Cell-Free Multistep Gene Regulatory Cascades Using Eukaryotic ON-Riboswitches Responsive to <i>in Situ</i> Expressed Protein Ligands

One of the most pressing challenges in cell-free synthetic biology is to assemble well-controlled genetic circuits. However, no complex circuits have been reported in eukaryotic cell-free systems, unlike the case in bacterial ones, despite several unique advantages of the former. We here developed protein-responsive upregulating riboswitches (ON-riboswitches) that function in wheat germ extract to create multistep gene regulatory cascades. Although the initial two types of ON-riboswitches we first designed were less efficient than desired, we improved one of them by incorporating hybridization switches to successfully construct a pair of highly efficient, protein-responsive ON-riboswitches. Both upregulated expression up to 20-fold through self-cleavage by a hammerhead ribozyme (HHR) in response to the corresponding protein ligands expressed <i>in situ</i>. We then combined them with similar types of HHR-based, small-molecule-responsive ON-riboswitches regulating protein ligand expression, to create four kinds of two-step regulatory cascades. Due to the high orthogonality of all the riboswitches used, we also succeeded in regulating two-step cascades concurrently and even in creating three-step cascades. Interestingly, the switching efficiency of each multistep cascade constructed was equivalent to that of the worst step within it. Therefore, more complex cascades with additional steps could be constructed using other efficient and orthogonal, protein-responsive ON-riboswitches with minimal loss of total switching efficiency, although the reaction conditions must be optimized to prevent a reduction of expression efficiencies. Riboswitch-based cascades fashioned through our proposed strategy would aid in the construction of eukaryotic genetic circuits for programmed cell-free systems or artificial cells with functionalities surpassing those of natural cells.