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

Area of Science:

• Synthetic Biology
• Molecular Engineering
• Biotechnology

Background:

• Scalable genetic circuits are crucial for advanced cellular functions.
• Synthetic RNA regulators offer advantages like low metabolic load and design flexibility.
• Current challenges include achieving wide dynamic ranges and multiplexed regulatory cascades in RNA circuits.

Purpose of the Study:

• To introduce the RATEX (Ribosome-Assisted Transcriptional EXpression controller) platform for scalable RNA-programmed circuit design.
• To integrate a translation-to-transcription converter with synthetic RNA regulators for enhanced control.
• To address limitations in dynamic range and multiplexing for synthetic RNA circuits.

Main Methods:

• Developed the RATEX platform by integrating a translation-to-transcription converter with synthetic RNA regulators.
• Utilized a library of translation regulators for gene regulation up to 1,492-fold.
• Leveraged natural ribosome-mediated sensing for environmental inputs like metabolites.
• Demonstrated multi-input logic gates (up to 6-input OR, hybrid 3-input) for RNA, metabolite, and small-molecule inputs.
• Implemented multiplexed signaling cascades for signal amplification and combinatorial control of RNA outputs.
• Applied RNA- and metabolite-sensing AND gates to control cellular morphology and spatial organization.

Main Results:

• The RATEX platform enables a compact and scalable RNA-programmed circuit architecture.
• Achieved significant gene regulation (up to 1,492-fold) using repurposed translation regulators.
• Successfully demonstrated multi-input logic processing with complex logic gates.
• Showcased signal amplification and multiplexed control of RNA outputs.
• Controlled cellular morphology and intracellular spatial organization using RATEX-based logic gates.

Conclusions:

• The RATEX platform provides a scalable and modular architecture for designing synthetic RNA circuits.
• Offers a broad design space for synthetic biology and biotechnology applications.
• Enables sophisticated control over cellular functions through RNA-programmed gene expression.