| Budget Amount *help |
¥18,070,000 (Direct Cost: ¥16,000,000、Indirect Cost: ¥2,070,000)
Fiscal Year 2007: ¥8,970,000 (Direct Cost: ¥6,900,000、Indirect Cost: ¥2,070,000)
Fiscal Year 2006: ¥9,100,000 (Direct Cost: ¥9,100,000)
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| Research Abstract |
Kinases and phosphatases regulate signal transduction through phosphorylation and dephosphorylation of various biological molecules. The techniques for detecting the activity of kinases and phosphatases are essential to the elucidation of signal transduction mechanism. The fluorescence ratio imaging is a sensitive, undestructive and accurate method for the detection of such enzymatic reactions inside living calls. In this research, we created fluorescent probes, which specifically react with phosphatase and change its fluorescence properties.
Our design strategy is based on the following requirements; (1) The probes should have a switch function, by which the excitation spectrum of the probe changes upon the reaction with phosphatase, and (2) the structure of the reactive site in the probe can be easily modified, so that the probe specifically reacts with a different kind of phosphatase, which recognizes a distinctive structure of the restive site. First, we chose 7-hydr xycoumarin as
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a basic structure of the probe. Importantly, our preliminary experiments show that the pK_a of the hydroxyl group is controlled by the anionic group sterically close to the hydroxyl group and affects excitation spectrum of the compound. We, therefore, introduced phosphate group into the proximal position of the hydroxyl group via the aliphatic ester linkage, so that removal of the phosphate group by phosphatase results in the alteration of pKa of the hydroxyl group, leading to the change of the excitation spectrum of the probe. In addition, the aliphatic linker allows modification of the structure close to the phosphate group. It was demonstrated that the probe specifically reacted with acid phosphatase and changed its excitation spectrum. Furthermore, the change of the excitation spectrum was large enough to conduct ratio measurements. These results provides valuable information about design of fluorescent ratio probes and our strategy has great potential toward application in the field of bio-imaging. Less
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