opens new opportunities for creating genetically encoded actuators for the optical manipulation of biological processes.
外文关键词：light-induced dimerization；nanobody；combinatorial antibody library；bacterial phytochrome；phage display；optogenetics
作者：Huang, ZM；Li, ZP；Zhang, X；Kang, SK；Dong, RZ；Sun, L；Fu, XN；Vaisar, D； Watanabe, K；Gu, LC
作者单位：Univ Washington；Minist Nat Resources
期刊名称：ACS SYNTHETIC BIOLOGY
期刊影响因子：4.411
出版年份：2020
出版刊次：12
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