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Quantitative Characterization of Translational Riboregulators Using an in Vitro Transcription–Translation System
ACS Synthetic Biology ( IF 4.7 ) Pub Date : 2018-04-04 00:00:00 , DOI: 10.1021/acssynbio.7b00387
Anis Senoussi 1 , Jonathan Lee Tin Wah 1 , Yoshihiro Shimizu 2 , Jérôme Robert 1 , Alfonso Jaramillo 3, 4, 5 , Sven Findeiss 6, 7 , Ilka M. Axmann 8 , André Estevez-Torres 1
Affiliation  

Riboregulators are short RNA sequences that, upon binding to a ligand, change their secondary structure and influence the expression rate of a downstream gene. They constitute an attractive alternative to transcription factors for building synthetic gene regulatory networks because they can be engineered de novo. However, riboregulators are generally designed in silico and tested in vivo, which provides little quantitative information about their performances, thus hindering the improvement of design algorithms. Here we show that a cell-free transcription-translation (TX–TL) system provides valuable information about the performances of in silico designed riboregulators. We first propose a simple model that provides a quantitative definition of the dynamic range of a riboregulator. We further characterize two types of translational riboregulators composed of a cis-repressed (cr) and a trans-activating (ta) strand. At the DNA level we demonstrate that high concentrations of taDNA poisoned the activator until total shut off, in agreement with our model, and that relative dynamic ranges of riboregulators determined in vitro are in agreement with published in vivo data. At the RNA level, we show that this approach provides a fast and simple way to measure dissociation constants of functional riboregulators, in contrast to standard mobility-shift assays. Our method opens the route for using cell-free TX–TL systems for the quantitative characterization of functional riboregulators in order to improve their design in silico.

中文翻译:

使用体外转录-翻译系统对翻译性核糖调节剂的定量表征

核糖调节剂是短的RNA序列,其在与配体结合后会改变其二级结构并影响下游基因的表达速率。它们可以替代转录因子来构建合成基因调控网络,因为它们可以从头设计。但是,核糖体调节剂通常是计算机设计的在体内进行了测试,这几乎没有提供有关其性能的定量信息,因此阻碍了设计算法的改进。在这里,我们展示了无细胞转录翻译(TX–TL)系统提供了有关计算机性能的宝贵信息设计的核糖调节器。我们首先提出一个简单的模型,该模型提供了定量调节核糖调节器动态范围的方法。我们进一步表征由顺式抑制(cr)和反式激活(ta)链组成的两种类型的翻译核糖调节子。在DNA水平上,我们证明了高浓度的taDNA会中毒激活剂,直到完全关闭为止,这与我们的模型一致,并且体外测定的核糖调节剂的相对动态范围与体内发表的结果一致数据。在RNA水平上,我们证明了该方法提供了一种快速而简单的方法来测量功能性核糖调节剂的解离常数,这与标准的迁移率漂移测定法不同。我们的方法为使用无细胞TX-TL系统进行功能性核糖调节蛋白的定量表征开辟了道路,以改善其计算机设计。
更新日期:2018-04-04
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