可用于活细胞的免标记核酸适配体传感器的构建

Monitoring dynamic process in living cells is very significant to the biological research. This project plans to construct a label-free aptasensor , which is feasible for the detection of targets in living cells. Firstly, the fluorescence switchable RNA aptamers of Tb3+(Eu3+) would be screened by the systematic evolution of ligands by exponential enrichment techbology. Then a modular aptameric sensor (MAS) was constructed by linking the fluorescence switchable aptamer and aptamer of target covalently. when the target is absent, Tb3+(Eu3+) was not recognized by MAS and there is no fluorescence. when the target is present, the structure of MAS was induced, Tb3+(Eu3+) was recognized by MAS and emit its' fluorescence. Based on the genetic engineering technology, MAS can be transformed into the living cells. And was detected by the time-resolved fluorescence technology.Qwing to the speicial fluorescence property of Tb3+(Eu3+), the background fluorescence can be removed by the time-resolved fluorescence technology. Aptamers can be generated against to many metabolites, which means the MAS is easily generalizable.Finallly, monitoring many moleculars in living cell in-situ and real time can be achieved by the project.The results obtained by this project will be beneficial to explore the structure and function of cell, law and nature of life.

活细胞的实时动态检测对生命科学研究非常重要。因此本项目计划构建可进入活细胞的免标记核酸适配体荧光传感器。首先，利用SELEX技术筛选对稀土离子Tb3+(Eu3+)荧光起开关作用的RNA适配体，然后将其与待测物适配体通过连接子组装为组合式适配体传感器（MAS），靶分子不存在时，MAS不与Tb3+(Eu3+)结合，无荧光；当靶分子出现时，诱导MAS结构发生变化，与Tb3+(Eu3+)结合发光。最后利用分子克隆技术将MAS转化进入活细胞内，并利用时间分辨荧光技术对靶分子进行检测。结合稀土离子的特殊荧光性质和适配体的靶分子范围广的优点，最终实现活细胞内多种代谢物的原位实时检测，克服目前活细胞检测所遇到的荧光背景高，可检测物质有限等问题，更好地认识细胞结构和功能、生命活动规律和本质。