适用于活细胞内蛋白特异性荧光标记的高时空分辨荧光探针的研究

The study of protein function in complex cellular environment is the forefront of today's life science research; it has strong requirement for the quantitative label of proteins in living cells. To solve the problems in the fields of protein specific fluorescent label in living cells and biological imaging, a new kind of protein specific fluorescent probes with high-precision positioning, high noise immunity and highflux fluorescent output that suitable for super-resolution fluorescence images are designed in this project. They are created by the superposition of three elements, specific label, photoactivation and photo-regulated luminescence, and inspired from our proposed photo-activatable coumarin phototrigger. The SNAP-tag ligand and 1,2,4,5-tetrazine molecules are introduced to realize the function of protein specific label; strong quench factors like disperse red that can quench coumarin by strong FRET mechanism are introduced to achieve the "light activation" function; CY5 dye and quinone electronic acceptors that can occur photoswitchable and photoactivatable luminescence with coumarin, respectively, are introduced into the photocleavage site of coumarin phototrigger to realize the final "photo-regulated luminescence" function. It is believable that this project would create excellent fluorescent probes with high specificity, high sensitivity and high spatial resolution for protein specific label in living cells, and provide a scientific basis for a comprehensive understanding of cell life processes and explaining the causes of human severe diseases.

复杂细胞环境中蛋白功能的研究是当今生命科学研究的前沿，对活细胞内蛋白质的标记与定量有强烈的需求。针对活细胞内蛋白质特异性荧光标记与生物成像领域亟待解决的问题，本项目在我们提出的激活型香豆素光扳机的基础上，设计能通过特异性标记、光激活和光调控发光三元要素的叠加，创制一类全新的高精度定位、高抗干扰性、高通量荧光输出并适用超分辨荧光影像的特异性蛋白标记荧光探针。一是通过引入SNAP-tag配体分子和1,2,4,5-四氮杂苯实现其蛋白特异性标记; 二是通过引入与香豆素具有强FRET淬灭作用的分散红等强淬灭因子来实现其"光激活"功能；三是通过在香豆素光可剪切位引入能与香豆素发生光调控转换发光的CY5染料和发生光激活发光的醌类电子受体以实现荧光探针的光调控发光性能。本研究将为活细胞内蛋白质提供高度特异、高灵敏度、高空间分辨率荧光探针，为全面认识细胞生命过程，阐释人类重要疾病的成因提供科学依据。

随着荧光技术的广泛应用以及生命科学的不断发展，荧光成像由于具有非物理接触、良好的时间控制性和以及对生物干扰小等优势，成为研究蛋白的位置、丰度、结构、功能等信息的重要手段。复杂细胞环境中蛋白功能的研究是当今生命科学研究的前沿，对活细胞内蛋白质的标记与定量有强烈的需求。针对活细胞内蛋白质特异性荧光标记与生物成像领域亟待解决的问题，项目设计合成了一系列人工荧光蛋白和小分子荧光染料的激活型荧光探针，以分子转子为基础，结合蛋白标签技术，实现了高荧光激活倍数、快速以及特异的蛋白标记，实现了体内、外蛋白的示踪与生物造影，满足不同蛋白功能研究的需要；同时基于课题组在光激活化学领域的研究，利用光扳机、光开关设计合成了一系列的光激活水凝胶生物材料，实现了水凝胶的原位光控构建，可控生物活性分子的引入，为水凝胶应用于3D细胞培养、组织工程和再生医学领域提供新的思路。