Laboratory of Bioanalytical Chemistry
Graduate School of Pharmaceutical Sciences, The University of Tokyo

Japanese / English

Subject of research

Living system is constituted based on incredible amount of bio-molecules that act in concert, therefore, revealing functions and interactions of bio-molecules lead us towards further understanding of living system. Single molecule fluorescence imaging is a powerful technique that enables us to observe functions and interactions of molecules at the level of the single molecules, thus obtained results are not in average of many molecules. To address biomolecular functions and interactions, my laboratory uses the-state-of-the art single molecule techniques with three distinguished approaches.

Group I: machinery molecules

Revealing the mechanism of machinery molecules using single molecule fluorescence imaging technique.

1. Investigating protein functional-structural dynamics using single molecule fluorescence imaging

We have been studying the mechanism of molecular chaperonin using single molecule fluorescence imaging technique. The GroEL, one of molecular chaperonins, mediates protein folding with its co-factor GroES in an ATP-dependent manner. We expect that elucidating the molecular mechanism leads us to be able to design biologically functional molecules and further to develop a new drug.

2. Analyzing functions and interactions of proteins using the next generation of single molecule imaging technique

Single molecule imaging using total internal reflection fluorescence microscopy has been widely used and enabled us to elucidate functions of various bio-molecules. However, there is a problem has to be overcome. This method can be successfully used under the condition where the concentration of fluorescent molecules is under 50 nM. Above that, fluorescent signal from target molecules is hindered due to increase in background noise originated from fluorescent molecules present in the evanescent field, consequently, only strong molecular interactions are observable with this method. In order for measuring weak molecular interactions, we have been recently developing a new single molecule imaging using a nano-hole technique. In this method, incident beam was passed onto nano-holes with a diameter of 100 nm, resulting in creating a very localized evanescent field. The area of excitation is about two orders of magnitude smaller compared to that of total internal reflection, therefore, this new method is expected to reveal weak molecular interactions and to contribute deeper understanding of bio-molecular interactions and functions.

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Group II: single molecule analysis in living cells

We have been analyzing gene expression patterns and molecular interactions in living cells using time-resolved single molecule fluorescence imaging. This brings us to reveal the molecular mechanism of signal transduction and information processing, further leading toward understanding of rules and theories for functional living system.

1. Investigating movement of mRNA and quantitating their expression levels in living cells

In order to understand the mechanism of gene expression, it is important to quantify the mRNA expression level and visualize their movement at the level of a single molecule in living cells. To do so, we have been developing new imaging methods with high sensitivity to accurately measure and observe the expression levels and gene movement in living cells. We believe that this technique enables to elucidate the function and mechanism of RNA interference in living cells.

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Group III: nano/micro device

Merging microfabricated and single molecule technologies opens a new era to further facilitate understanding of bio-molecular functions.

1. Developing on-chip bio-molecule sorting system

Bio-molecules play a central roles, further understanding of these bio-molecules are essential for basic medicine and drug discovery. These bio-molecules don't always act alone, but they may form supermolecular complexes like an organelle for their function. In conventional ways, organelles and bio-molecules have been difficult to purify and identify its components, especially at low concentration. For this reason, high purification technologies for separation and purification of bio-molecules have been required. We have been developing a micro fabricated fluorescence-activated bio-molecule sorter system using thrmoreversible gelation polymer which functions as a valve, and their opening and closing are controlled by laser-induced heating under epifluorescence microscopy. Identifying purified bio-molecular complexes using mass spectrometry lead us to reveal bio-molecular interactions even with small sample volumes with low concentrations.

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