Simultaneous Detection\nof Multiple Analytes at Ambient\nTemperature Using Eukaryotic Artificial Cells with Modular and Robust\nSynthetic Riboswitches

Cell-free systems, which can express an easily detectable\noutput\n(protein) with a DNA or mRNA template, are promising as foundations\nof biosensors devoid of cellular constraints. Moreover, by encasing\nthem in membranes such as natural cells to create artificial cells,\nthese systems can avoid the adverse effects of environmental inhibitory\nmolecules. However, the bacterial systems generally used for this\npurpose do not function well at ambient temperatures. We here encapsulated\na eukaryotic cell-free system consisting of wheat germ extract (WGE)\nand a DNA template encoding an analyte-responsive regulatory RNA (called\na riboswitch) into giant unilamellar vesicles (GUVs) to create eukaryotic\nartificial cell-based sensors that function well at ambient temperature.\nFirst, we improved our previously reported eukaryotic synthetic riboswitches\nand WGE for use in GUVs by chimerizing two internal ribosome entry\nsites and optimizing magnesium concentrations, respectively, both\nof which increased the expression efficiency in GUVs several fold.\nThen, a DNA template encoding one of these riboswitches followed by\na reporter protein was encapsulated with the optimized GUV-friendly\nWGE. Importantly, our previously established versatile method allowed\nfor the rational design of highly efficient eukaryotic riboswitches\nthat are responsive to a user-defined analyte. In fact, we utilized\nthis method to successfully create three types of artificial cells,\neach of which responded to a specific, membrane-permeable analyte\nwith wide-range, analyte-dose dependency and high sensitivity at ambient\ntemperature. Finally, due to their orthogonality and robustness, we\nwere able to mix a cocktail of these artificial cells to achieve simultaneous\ndetection of the three analytes without significant barriers.