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。我们已经开发了光学探针，可以通过基因编码的细胞内荧光和生物发光探针或指示器在活细胞中监测细胞内信号，其中包括鸟苷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）等第二信使，蛋白质磷酸化，蛋白质-蛋白质相互作用和蛋白质定位。建立了一种放大耦合的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，检测限为0.1 nM。基于FRET的PIP3荧光指示器允许对PIP3浓度进行局部分析。我们分析了单个活细胞中PIP3产生的时空调控。另一种脂质第二信使二酰基（diacylly…More）的细胞内荧光指示器也有报道。我们开发了基因编码荧光指示器，用于可视化活细胞中的蛋白质磷酸化。由于磷酸化，在两个荧光单位之间诱导了FRET。磷酸酶激活后，磷酸化的底物结构域被去磷酸化，FRET信号减弱。为了监测蛋白质相互作用，建立了一种基于分裂报告基因重构的新方法。分裂- gfp重组系统的应用之一是在单个活细胞中可视化内源性mRNA的探针。我们开发了遗传编码的RNA探针，用于表征单个活细胞中mtRNA的定位和动态。我们发现ND6 mtRNA定位于线粒体内，并特别集中在mtDNA上。我们展示了分裂荧光素酶重组系统的特定蛋白质运输到胞内细胞器。我们开发了一种基因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。我们还观察了活细胞和小鼠中雄激素受体（AR）、糖皮质激素受体（GR）和STAT3进入细胞核的运动。这些方法允许高通量筛选化学物质与多效价板和成像的蛋白质动态在活的小鼠。核碱基分子尖端是用腺嘌呤、鸟嘌呤、胞嘧啶和尿嘧啶的硫醇衍生物对底层金属尖端进行化学修饰而制成的，最外面的单个核碱基吸附物探针通过分子间电子隧道进出样品核碱基分子。我们发现，与非互补核碱基相比，样品核碱基与其互补核碱基分子尖端之间的电子隧穿要容易得多。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。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