Full Design Automation of Multi-State RNA Devices to Program Gene Expression Using Energy-Based Optimization

Small RNAs (sRNAs) can operate as regulatory agents to control protein expression by interaction with the 5′ untranslated region of the mRNA. We have developed a physicochemical framework, relying on base pair interaction energies, to design multi-state sRNA devices by solving an optimization problem with an objective function accounting for the stability of the transition and final intermolecular states. Contrary to the analysis of the reaction kinetics of an ensemble of sRNAs, we solve the inverse problem of finding sequences satisfying targeted reactions. We show here that our objective function correlates well with measured riboregulatory activity of a set of mutants. This has enabled the application of the methodology for an extended design of RNA devices with specified behavior, assuming different molecular interaction models based on Watson-Crick interaction. We designed several YES, NOT, AND, and OR logic gates, including the design of combinatorial riboregulators. In sum, our de novo approach provides a new paradigm in synthetic biology to design molecular interaction mechanisms facilitating future high-throughput functional sRNA design.

小RNA（small RNAs, sRNAs）可作为调控因子，通过与信使RNA（messenger RNA, mRNA）的5′非翻译区相互作用，调控蛋白质的表达。本研究开发了一种基于碱基配对相互作用能的物理化学框架，通过求解以过渡态与最终分子间复合物稳定性为目标函数的优化问题，实现多状态sRNA器件的设计。相较于针对sRNA群体反应动力学的常规分析，本研究求解了反向设计问题，即筛选出可满足靶向反应需求的RNA序列。研究证实，所提出的目标函数与一组突变体的实测核糖调控活性具有良好相关性。这一成果使得该方法能够依托基于沃森-克里克（Watson-Crick）相互作用的不同分子互作模型，拓展设计具备特定功能的RNA器件。本研究共设计了多种YES、NOT、AND及OR逻辑门器件，其中涵盖组合式核糖调控元件的设计。综上，本研究提出的从头（de novo）设计方法为合成生物学领域的分子互作机制构建提供了全新范式，可为未来高通量功能性sRNA的设计开发提供助力。