Takahashi Hajime, Ikemoto Yuri, Ogawa Atsushi
Proteo-Science Center, Ehime University, 2-5 Bunkyo, Matsuyama, Ehime 790-8577, Japan.
ACS Synth Biol. 2025 Mar 21;14(3):771-780. doi: 10.1021/acssynbio.4c00696. Epub 2024 Dec 27.
Cell-free systems, which can express an easily detectable output (protein) with a DNA or mRNA template, are promising as foundations of biosensors devoid of cellular constraints. Moreover, by encasing them in membranes such as natural cells to create artificial cells, these systems can avoid the adverse effects of environmental inhibitory molecules. However, the bacterial systems generally used for this purpose do not function well at ambient temperatures. We here encapsulated a eukaryotic cell-free system consisting of wheat germ extract (WGE) and a DNA template encoding an analyte-responsive regulatory RNA (called a riboswitch) into giant unilamellar vesicles (GUVs) to create eukaryotic artificial cell-based sensors that function well at ambient temperature. First, we improved our previously reported eukaryotic synthetic riboswitches and WGE for use in GUVs by chimerizing two internal ribosome entry sites and optimizing magnesium concentrations, respectively, both of which increased the expression efficiency in GUVs several fold. Then, a DNA template encoding one of these riboswitches followed by a reporter protein was encapsulated with the optimized GUV-friendly WGE. Importantly, our previously established versatile method allowed for the rational design of highly efficient eukaryotic riboswitches that are responsive to a user-defined analyte. In fact, we utilized this method to successfully create three types of artificial cells, each of which responded to a specific, membrane-permeable analyte with wide-range, analyte-dose dependency and high sensitivity at ambient temperature. Finally, due to their orthogonality and robustness, we were able to mix a cocktail of these artificial cells to achieve simultaneous detection of the three analytes without significant barriers.
无细胞系统能够利用DNA或mRNA模板表达易于检测的输出（蛋白质），有望成为不受细胞限制的生物传感器基础。此外，通过将它们包裹在诸如天然细胞的膜中来制造人工细胞，这些系统可以避免环境抑制分子的不利影响。然而，通常用于此目的的细菌系统在环境温度下功能不佳。我们在此将由小麦胚芽提取物（WGE）和编码分析物响应调节RNA（称为核糖开关）的DNA模板组成的真核无细胞系统封装到巨型单层囊泡（GUV）中，以创建在环境温度下功能良好的基于真核人工细胞的传感器。首先，我们分别通过嵌合两个内部核糖体进入位点和优化镁浓度，改进了我们先前报道的用于GUV的真核合成核糖开关和WGE，这两者都使GUV中的表达效率提高了几倍。然后，将编码这些核糖开关之一并随后接报告蛋白的DNA模板与优化的对GUV友好的WGE一起封装。重要的是，我们先前建立的通用方法允许合理设计对用户定义的分析物有响应的高效真核核糖开关。事实上，我们利用这种方法成功创建了三种类型的人工细胞，每种细胞在环境温度下对特定的、可透过膜的分析物具有宽范围、分析物剂量依赖性和高灵敏度的响应。最后，由于它们的正交性和稳健性，我们能够混合这些人工细胞的混合物，以实现对三种分析物的同时检测而没有明显障碍。
