ABSTRACT

Gene regulation in prokaryotes often depends on RNA elements such as riboswitches or RNA thermometers located in the 5′ untranslated region of mRNA. Rearrangements of the RNA structure in response, e.g., to the binding of small molecules or ions control translational initiation or premature termination of transcription and thus mRNA expression. Such structural responses are amenable to computational modelling, making it possible to rationally design synthetic riboswitches for a given aptamer. Starting from an artificial aptamer, we construct the first synthetic transcriptional riboswitches that respond to the antibiotic neomycin. We show that the switching behaviour in vivo critically depends not only on the sequence of the riboswitch itself, but also on its sequence context. We therefore developed in silico methods to predict the impact of the context, making it possible to adapt the design and to rescue non-functional riboswitches. We furthermore analyse the influence of 5′ hairpins with varying stability on neomycin riboswitch activity. Our data highlight the limitations of a simple plug-and-play approach in the design of complex genetic circuits and demonstrate that detailed computational models significantly simplify, improve, and automate the design of transcriptional circuits. Our design software is available under a free licence on GitHub (https://github.com/xileF1337/riboswitch_design).

摘要

原核生物中的基因调控通常依赖于 RNA 元件，例如位于 mRNA 5' 非翻译区的核糖开关或 RNA 温度计。例如响应小分子或离子的结合而发生的RNA结构重排控制转录的翻译起始或提前终止，从而控制mRNA表达。这种结构反应适合计算建模，使得为给定适体合理设计合成核糖开关成为可能。从人工适体开始，我们构建了第一个对抗生素新霉素做出反应的合成转录核糖开关。我们表明，体内的开关行为不仅取决于核糖开关本身的序列，还取决于其序列背景。因此，我们开发了计算机方法来预测环境的影响，从而可以调整设计并挽救非功能性核糖开关。我们进一步分析了不同稳定性的 5' 发夹对新霉素核糖开关活性的影响。我们的数据强调了简单的即插即用方法在复杂遗传电路设计中的局限性，并证明详细的计算模型可以显着简化、改进和自动化转录电路的设计。我们的设计软件可在 GitHub (https://github.com/xileF1337/riboswitch_design) 上获得免费许可。