The ability of RNA to sense, regulate, and store information is an attractive attribute for a variety of functional applications including the development of regulatory control devices for synthetic biology. RNA folding and function is known to be highly context sensitive, which limits the modularity and reuse of RNA regulatory devices to control different heterologous sequences and genes. We explored the cause and effect of sequence context sensitivity for translational ON riboswitches located in the 5′ UTR, by constructing and screening a library of N-terminal synonymous codon variants. By altering the N-terminal codon usage we were able to obtain RNA devices with a broad range of functional performance properties (ON, OFF, fold-change). Linear regression and calculated metrics were used to rationalize the major determining features leading to optimal riboswitch performance, and to identify multiple interactions between the explanatory metrics. Finally, partial least squared (PLS) analysis was employed in order to understand the metrics and their respective effect on performance. This PLS model was shown to provide good explanation of our library. This study provides a novel multivariant analysis framework to rationalize the codon context performance of allosteric RNA-devices. The framework will also serve as a platform for future riboswitch context engineering endeavors.

中文翻译：

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使动态 RNA 调节装置的上下文相关性能合理化

RNA 感知、调节和存储信息的能力是各种功能应用的一个有吸引力的属性，包括开发合成生物学的调节控制装置。众所周知，RNA 折叠和功能是高度上下文敏感的，这限制了 RNA 调节装置的模块化和重用，以控制不同的异源序列和基因。我们通过构建和筛选 N 端同义密码子变体文库，探索了位于 5' UTR 的翻译 ON 核糖开关的序列上下文敏感性的原因和影响。通过改变 N 末端密码子的使用，我们能够获得具有广泛功能性能特性（开、关、倍数变化）的 RNA 装置。线性回归和计算指标用于合理化导致最佳核糖开关性能的主要决定性特征，并确定解释性指标之间的多重相互作用。最后，采用偏最小二乘 (PLS) 分析来了解指标及其对性能的各自影响。这个 PLS 模型被证明可以很好地解释我们的库。本研究提供了一个新的多变量分析框架，以合理化变构 RNA 装置的密码子上下文性能。该框架还将作为未来核糖开关上下文工程工作的平台。采用偏最小二乘 (PLS) 分析来了解指标及其对性能的各自影响。这个 PLS 模型被证明可以很好地解释我们的库。本研究提供了一个新的多变量分析框架，以合理化变构 RNA 装置的密码子上下文性能。该框架还将作为未来核糖开关上下文工程工作的平台。采用偏最小二乘 (PLS) 分析来了解指标及其对性能的各自影响。这个 PLS 模型被证明可以很好地解释我们的库。本研究提供了一个新的多变量分析框架，以合理化变构 RNA 装置的密码子上下文性能。该框架还将作为未来核糖开关上下文工程工作的平台。