基于无酶核酸放大反应的三元DNA纳米探针用于活动物内RNA成像研究

The ability for RNA imaging in living animals creates new possibilities of deciphering biology and early diagnosis of diseases including cancer. However, current fluorescent probes for RNA imaging have only been realized in living cells or sliced tissues from living animals. To our knowledge, RNA imaging in living animals has not been reported yet. A key issue is that the nucleic acid probe can be degraded by nuclease in cells or body blood and the targeted cellular delivery of nucleic acid is inefficiency. Another issue is that the lacking of signal amplification limit their detection of low-abundance RNA targets. To address the above issues, a nonenzymatic nucleic acid amplification reaction circuit based cell-targeting Y-shaped tripartite DNA nanoprobe will be developed in this project, providing an effective platform for RNA imaging in living animals. Systematic studies will be performed on the synthesis yield of DNA nanostructure and the sensitivity of RNA detection in vitro; Moreover, the efficiency of targeted nucleic acid delivery via receptor mediated endocytosis and the stability of chemical modified DNA probe in fluid blood will be studied, enabling in vivo hybridization chain reaction and catalytical hairpin assembly. Depending on the preliminary research, the ability of tripartite DNA nanoprobe for low abundance RNA imaging in living mouse will be investigated. This project will provide a useful tool for ultrasensitive RNA imaging in living animals and related early cancer clinical diagnosis and precise treatment.

实现活动物内RNA成像为深层次理解包括癌症在内的疾病的生理学及早期诊断提供了新的可能。当前基于荧光分子探针的RNA成像方法仅实现了对活细胞或者活动物组织切片RNA成像，活动物内源性RNA成像还未被报道过。一个关键问题是核酸在血液中易被核酸酶降解以及靶向细胞输送效率不足；另外由于缺少信号放大功能限制了对低丰度RNA的灵敏检测。针对这些问题，本项目拟基于无酶核酸放大反应，设计合成靶向Y型三元DNA纳米探针用于活动物内RNA灵敏成像。系统研究DNA纳米结构的合成效率和RNA检测灵敏度，考察基于受体介导内吞的靶向核酸输送效率以及化学修饰DNA探针在血液等复杂体系中的稳定性，实现杂交链反应和催化发夹组装反应在活体内应用。在此基础上，以肿瘤相关RNA为目标，通过尾静脉注射，探索DNA纳米探针对活小鼠内RNA成像和实时监测，建立灵敏高效活体内RNA成像新技术，以期为癌症早期诊断和治疗提供有效的研究手段。

活体水平RNA灵敏成像分析为深层次理解包括癌症在内的疾病提供了更多的生物学信息。当前两个主要问题限制了DNA分子探针在该领域的应用：一是DNA探针的抗酶降解稳定性以及靶向细胞递送；二是对低丰度RNA的检测灵敏度。本项目系统设计合成了DNA高分子纳米结构，并探讨其对DNA分子探针的抗酶降解稳定性、反应动力学的提升效能；通过表面核酸适配体工程化，以实现高效的靶向细胞递送；基于邻近驱动的DNA级联反应，通过信号放大提升了检测灵敏度。在此基础上，发展了活细胞和活动物水平RNA高效灵敏成像新方法。此外，我们进一步探讨了基于DNA模板生物正交反应以及DNA表观遗传修饰的DNA分子探针生物传感新策略与新方法。总的来说，该项目基本构建了活细胞和活动物水平RNA以及其他生物分子灵敏精准成像技术平台，取得了一系列创新性研究成果，具备较好的应用前景。相关研究成果发表在Angew. Chem. Int. Ed., Anal. Chem., Chem. Commun.等化学测量学领域顶级期刊。