基于核酶开关的miRNA荧光适配体探针设计及在药物性肝损伤检测中的应用

Drug-induced liver injury is a major problem in drug development and clinical use. Biomarkers that can specifically and sensitively predict hepatotoxicity and their detection techniques are very important for drug development and drug safety. Due to the specific distribution in liver and stability, microRNA is a new ideal marker for the evaluation of liver injury. The development of the detection methods, especially the real-time dynamic analysis of intracellular miRNA , will be helpful for the study of its biological function during the occurrence and development of liver injury, as well as the preclinical evaluation of drug safety and clinical diagnosis. Herein we will try to develop a novel strategy for in-situ imaging of miRNA, combining genetically encoded fluorescent aptamers with riboswitch to solve the detection difficulties such as low content of miRNA in cells. Signal amplification can be achieved through the cyclic utilization of target miRNA mediated by riboswitch that recognizes the target sequence, and at the same time, the structure of fluorescent aptamers can be reconstructed to generate fluorescence. Taking typical drug-induced liver injury cell model as an example, this method will be applied in rapid visual evaluation of drug hepatotoxicity, which is expected to provide a new technique for the detection of miRNA in living cells and a new strategy for drug safety evaluation.

药物性肝损伤是药物研发及临床使用中存在的主要问题，能特异及敏感地预测肝毒性的生物标志物及其检测技术对于新药开发及安全用药十分重要。MiRNA由于具有组织特异性分布及稳定性等特点，是评价肝损伤的新型理想标志物，开发其检测方法特别是活细胞内miRNA的实时动态分析对其在肝损伤发生及发展过程中生物功能的研究、药物临床前安全性评价及临床诊断应用均有重要意义。本项目针对miRNA含量较低等检测难点，将可基因编码的荧光适配体和功能性剪切核酶结合，充分利用核酸结构的特性，通过识别目标序列的核酶开关介导目标miRNA循环利用进行信号放大，同时实现荧光适配体的结构重建产生荧光信号；从而构建基于核酶开关的基因编码荧光适配体探针及miRNA活细胞原位成像新技术，以典型药物性肝损伤细胞模型为例，探究该方法在药物肝毒性快速可视化评价中的应用，有望为活细胞水平miRNA的检测提供新技术，为药物安全性评价提供新策略。

肝损伤标志物的发现及其检测技术的开发对于新药研究及临床安全用药具有重要意义。miRNA由于稳定性较好、肝脏分布特异性高、肝损伤高度相关等优点，具有作为肝毒性生物标志物的潜力，有望成为临床前药物评价及肝损伤早期预测的新依据。本项目针对目前大多数miRNA检测方法受限于离体检测、无法对细胞内miRNA进行定位及动态分析等局限，借助核酶及荧光适配体的优良特性，构建了可基因编码的荧光适配体探针，为活细胞水平肝损伤相关标志物的检测提供了新方法和新策略。主要的研究进展包括：1）成功设计并优化得到了目标miRNA启动的荧光适配体结构重建信号传感以及核酶开关介导信号放大的核酸探针，并构建了肝损伤相关miRNA的荧光检测方法。2）成功构建了基于配体荧光分子结构改造策略的肝脏代谢酶活性检测新策略和新方法。截至目前，本课题已基本完成课题任务书的研究内容，本项目共发表SCI论文2篇，培养硕士生2名，本科生1名。