Aptamer-Mediated Nanoparticle-Based Protein Labeling Platform for Intracellular Imaging and Tracking Endocytosis Dynamics
Aptamer-Mediated Nanoparticle-Based Protein Labeling Platform for Intracellular Imaging and Tracking Endocytosis Dynamics
复制标题
用于细胞内成像和跟踪内吞作用动力学的适体介导的基于纳米颗粒的蛋白质标记平台
DOI：
10.1021/ac202810b
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发表时间：
2012-04-03
影响因子：
7.4
通讯作者：
Huang, Cheng Zhi
中科院分区：
文献类型：
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作者：
Chen, Li Qiang;Xiao, Sai Jin;Huang, Cheng Zhi
虽然纳米粒子已被广泛用作细胞成像的光学对比，但纳米探针的复杂的预功能化步骤和低标记效率极大地限制了其在细胞蛋白质成像中的应用。在这项研究中，我们开发了一种新的和通用的策略，采用适体不仅作为蛋白质识别的识别器，而且作为nanoreporter靶向特异性标记感兴趣的膜蛋白并跟踪其内吞途径的接头。利用这一策略，三种纳米颗粒，包括金纳米颗粒，银纳米颗粒，和量子点（QD），已成功地靶向感兴趣的膜蛋白，如核仁素或朊病毒蛋白（PrPC）。以下采用荧光免疫共定位试验对亚细胞分布的研究表明，PrPC-适体-QD复合物最有可能通过经典的网格蛋白依赖性/受体介导的途径内化至细胞质中。进一步的单粒子跟踪和轨迹分析表明，PrPC-适配体-QD复合物呈现出复杂的动力学过程，包括膜扩散、囊泡运输和受限扩散3种类型的运动，并且所有类型的运动都与PrPC内吞的不同阶段相关。与传统的多层膜方法相比，我们提出的适体介导的方法步骤简单，避免了任何复杂的探针预修饰和纯化。特别是，新的双色标记策略是独特的和重要的，由于其上级的优势，同时针对两个信号报告在一个单一的蛋白质，仅使用一个适体。更重要的是，我们已经构建了一个通用的和通用的适体介导的蛋白质标记纳米平台，显示了巨大的前景，为未来的生物医学标记和细胞内蛋白质的动态分析。
Although nanoparticles have been widely used as optical contrasts for cell imaging, the complicated prefunctionalized steps and low labeling efficiency of nanoprobes greatly inhibit their applications in cellular protein imaging. In this study, we developed a novel and general strategy that employs an aptamer not only as a recognizer for protein recognition but also as a linker for nanoreporter targeting to specifically label membrane proteins of interest and track their endocytic pathway. With this strategy, three kinds of nanoparticles, including gold nanoparticles, silver nanoparticles, and quantum dots (QDs), have been successfully targeted to the membrane proteins of interest, such as nucleolin or prion protein (PrPC). The following investigations on the subcellular distribution with fluorescent immunocolocalization assay indicated that PrPC-aptamer-QD complexes most likely internalized into cytoplasm through a classical clathrin-dependent/receptor-mediated pathway. Further single-particle tracking and trajectory analysis demonstrated that PrPC-aptamer-QD complexes exhibited a complex dynamic process, which involved three types of movements, including membrane diffusion, vesicle transportation, and confined diffusion, and all types of these movements were associated with distinct phases of PrPC endocytosis. Compared with traditional multilayer methods, our proposed aptamer-mediated strategy is simple in procedure, avoiding any complicated probe premodification and purification. In particular, the new double-color labeling strategy is unique and significant due to its superior advantages of targeting two signal reporters simultaneously in a single protein using only one aptamer. What is more important, we have constructed a general and versatile aptamer-mediated protein labeling nanoplatform that has shown great promise for future biomedical labeling and intracellular protein dynamic analysis.