Center for Integrated Protein Science at the Department of Chemistry Technische Universität München, Lichtenbergstraße 4, 85748, Garching, Germany.
Assay Development and Screening Platform at the Institute for Molecular Toxicology and Pharmacology, Helmholtz Zentrum München für Gesundheit und Umwelt, Ingolstädter Landstraße 1, 85764, Neuherberg, Germany.
Sci Rep. 2017 Aug 10;7(1):7732. doi: 10.1038/s41598-017-07870-w.
Riboswitches are bacterial RNA elements that regulate gene expression in response to metabolite or ion abundance and are considered as potential drug targets. In recent years a number of methods to find non-natural riboswitch ligands have been described. Here we report a high-throughput in vivo screening system that allows identifying OFF-riboswitch modulators in a 384 well bioluminescence assay format. We use a reverse reporter gene setup in Bacillus subtilis, consisting of a primary screening assay, a secondary assay as well as counter assays to detect compounds in a library of 1,280 molecules that act on the guanine-responsive xpt riboswitch from B. anthracis. With this in vivo high-throughput approach we identified several hit compounds and could validate the impact of one of them on riboswitch-mediated gene regulation, albeit this might not be due to direct binding to the riboswitch. However, our data demonstrate the capability of our screening assay for bigger high-throughput screening campaigns. Furthermore, the screening system described here can not only be generally employed to detect non-natural ligands or compounds influencing riboswitches acting as genetic OFF switches, but it can also be used to investigate natural ligands of orphan OFF-riboswitches.
Riboswitches 是一类细菌 RNA 元件，能够响应代谢物或离子丰度的变化来调节基因表达，它们被认为是潜在的药物靶点。近年来，已经有许多方法被用于寻找非天然的 riboswitch 配体。在这里，我们报道了一种高通量的体内筛选系统，可以在 384 孔生物发光测定法中鉴定 OFF-riboswitch 调节剂。我们在枯草芽孢杆菌中使用了一个反向报告基因系统，该系统包括一个初步筛选测定法、一个次级测定法以及一个反式测定法，以检测来自炭疽芽孢杆菌的鸟嘌呤响应 xpt riboswitch 的 1280 个化合物文库中的化合物。使用这种体内高通量方法，我们鉴定了几种有效化合物，并验证了其中一种化合物对 riboswitch 介导的基因调控的影响，尽管这可能不是由于其与 riboswitch 的直接结合所致。然而，我们的数据证明了我们的筛选测定法在更大规模的高通量筛选中的能力。此外，这里描述的筛选系统不仅可以普遍用于检测非天然配体或影响作为遗传 OFF 开关的 riboswitch 的化合物，还可以用于研究孤儿 OFF-riboswitch 的天然配体。
