New Analytical Methods for Molecular Imaging in Single Live Cells and Interfacial Molecular Assemblies

单个活细胞和界面分子组装体分子成像的新分析方法

基本信息

批准号：
15105003
负责人：
UMEZAWA Yoshio
金额：
$ 67.14万
依托单位：
Musashino University (2007)The University of Tokyo (2003-2006)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (S)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2007
项目状态：
已结题

项目摘要

The purpose of the present research is to develop new analytical methods for molecular imaging.We have developed optical probes that the intracellular signaling can be monitored in vivo in living cells by genetically encoded intracellular fluorescent and bioluminescent probes or indicators, which include second messengers such as guanosine 3',5'-cyclic monophosphate(cGMP), inositol 1,4,5-trisphosphate(IP3), phosphatidylinositol 3,4,5-trisphosphate(PIP3), and nitric oxide(NO), protein phosphorylations, protein-protein interactions, and protein localizations. An amplifier-coupled fluorescent indicator for NO was developed to visualize physiological nanomolar dynamics of NO in living cells with a detection limit of 0.1 nM. Fluorescent indicators for PIP3 based on FRET allowed localized analysis of PIP3 concentrations. We analyzed the spatio-temporal regulation of the PIP3 production in single living cells. A intracellular fluorescent indicator for another lipid second messenger, diacylgly … More cerol was also reported.We developed genetically encoded fluorescent indicators for visualizing protein phosphorylations in living cells. As a result of the phosphorylation, FRET was induced between the two fluorescent units. Upon activation of the phosphatases, the phosphorylated substrate domain is dephosphorylated and the FRET signal is decreased.To monitor protein-protein interactions, a new method was developed by based on split-reporter reconstitution. One of the applications of the split-GFP reconstitution system is a probe for visualizing endogenous mRNA in single living cells. We developed genetically-encoded RNA probes for characterizing localization and dynamics of mtRNA in single living cells. We showed that ND6 mtRNA is localized within mitochondria and concentrated particularly on mtDNA. We demonstrated split-luciferase reconstitution system for a specific protein transported into the intracellular organelles. We developed a genetically encoded bioluminescence indicator for monitoring the release of proteins from the mitochondria in living cells. We also visualized movements of androgen receptor(AR), glucocorticoid receptor(GR) and STAT3 into the nucleus in living cells and mice. These methods allowed high-throughput screening of chemicals with multi-titer plates and imaging of the protein dynamics in living mice.Nucleobase molecular tips were prepared by chemical modification of underlying metal tips with thiol derivatives of adenine, guanine, cytosine, and uracil and the outmost single nucleobase adsorbate probes intermolecular electron tunneling to or from a sample nucleobase molecule. We found that the electron tunneling between a sample nucleobase and its complementary nucleobase molecular tip was much facilitated compared with its noncomplementary counterpart. Methods of analysis described above have been promised to become key analytical methods for studying chemistry and biology. Less

本研究的目的是开发新的分子成像分析方法。我们开发了光学探针，可以通过基因编码的细胞内荧光和生物发光探针或指示剂在活细胞体内监测细胞内信号传导，其中包括第二信使，例如鸟苷3',5'-环单磷酸(cGMP)、肌醇1,4,5-三磷酸(IP3)、磷脂酰肌醇3,4,5-三磷酸 (PIP3) 和一氧化氮 (NO)、蛋白质磷酸化、蛋白质-蛋白质相互作用和蛋白质定位开发了一种放大器耦合的 NO 荧光指示剂，用于可视化活细胞中 NO 的生理纳摩尔动力学。基于 FRET 的 PIP3 荧光指示剂的检测限为 0.1 nM，我们分析了 PIP3 的时空调节。还报道了另一种脂质第二信使二酰甘油的细胞内荧光指示剂。我们开发了用于可视化活细胞中蛋白质磷酸化的基因编码荧光指示剂。磷酸酶激活后，磷酸化底物结构域去磷酸化，FRET 信号减弱。为了监测蛋白质-蛋白质相互作用，开发了一种新方法。基于 split-reconstitution 的 split-GFP 重建系统的应用之一是用于可视化单个活细胞中内源 mRNA 的探针。研究表明，ND6 mtRNA 位于线粒体内，特别集中在 mtDNA 上。我们展示了用于将特定蛋白质转运至细胞内细胞器的分裂荧光素酶重建系统。用于监测活细胞线粒体中蛋白质释放的生物发光指示剂我们还可视化了雄激素受体 (AR)、糖皮质激素受体 (GR) 和 STAT3 进入活细胞和小鼠细胞核的运动。使用多滴定板对化学物质进行分析，并对活体小鼠中的蛋白质动力学进行成像。核碱基分子尖端是通过用腺嘌呤、鸟嘌呤、胞嘧啶和胞嘧啶的硫醇衍生物对底层金属尖端进行化学修饰来制备的。尿嘧啶和最外面的单个核碱基吸附探针与样品核碱基分子之间的电子隧道效应我们发现，与上述的非互补对应物相比，样品核碱基与其互补核碱基分子尖端之间的电子隧道效应更加容易。 Less 有望成为研究化学和生物学的关键分析方法。