基于蛋白邻位巯基效应的荧光激活型探针的构建及其应用研究

Fluorescent labeling technique has become an important tool for protein research. In this field, organic dyes, with the advantages of multicolor, high brightness and high stability, have become an indispensable and important tool for protein labeling as an excellent alternative to fluorescent proteins. Aiming at the defect of traditional organic dyes with unspecific labeling of proteins and fluorescent turn-on, this program proposes to develop a new kind of fluorogenic probes in which the probes covalently bond to target protein’s proximity thiol as well as fluorescent turn-on. The fluorogenic probes are constructed by conjugating the fluorophores and protein ligands with an acryl group, which as acts as both a fluorescence quencher and an electrophile. When the acryl group reacts with proximity thiol through a Michael reaction, the probes are covalently bound to the target protein accompanying with the fluorescence turn-on. Compared with the current techniques, this method is not only suitable for fusion proteins, but also for some endogenous proteins, which can realize the modular selection of fluorophores and ligands. In addition, these fluorogenic probes would have rapid labeling rate and high specificity, and without any leaving group liberation during the labeling process, which are expected to realize proteins trace, quantification and detection. These probes also have the potential applications in drug-assisted screening and guided tumor resection and would provide a new tool for understanding the life process of proteins and explicating the related major diseases and pathogenesis.

荧光标记技术已成为蛋白质研究的重要工具。有机染料具有多色、高亮度与高稳定性等优势，与荧光蛋白标签相得益彰，成为蛋白荧光标记中不可或缺的重要工具。本项目申请针对传统有机染料蛋白标记的“位点”与“点亮”特异性的缺陷，提出发展蛋白“邻位巯基”共价标记并同步激活荧光的有机染料探针。拟采用丙烯酰基连接荧光团和蛋白配体构建荧光生成型探针，其中，丙烯酰基不但作为染料的荧光猝灭基团，同时，还作为与邻位巯基反应的官能团，与蛋白邻位巯基发生快速特异性的迈克尔加成反应，实现探针共价标记与荧光“点亮”。与当前相关技术相比，该方法不仅适用于融合蛋白，还适用于部分内源性蛋白，能实现荧光团与配体的模块化构建；另外，该类探针标记速度快、特异性强，标记过程无游离基团的释放；因此，有望实现对所标记蛋白的示踪、定量和检测，并适用于药物辅助筛选和肿瘤的指导性切除，为认识蛋白质的生命过程，阐述相关重大疾病和发病机理提供新的工具。

荧光标记技术已成为蛋白质研究的重要工具。本项目旨在设计合成特异性强、标记 快、生物相容性好以及背景低的荧光探针，实现蛋白和生物活性分子的特异性标记、示踪以及定量检测，为认识蛋白质的生命过程，阐述相关重大疾病和发病机理提供新的工具。通过该项目的实施，目前取得如下主要成果：1）基于分子转子类生色团，发展了一批枝丫状荧光RNA探针，该类荧光RNA具有波长多色可调（从青光到红光光谱）、量子产率高、结合能力强、稳定性好、适用于哺乳动物细胞标记等优势，能够实现细胞内RNA的超分辨成像，为RNA研究提供了一项新工具，有望用于疾病监测与诊断方面；2）基于TICT的机制构建了一系列光学性质可控调节（波长从蓝光至红光谱系）的荧光探针分子，结合蛋白特异性标签后，所得探针分子可同时实现细胞内外特异性的蛋白标记、示踪及定量检测；3）基于激活荧光机制构建了一系列光学性质可调（波长从绿光到近红外光）的荧光探针，在与目标蛋白结合前几乎没有荧光，而当与目标蛋白结合时，探针因结构的变换发出荧光，从而实现对目标蛋白的共价荧光标记；4）通过特异性筛选、染料分子的优化得到cpPepper 荧光RNA，该类荧光RNA具有波长可调，量子产率高、结合能力强、稳定性好、离子依赖度低，以及哺乳动物细胞内阳性率高等优势，适用于对细胞内目标RNA的定位与示踪。这些探针有望实现对所标记蛋白的示踪、定量和检测，并适用于药物辅助筛选和肿瘤的指导性切除，为认识蛋白质的生命过程，阐述相关重大疾病和发病机理提供新的工具。