Beijing Institute of Life Sciences, Chinese Academy of Sciences, Beijing 100101, China.
Department of Pulmonary and Critical Care Medicine, Department of Inflammation and Clinical Allergology, the Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, China.
Nucleic Acids Res. 2023 Sep 8;51(16):8322-8336. doi: 10.1093/nar/gkad620.
Sensors to measure the abundance and signaling of intracellular molecules are crucial for understanding their physiological functions. Although conventional fluorescent protein-based sensors have been designed, RNA-based sensors are promising imaging tools. Numerous RNA-based sensors have been developed. These sensors typically contain RNA G-quadruplex (RG4) motifs and thus may be suboptimal in living cells. Here we describe RNA-based sensors based on Pepper, a fluorogenic RNA without an RG4 motif. With Pepper, we engineered various sensors for metabolites, synthetic compounds, proteins and metal ions in vitro and in living cells. In addition, these sensors show high activation and selectivity, demonstrating their universality and robustness. In the case of sensors responding to S-adenosylmethionine (SAM), a metabolite produced by methionine adenosyltransferase (MATase), we showed that our sensors exhibited positively correlated fluorescence responding to different SAM levels. Importantly, we revealed the SAM biosynthesis pathway and monitored MATase activity and gene expression spatiotemporally in living individual human cells. Additionally, we constructed a ratiometric SAM sensor to determine the inhibition efficacy of a MATase inhibitor in living cells. Together, these sensors comprising Pepper provide a useful platform for imaging diverse cellular targets and their signaling pathway.
用于测量细胞内分子丰度和信号转导的传感器对于理解其生理功能至关重要。尽管已经设计出了基于传统荧光蛋白的传感器，但基于 RNA 的传感器是很有前途的成像工具。已经开发出了许多基于 RNA 的传感器。这些传感器通常包含 RNA G-四联体（RG4）基序，因此在活细胞中可能不是最佳的。在这里，我们描述了基于 Pepper 的 RNA 传感器，Pepper 是一种没有 RG4 基序的荧光 RNA。利用 Pepper，我们在体外和活细胞中为代谢物、合成化合物、蛋白质和金属离子设计了各种传感器。此外，这些传感器表现出高的激活和选择性，证明了它们的通用性和稳健性。在响应 S-腺苷甲硫氨酸（SAM）的传感器的情况下，SAM 是由蛋氨酸腺苷转移酶（MATase）产生的代谢物，我们表明我们的传感器对不同的 SAM 水平表现出正相关的荧光响应。重要的是，我们揭示了 SAM 生物合成途径，并在活的单个人类细胞中时空监测 MATase 活性和基因表达。此外，我们构建了一个比率型 SAM 传感器来确定 MATase 抑制剂在活细胞中的抑制效果。总之，这些包含 Pepper 的传感器为成像各种细胞靶标及其信号通路提供了一个有用的平台。
