VIPERnano: Improved Live Cell Intracellular Protein Tracking.
VIPERnano: Improved Live Cell Intracellular Protein Tracking.
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VIPERnano：改进的活细胞胞内蛋白质追踪。
DOI：
10.1021/acsami.9b12679
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发表时间：
2019
影响因子：
9.5
通讯作者：
Reich,Norbert
中科院分区：
文献类型：
--
作者：
Morgan,Erin;Doh,Julia;Beatty,Kimberly;Reich,Norbert
追踪活细胞中的细胞内蛋白质面临许多挑战。最广泛使用的方法是荧光蛋白融合，可以追踪天然细胞环境中的蛋白质，并在细胞生物学领域取得了重大发现。融合蛋白增加了目标蛋白的空间体积，并对天然蛋白功能产生负面影响。使用外源探针（例如抗体或蛋白质标记）是有问题的，因为它们不能自行穿过质膜，因此不能标记细胞中的细胞内靶标。我们开发了一个标记平台 VIPERnano，用于使用目标蛋白质的肽融合标签 (CoilE) 以及荧光标记探针肽 (CoilR) 的递送来对细胞内蛋白质进行活细胞成像。 CoilR 和 CoilE 与目标蛋白形成 α-螺旋异二聚体，从而生成标记蛋白。使用空心金纳米壳 (HGN) 作为主要递送载体将 CoilR 递送到细胞中。该技术依赖于 HGN 表面 CoilR 肽的缀合和光激活释放。我们展示了光激活 VIPER 纳米递送和标记两种细胞内蛋白质，位于线粒体或细胞核中。该技术能够研究细胞内蛋白质动力学和空间跟踪，同时减少与目标蛋白质相关的标签的空间体积。
Tracking intracellular proteins in live cells has many challenges. The most widely used method, fluorescent protein fusions, can track proteins in their native cellular environment and has led to significant discoveries in cell biology. Fusion proteins add steric bulk to the target protein and can negatively affect native protein function. The use of exogenous probes such as antibodies or protein labels is problematic because these cannot cross the plasma membrane on their own and thus cannot label intracellular targets in cells. We developed a labeling platform, VIPERnano, for live cell imaging of intracellular proteins using a peptide fusion tag (CoilE) to the protein of interest and delivery of a fluorescently labeled probe peptide (CoilR). CoilR and CoilE form an α-helical heterodimer with the protein of interest, rendering a labeled protein. Delivery of CoilR into the cell uses hollow gold nanoshells (HGNs) as the primary delivery vehicle. The technology relies on the conjugation and light-activated release of the CoilR peptide on the surface of the HGNs. We demonstrate light-activated VIPERnanodelivery and labeling with two intracellular proteins, localized either in the mitochondria or the nucleus. This technology has the ability to study intracellular protein dynamics and spatial tracking while lessening the steric bulk of tags associated with the protein of interest.