Versatile RNA-sensing transcriptional regulators for engineering genetic networks

TLDR

RNA molecules can sense small molecules and regulate genes, enabling synthetic biology applications, but existing RNA‑based regulatory networks have relied on proteins to transmit signals. This study aims to eliminate protein‑mediated signal propagation and extend the RNA toolkit by engineering three novel features of the pT181 antisense‑RNA transcription attenuation system. The authors engineered the pT181 antisense‑RNA system to produce target‑specific variants that independently regulate multiple genes, to arrange these variants in tandem for signal integration and logic, and to link them into an RNA‑mediated transcriptional cascade that propagates signals without proteins. Combining these features into a single RNA‑based mechanism could simplify genetic network design by enabling direct RNA signal propagation.

Abstract

The widespread natural ability of RNA to sense small molecules and regulate genes has become an important tool for synthetic biology in applications as diverse as environmental sensing and metabolic engineering. Previous work in RNA synthetic biology has engineered RNA mechanisms that independently regulate multiple targets and integrate regulatory signals. However, intracellular regulatory networks built with these systems have required proteins to propagate regulatory signals. In this work, we remove this requirement and expand the RNA synthetic biology toolkit by engineering three unique features of the plasmid pT181 antisense-RNA-mediated transcription attenuation mechanism. First, because the antisense RNA mechanism relies on RNA-RNA interactions, we show how the specificity of the natural system can be engineered to create variants that independently regulate multiple targets in the same cell. Second, because the pT181 mechanism controls transcription, we show how independently acting variants can be configured in tandem to integrate regulatory signals and perform genetic logic. Finally, because both the input and output of the attenuator is RNA, we show how these variants can be configured to directly propagate RNA regulatory signals by constructing an RNA-meditated transcriptional cascade. The combination of these three features within a single RNA-based regulatory mechanism has the potential to simplify the design and construction of genetic networks by directly propagating signals as RNA molecules.