Design and Construction of Generalizable RNA-Protein Hybrid Controllers by Level-Matched Genetic Signal Amplification

For synthetic biology applications, protein-based transcriptional genetic controllers are limited in terms of orthogonality, modularity, and portability. Although ribozyme-based switches can address these issues, their current two-stage architectures and limited dynamic range hinder their broader incorporation into systems-level genetic controllers. Here, we address these challenges by implementing an RNA-protein hybrid controller with a three-stage architecture that introduces a transcription-based amplifier between an RNA sensor and a protein actuator. To facilitate the construction of these more complex circuits, we use a model-guided strategy to efficiently match the activities of stages. The presence of the amplifier enabled the three-stage controller to have up to 200-fold higher gene expression than its two-stage counterpart and made it possible to implement higher-order controllers, such as multilayer Boolean logic and feedback systems. The modularity inherent in the three-stage architecture along with the sensing flexibility of RNA devices presents a generalizable framework for designing and building sophisticated genetic control systems.