RATEX: A Scalable RNA-Based Platform for Logical and Multi-Layered Cellular Programming.

Scalable genetic circuits are essential for implementing complex functions in living cells. Toward this goal, RNA regulators can provide a much-needed parts library with added benefits of low metabolic load, design flexibility, and logic capacity. However, despite the great potential of synthetic RNA circuits, constructing such circuits with wide dynamic ranges and multiplexed regulatory cascades remains a challenge. To address this, we introduce RATEX (Ribosome-Assisted Transcriptional EXpression controller) by integrating a translation-to-transcription converter with synthetic RNA regulators, enabling a compact and scalable RNA-programmed circuit architecture. The RATEX platform repurposes a large library of well-characterized translation regulators with up to 1,492-fold gene regulation, while leveraging natural ribosome-mediated sensing of diverse environmental inputs, such as metabolites. We demonstrated multi-input logic processing with up to a 6-input OR logic gate for RNA inputs and hybrid 3-input logic gates to sense diverse metabolite and small-molecule inputs alongside RNA signals. Signal amplification with multiplexed combinatorial control of RNA outputs was achieved through multiplexed signaling cascades. Finally, the RNA- and metabolite-sensing 3-input AND gates were used to control cellular morphology and intracellular spatial organization. Together, the RATEX platform, with its scalable and modular architecture, offers a broad potential design space for synthetic biology and biotechnology.