| Research Abstract |
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
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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
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