Pérez-Ropero Guillermo, Dolcemascolo Roswitha, Pérez-Ràfols Anna, Andersson Karl, Danielson U Helena, Rodrigo Guillermo, Buijs Jos
Ridgeview Instruments AB, 75237 Uppsala, Sweden.
Department of Chemistry-BMC, Uppsala University, 75123 Uppsala, Sweden.
Biosensors (Basel). 2025 Mar 8;15(3):175. doi: 10.3390/bios15030175.
Reporter systems are widely used to study biomolecular interactions and processes in vivo, representing one of the basic tools used to characterize synthetic regulatory circuits. Here, we developed a method that enables the monitoring of RNA-protein interactions through a reporter system in bacteria with high temporal resolution. For this, we used a Real-Time Protein Expression Assay (RT-PEA) technology for real-time monitoring of a fluorescent reporter protein, while having bacteria growing on solid media. Experimental results were analyzed by fitting a three-variable Gompertz growth model. To validate the method, the interactions between a set of RNA sequences and the RNA-binding protein (RBP) Musashi-1 (MSI1) were evaluated, as well as the allosteric modulation of the interaction by a small molecule (oleic acid). This new approach proved to be suitable to quantitatively characterize RNA-RBP interactions, thereby expanding the toolbox to study molecular interactions in living bacteria, including allosteric modulation, with special relevance for systems that are not suitable to be studied in liquid media.
报告系统被广泛用于研究体内生物分子相互作用和过程，是用于表征合成调控回路的基本工具之一。在此，我们开发了一种方法，能够通过报告系统在细菌中以高时间分辨率监测RNA-蛋白质相互作用。为此，我们使用实时蛋白质表达分析（RT-PEA）技术实时监测荧光报告蛋白，同时使细菌在固体培养基上生长。通过拟合三变量Gompertz生长模型对实验结果进行分析。为了验证该方法，评估了一组RNA序列与RNA结合蛋白（RBP）Musashi-1（MSI1）之间的相互作用，以及小分子（油酸）对该相互作用的变构调节。这种新方法被证明适用于定量表征RNA-RBP相互作用，从而扩展了研究活细菌中分子相互作用的工具箱，包括变构调节，这对于不适于在液体培养基中研究的系统具有特殊意义。
