Rational design of a small molecule-responsive intramer controlling transgene expression in mammalian cells
Rational design of a small molecule-responsive intramer controlling transgene expression in mammalian cells
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DOI：
10.1093/nar/gkr829
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发表时间：
2011-12-01
影响因子：
14.9
通讯作者：
Fussenegger, Martin
中科院分区：
文献类型：
--
作者：
Auslaender, David;Wieland, Markus;Fussenegger, Martin
结合蛋白质或小分子的适体已被证明是用于构建人工基因开关的通用且强大的构建块。在这项研究中，我们提出了一种新的基于适体的构建体调节泰特关闭系统在四环素的非依赖性的方式，从而实现控制转基因的表达。为此目的，TetR蛋白抑制适体被工程化用于哺乳动物细胞中，使得能够对四环素依赖性反式激活因子（tTA）进行RNA响应性控制。通过合理地连接茶碱适体作为传感器，抑制性TetR适体和因此tTA活性变得依赖于传感器适体的配体。小分子茶碱的加入导致在体外与相应蛋白质的结合增强，并抑制哺乳动物细胞系中报告基因的表达。通过使用适体作为衔接子，以控制蛋白质活性的预定的小分子，我们提出了一个简单而直接的方法，为未来的应用在化学生物学领域。此外，广泛使用的泰特系统的基于适体的控制引入了新的调节层，从而促进了更复杂的基因网络的构建。
Aptamers binding proteins or small molecules have been shown to be versatile and powerful building blocks for the construction of artificial genetic switches. In this study, we present a novel aptamer-based construct regulating the Tet Off system in a tetracycline-independent manner thus achieving control of transgene expression. For this purpose, a TetR protein-inhibiting aptamer was engineered for use in mammalian cells, enabling the RNA-responsive control of the tetracycline-dependent transactivator (tTA). By rationally attaching the theophylline aptamer as a sensor, the inhibitory TetR aptamer and thus tTA activity became dependent on the ligand of the sensor aptamer. Addition of the small molecule theophylline resulted in enhanced binding to the corresponding protein in vitro and in inhibition of reporter gene expression in mammalian cell lines. By using aptamers as adaptors in order to control protein activity by a predetermined small molecule, we present a simple and straightforward approach for future applications in the field of Chemical Biology. Moreover, aptamer-based control of the widely used Tet system introduces a new layer of regulation thereby facilitating the construction of more complex gene networks.