Chen Dian, Li Jun, Wu You, Hong Liang, Liu Yu
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
School of Physics and Astronomy, Shanghai Jiao Tong University, Shanghai 200240, China.
Sci Adv. 2025 Apr 4;11(14):eadt8165. doi: 10.1126/sciadv.adt8165. Epub 2025 Apr 2.
Cyclic diadenosine monophosphate (C-di-AMP) synthases are key enzymes for synthesizing c-di-AMP, a potent activator of the stimulator of interferon genes (STING) immune pathway. However, characterizing these enzymes has been hampered by the lack of effective sensors. While c-di-AMP riboswitches, as natural aptamers, hold the potential as RNA biosensors, their poorly comprehended structural dynamics and inherent "OFF" genetic output pose substantial challenges. To address these limitations, we synthesized over 10 fluorophore-labeled samples to probe the conformational changes of the riboswitch at the single-molecule level. By integrating these dynamic findings with steady-state fluorescence titration, mutagenesis, in vivo assays, and strand displacement strategy, we transformed the natural aptamer into a c-di-AMP biosensor. This engineered biosensor reversed its genetic output from "OFF" to "ON" upon c-di-AMP binding, exhibiting a 50-fold improvement in the c-di-AMP detection limit. Leveraging this refined biosensor, we developed a robust strategy for high-throughput in vivo evolution of c-di-AMP synthases.
环二磷酸腺苷（c-di-AMP）合酶是合成c-di-AMP的关键酶，c-di-AMP是干扰素基因刺激因子（STING）免疫途径的有效激活剂。然而，由于缺乏有效的传感器，对这些酶的特性研究受到了阻碍。虽然c-di-AMP核糖开关作为天然适配体，有潜力成为RNA生物传感器，但其结构动力学理解不足以及固有的“关闭”基因输出带来了重大挑战。为了解决这些限制，我们合成了10多个荧光团标记的样品，以在单分子水平上探测核糖开关的构象变化。通过将这些动态研究结果与稳态荧光滴定、诱变、体内试验和链置换策略相结合，我们将天然适配体转化为了c-di-AMP生物传感器。这种工程化的生物传感器在结合c-di-AMP后，其基因输出从“关闭”变为“开启”，c-di-AMP检测限提高了50倍。利用这种优化的生物传感器，我们开发了一种强大的策略，用于c-di-AMP合酶的高通量体内进化。
