Technische Universität Darmstadt, Department of Biology, Schnittspahnstrasse 10, 64287 Darmstadt, Germany.
Goethe-University Frankfurt, Institute for Organic Chemistry and Chemical Biology, Max-von-Laue-Strasse 9, 60438 Frankfurt (M), Germany.
Nucleic Acids Res. 2019 Feb 28;47(4):2029-2040. doi: 10.1093/nar/gky1225.
Regulation of complex biological networks has proven to be a key bottleneck in synthetic biology. Interactions between the structurally flexible RNA and various other molecules in the form of riboswitches have shown a high-regulation specificity and efficiency and synthetic riboswitches have filled the toolbox of devices in many synthetic biology applications. Here we report the development of a novel, small molecule binding RNA aptamer, whose binding is dependent on light-induced change of conformation of its small molecule ligand. As ligand we chose an azobenzene because of its reliable photoswitchability and modified it with chloramphenicol for a better interaction with RNA. The synthesis of the ligand 'azoCm' was followed by extensive biophysical analysis regarding its stability and photoswitchability. RNA aptamers were identified after several cycles of in vitro selection and then studied regarding their binding specificity and affinity toward the ligand. We show the successful development of an RNA aptamer that selectively binds to only the trans photoisomer of azoCm with a KD of 545 nM. As the aptamer cannot bind to the irradiated ligand (λ = 365 nm), a light-selective RNA binding system is provided. Further studies may now result in the engineering of a reliable, light-responsible riboswitch.
调控复杂的生物网络已被证明是合成生物学的一个关键瓶颈。结构灵活的 RNA 与各种其他分子（如核糖开关）之间的相互作用表现出了高度的调控特异性和效率，而合成的核糖开关则为许多合成生物学应用中的器件工具箱提供了补充。在这里，我们报告了一种新型小分子结合 RNA 适体的开发，其结合依赖于小分子配体构象的光诱导变化。我们选择了偶氮苯作为配体，因为它具有可靠的光致开关性，并对其进行了氯霉素修饰，以增强与 RNA 的相互作用。在对配体“azoCm”进行了广泛的物理化学分析，包括其稳定性和光致开关性之后，我们完成了其合成。经过几轮体外选择，我们鉴定出了 RNA 适体，并研究了它们与配体的结合特异性和亲和力。我们成功开发了一种 RNA 适体，它只能选择性地与 azoCm 的反式光异构体结合，KD 值为 545 nM。由于适体不能与照射的配体（λ = 365nm）结合，因此提供了一种光选择性的 RNA 结合系统。进一步的研究可能会导致可靠的、光响应的核糖开关的工程设计。
