Wayment-Steele Hannah, Wu Michelle, Gotrik Michael, Das Rhiju
Department of Chemistry, Stanford University, Stanford, CA, United States.
Program in Biomedical Informatics, Stanford University, Stanford, CA, United States.
Methods Enzymol. 2019;623:417-450. doi: 10.1016/bs.mie.2019.05.028. Epub 2019 Jun 18.
Riboswitches are RNA elements that recognize diverse chemical and biomolecular inputs, and transduce this recognition process to genetic, fluorescent, and other engineered outputs using RNA conformational changes. These systems are pervasive in cellular biology and are a promising biotechnology with applications in genetic regulation and biosensing. Here, we derive a simple expression bounding the activation ratio-the proportion of RNA in the active vs. inactive states-for both ON and OFF riboswitches that operate near thermodynamic equilibrium: 1+[I]/K, where [I] is the input ligand concentration and K is the intrinsic dissociation constant of the aptamer module toward the input ligand. A survey of published studies of natural and synthetic riboswitches confirms that the vast majority of empirically measured activation ratios have remained well below this thermodynamic limit. A few natural and synthetic riboswitches achieve activation ratios close to the limit, and these molecules highlight important principles for achieving high riboswitch performance. For several applications, including "light-up" fluorescent sensors and chemically-controlled CRISPR/Cas complexes, the thermodynamic limit has not yet been achieved, suggesting that current tools are operating at suboptimal efficiencies. Future riboswitch studies will benefit from comparing observed activation ratios to this simple expression for the optimal activation ratio. We present experimental and computational suggestions for how to make these quantitative comparisons and suggest new molecular mechanisms that may allow non-equilibrium riboswitches to surpass the derived limit.
核糖开关是一种RNA元件,能够识别多种化学和生物分子输入,并利用RNA构象变化将这种识别过程转化为遗传、荧光和其他工程输出。这些系统在细胞生物学中普遍存在,是一种有前途的生物技术,可应用于基因调控和生物传感。在这里,我们推导出一个简单的表达式,用于界定在热力学平衡附近运行的开启型和关闭型核糖开关的激活率(即处于活性状态与非活性状态的RNA比例):1 + [I]/K,其中[I]是输入配体浓度,K是适体模块对输入配体的固有解离常数。对已发表的天然和合成核糖开关研究的调查证实,绝大多数通过实验测量的激活率仍远低于这个热力学极限。少数天然和合成核糖开关的激活率接近该极限,这些分子突出了实现高核糖开关性能的重要原则。对于包括“点亮”荧光传感器和化学控制的CRISPR/Cas复合物在内的几种应用,尚未达到热力学极限,这表明当前工具的运行效率并非最优。未来的核糖开关研究将受益于将观察到的激活率与这个最优激活率的简单表达式进行比较。我们提出了关于如何进行这些定量比较的实验和计算建议,并提出了可能使非平衡核糖开关超越推导极限的新分子机制。