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

本研究的目的是开发新的分子成像分析方法。我们开发的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体细胞内监测细胞内的信号，这些探针或指示剂包括第二信使，如鸟苷3‘，5’-环一磷酸(CGMP)、三磷酸肌醇(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。开发了一种放大耦合的NO荧光指示剂，用于显示活细胞中NO的生理性纳摩尔动力学，检测下限为0.1 nM。基于FRET的PIP3荧光指示剂允许对PIP3浓度进行本地化分析。我们分析了单个活细胞中PIP3产生的时空调节。另一种脂类第二信使二酰甘氨酸…的细胞内荧光指示剂我们开发了基因编码的荧光指示剂，用于可视化活细胞中的蛋白质磷酸化。作为磷酸化的结果，FRET在两个荧光单位之间被诱导。在磷酸酶被激活后，磷酸化底物结构域被去磷酸化，FRET信号减弱。为了监测蛋白质之间的相互作用，开发了一种基于分裂报告重构的新方法。Split-GFP重组系统的应用之一是用来显示单个活细胞中内源性mRNA的探针。我们开发了遗传编码的RNA探针，用于表征mtRNA在单个活细胞中的定位和动态。我们发现ND6mtRNA定位于线粒体中，并集中在mtDNA上。我们展示了一种特定蛋白质转运到细胞内细胞器的裂解荧光素酶重组系统。我们开发了一种遗传编码的生物发光指示器，用于监测活细胞线粒体中蛋白质的释放。我们还观察了雄激素受体(AR)、糖皮质激素受体(GR)和STAT3在活细胞和小鼠中进入细胞核的运动。这些方法可以用多效价平板高通量筛选化学物质，并对活体小鼠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、胞嘧啶和尿嘧啶的硫醇衍生物化学修饰底层金属尖端和最外面的单核苷酸吸附探针来制备的，分子间电子隧穿进出样本核碱基分子。我们发现，与非互补碱基分子尖端相比，样品碱基与其互补碱基分子尖端之间的电子隧穿更加容易。上述分析方法有望成为研究化学和生物学的关键分析方法。较少

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