活细胞单分子成像内源RNA技术

With the ever-growing number of newly discovered RNA molecules and functions, it has become critical to be able to visualize RNA intracellular distribution and dynamics at the single-molecule level in their native live-cell context in order to decipher their physiological significances. Conventional molecular beacons have the great potential for imaging specific RNAs in living cells, but their tendency to generate false-positive signals as a result of nuclease degradation and/or nonspecific binding limits sensitive and accurate intracellular RNA imaging. In this proposal we will develop a novel molecular beacon platform named Ratiometric Molecular Beacon, RMB, that enables sensitive detection of endogeous RNA at the single-molecule level. Analogous to molecular beacons, RMBs are oligonucleotide-based probes that can form a stem-loop structure and are labeled with a fluorophore at one end and a quencher at the other. Moreover, the oligonucleotide will also be labeled with a second optically distinct ‘reference’ dye, which will be selected and positioned such that it is unquenched regardless of the conformation of the probe. The reference dye will expand upon the versatility of the molecular beacon by improving the ability to monitor transfection efficiency and probe localization. To avoid probe degradation and nonspecific binding, which are the cause of false-positive signals, we plan to systematically optimize the oligonucleotide backbone composition with various combinations of chemical modifications. Additionally, fluorophores will also be.chosen such that the highest signal-to-background signal can be achieved in living cells. The optimal configuration will be investigated for its ability to accurately image single RNA transcripts and measure RNA dynamics in living cells without affecting gene expression and cell viability. With the increasing need for high resolution imaging of specific RNAs at the single-molecule level, RMBs could be a useful tool in furthering our understanding of the role of RNA in health and disease.

研究RNA分子在活细胞内的表达、转运和定位，以及完全掌握它们与细胞正常生理以及疾病发生发展的关系，至今仍然是一项挑战。建立一种在活细胞中监控单一RNA的时程表达与空间分布技术对于上述问题的解决有着至关重要的作用。分子信标(MB)是目前在活细胞内研究内源RNA的主要手段之一，但是MB容易被细胞核吸收，并被核内的核酸酶降解或与蛋白结合，此类非特异性作用将造成报告荧光基团和淬灭基团分离从而引起假阳性信号。虽然通过连接大分子防止MB进入细胞核的方法已被证实可以有效降低假阳性信号，但同时也限制了RNA在细胞核或其它细胞器中的检测。因此，我们计划通过优化MB的化学组成、结构等，发展一种能防止假阳性信号从而能灵敏检测RNA的新型分子探针“比率荧光分子信标”（RMB），并测试RMB在活细胞中对内源RNA的单分子成像能力。我们期望RMB技术的发展能帮助研究者对揭示RNA新功能以及疾病检测等相关研究做出贡献。

陈匡时课题组在本项目中主要开展了活细胞中标记RNA的分子探针技术研发、HIV-1病毒颗粒组装过程中生物大分子直接相互作用的角色探究等工作。主要研究成果包括：通过超分辨率显微成像技术解析RNA与HIV-1 Gag蛋白互作在病毒组装中的角色；发展新型蛋白质互作成像技术并在纳米尺度下解析HIV-1病毒组装时Gag蛋白与宿主细胞蛋白的互作过程；开发可干扰HIV-1病毒组装的RNA纳米材料；通过对CRISPR体系中的sgRNA进行改造并标记，发展活细胞单分子DNA标记技术。通过这些研究，课题组提出了关于HIV-1病毒在宿主细胞中复制方式的新理论，为相关研究提供了新工具，也为逆转录病毒相关疾病的治疗提供了新思路。