Qin Pei-Pei, Chen Pin-Ru, Tan Liu, Chu Xiaohe, Ye Bang-Ce, Yin Bin-Cheng
Institute of Engineering Biology and Health, Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, College of Pharmaceutical Sciences, Zhejiang University of Technology, No.18 Chao Wang Road, Gongshu District, Hangzhou 310014, China.
Lab of Biosystem and Microanalysis, State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, No.130 Meilong Road, Xuhui District, Shanghai 200237, China.
Nucleic Acids Res. 2025 Jan 7;53(1). doi: 10.1093/nar/gkae1146.
RNA editing leveraging ADARs (adenosine deaminases acting on RNA) shows promising potential for in vivo biosensing beyond gene therapy. However, current ADAR sensors sense only a single target of RNA transcripts, thus limiting their use in different biosensing scenarios. Here, we report a hairpin RNA sensor that exploits new mechanisms to generate intramolecular duplex substrates for efficient ADAR recruitment and editing and apply it to detection of various intracellular molecules, including messenger RNA, small molecules and proteins. We utilize the base pairing interactions between neighbouring bases for enhanced stability, as well as the reverse effects to sense RNA transcripts and single-nucleotide variants with high sensitivity and specificity, irrespective of sequence requirement for complementarity to an UAG stop codon. In addition, we integrate RNA aptamers into the hairpin RNA sensor to realize the detection of the primary energy-supplying molecule, ATP, and a transcription factor, nuclear factor-kappa B (NF-κB), in live cells via a simple conformational change for programming the activation of hairpin RNA. This sensor not only broadens the detection of applicable molecules, but also offers potential for diverse cell manipulation.
利用作用于RNA的腺苷脱氨酶（ADARs）进行RNA编辑在基因治疗之外的体内生物传感方面显示出有前景的潜力。然而，目前的ADAR传感器只能检测RNA转录本的单一靶点，因此限制了它们在不同生物传感场景中的应用。在此，我们报道了一种发夹RNA传感器，该传感器利用新机制生成分子内双链底物，以有效招募和编辑ADAR，并将其应用于检测各种细胞内分子，包括信使RNA、小分子和蛋白质。我们利用相邻碱基之间的碱基配对相互作用来增强稳定性，并利用反向效应以高灵敏度和特异性检测RNA转录本和单核苷酸变体，而无需与UAG终止密码子互补的序列要求。此外，我们将RNA适体整合到发夹RNA传感器中，通过简单的构象变化来编程发夹RNA的激活，从而在活细胞中实现对主要能量供应分子ATP和转录因子核因子-κB（NF-κB）的检测。这种传感器不仅拓宽了可检测分子的范围，还为多种细胞操作提供了潜力。
