新型分子信标研究及其在活细胞内HIV-1病毒RNA成像的应用

Human immunodeficiency virus type-1 (HIV-1) latency in resting primary CD4+ T cells is one of the major obstacles to curing HIV infection. Within an infected, resting T-cell, the expression of the viral RNA is suppressed below the threshold level detectable by currently available techniques. Therefore, the ability to sensitively detect the synthesis, localization, processing, and transport of the viral genome is of critical importance to the improvement of disease diagnostics and management. One tool that has a great potential for monitoring HIV-1 viral RNA activities in living cells is the molecular beacon (MB). However, it is becoming increasingly evident that MBs can generate high false-positive signals in living cells, thereby limiting their applications for the detection of highly-abundant RNA molecules only. Recently we have developed a novel class of molecular beacons, known as the Ratiometric Biomolecular Beacons (RBMB), and demonstrated its superior capability for RNA detection over conventional molecular beacons. The overall goal of this proposal is to use RBMBs to monitor the expression level of HIV-1 viral RNA in single living cells. Specific studies will be performed to optimize RBMBs in order to improve their sensitivity, specificity and functionality for RNA detection in living cells. In addition, we will investigate whether RBMBs can be used to detect and monitor changes in the expression level of HIV-1 viral RNA in response to therapeutics or external stimuli (i.e. T-cell activation). Further, we will also develop flow-cytometry based strategies for the high-throughput analysis of HIV-1 viral RNA by RBMB. The long-term goal of this research is to develop a highly robust and versatile technology that can be used for the rapid analysis of HIV-1 viral RNA in a laboratory setting as well as a clinical tool for disease detection, diagnosis, and prognosis.

HIV病毒在休眠T细胞内潜伏是目前治疗药物无法根除病毒的主要原因。在潜伏期中，HIV 病毒基因组的表达受到T细胞休眠机制的控制，能有效躲避药物的杀伤，并且难以被传统检查手段发现。因此，实时地、灵敏地检测HIV 病毒RNA的合成、转运、表达及定位将极大地推动对艾滋病的诊断。虽然目前可通过纳米探针如分子信标检测RNA在活细胞中表达水平，但是纳米探针的应用面临背景信号高和灵敏度低的缺点。我们之前开发的荧光比例双链分子信标(RBMB)已克服了传统分子信标的缺点，能够在活细胞中更加灵敏的检测RNA。本研究计划的核心内容是优化RBMB，从而特异地、灵敏地检测HIV-1 RNA。使用改进后的RBMB，我们将检测HIV病毒基因组在活细胞中的变化，并致力于开发其在高通量分析方法（例如：流式细胞仪）中的运用。通过优化RBMB技术，我们期望开发以RBMB技术为核心的艾滋病临床检测工具，以服务于疾病的诊断与治疗。

陈匡时课题组在本项目中主要开展了在活细胞中标记RNA的分子探针技术研发工作，并研究了HIV-1 RNA 的转运与定位在HIV-1 病毒复制中所扮演的角色。通过优化分子探针的骨架，成功地研制出一种能在细胞中具有高生物相容性的分子信标探针，解决了长久以来RNA分子探针在活细胞中低生物相容性的问题，也证实了该探针能在单分子水平下成像RNA，还可通过结合单颗粒示踪技术观察被标记RNA在细胞质与细胞核内的转运与定位。除了建立了一系列在活细胞中单分子成像RNA 的技术外，课题组还结合荧光、电镜、生化等手段研究了RNA-蛋白交互作用在HIV-1 以及MLV 逆转录病毒在宿主细胞中组装的作用。研究发现逆转录病毒组装依赖病毒RNA,并且microRNA与逆转录病毒蛋白Gag 可形成具有破坏病毒颗粒组装能力的microRNA-Gag 复合体，从而干扰病毒RNA的包装。此外，还发现microRNA-Gag蛋白复合体的形成能进一步诱导细胞自噬的发生,在病毒组装失败并且被细胞内吞后，通过自噬小体的介导将内吞后的病毒传递至溶酶体中并将其降解。通过这些研究，对HIV-1病毒在宿主细胞中的复制方法提出了新理论，也为逆转录病毒的治疗方法提供了新思路。.迄今为止，在《PNAS》、《Biomaterials》、《Protein & Cell》、《Scientific Reports》等发表标注该项目资助的论文共7篇。.人才培养：在读博士生中2人获得北京大学校长奖学金，1人获得研究生国家奖学金。.国际合作交流： 邀请著名生物学家、美国国家科学院、医学院院士Jennifer Lippincott-Schwartz博士来我校交流和讲学。