基于遗传编码RNA适配体生物传感器用于活细胞内RNA甲基化修饰的可视化研究

The visualization of RNA base methylations is of great significance to the understanding epitranscriptomic modifications. The project focuses on the great challenge in real-time, in situ, visualized and selective imaging the RNA base methylations in living cells. Exploiting the fact that methyl groups in RNA can impair base pairing or destabilize of secondary structure in RNA, we will combine the genetic engineering techniques and the versatile molecular recognition functions and the powerful imaging ability of RNA “light-up probes to develop novel approach and technology to image the RNA methyl-modifications dynamically and in real-time in specific sites in living cells. Taking the methylation by KsgA methyltransferase as model, we will utilize the responsive probe that sensitive to methylation of their complementary RNA sequences to a novel genetically encoded light-up methy-RNA sensor while using m6A-containing secondary structure RNA sequence as example, we will take advantage of the N6-methyladenosine-deependent RNA structure switch to light-up RNA aptamer for the detection of methylation in RNA. These sensor may have great potential as an efficient platform for epitranscriptome research.

表观转录组RNA甲基化的可视化分析意义重大。本项目围绕RNA甲基化检测仍面临着不能活细胞实时示踪成像的难题，基于RNA特定位点的甲基化修饰可以改变双链杂交的稳定性或者改变RNA的二级构象，结合基因工程技术及RNA核酸适体强大的识别功能和荧光成像能力，拟发展两种活细胞内RNA甲基化修饰的实时、动态成像新方法。以大肠杆菌中KsgA作用的rRNA底物为模型，发展基于RNA甲基化敏感的双链杂交激活点亮RNA适配体用于活细胞内RNA甲基化修饰成像新方法研究；以哺乳动物细胞中包含N6-A二级结构的RNA底物为模型，发展基于甲基化构像变化激活点亮RNA适配体用于活细胞内RNA甲基化碱基成像新方法研究。以期为表观转录组学中RNA的化学修饰的可视化研究提供新的技术平台。

表观转录组RNA甲基化的可视化分析意义重大。我们围绕RNA甲基化修饰的动态可视化检测问题，利用基因工程技术及RNA核酸适体强大的识别功能和荧光成像能力，发展基于遗传编码RNA适配体传感及活细胞成像新方法，重点探索了基于多启动子转录事件控制RNA适体及其表达可行性，以及基于甲基化作为转换器来改变 RNA 二级结构发展RNA甲基化可视化检测的合理性，据此建立活细胞内RNA甲基化修饰的实时、动态成像新方法。项目严格按照研究计划进行，相关的工作在进一步整理发表。同时，我们也围绕生化分析与生物传感的前沿问题，以生物标志物分子及细胞内重要分子信号检测的新原理与新方法研究为基点，利用新型纳米材料的独特性质，发展活细胞生物传感与成像研究。项目执行期间，相关研究成果已经形成SCI收录学术论文4篇，均标注本项目资助。培养博士生2名，研究生6名。