VPg-based bidirectional synthetic mRNA circuits enable orthogonal protein regulation for high-resolution cell separation.

Synthetic mRNA circuits commonly sense input to produce binary output signals for cell separation. Based on virus-origin cap-independent translation initiation machinery and RBP-aptamer interaction, we designed smart synthetic mRNA-based circuits that sense single input molecules to bidirectionally tune output signals in an orthogonal manner, enabling high-resolution separation of cell populations.

Dual Input-Controlled Synthetic mRNA Circuit for Bidirectional Protein Expression Regulation.

Synthetic mRNA circuits manipulate cell fate by controlling output protein expression via cell-specific input molecule detection. Most current circuits either repress or enhance output production upon input binding. Such binary input-output mechanisms restrict the fine-tuning of protein expression to control complex cellular events. Here we designed mRNA circuits using enhancer/repressor modules that were independently controlled by different input molecules, resulting in bidirectional output regulation; the maximal enhancement over maximal repression was 57 fold. The circuit either enhances or represses protein production in different cells based on the difference in the expression of two microRNAs. This study examined novel bidirectional circuit designs capable of fine-tuning protein production by sensing multiple input molecules. It also broadened the scope of cell manipulation by synthetic mRNA circuits, facilitating the development of mRNA circuits for precise cell manipulation and providing cell-based solutions to biomedical problems.