| Project/Area Number |
16350089
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Chemistry related to living body
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| Research Institution | Kyusyu Institute of Technology (2005)
Kyushu University (2004) |
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Principal Investigator |
TAKENAKA Shigeori Kyusyu Institute of Technology, Department of Materials Science, Professor, 工学部, 教授 (60188208)
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| Co-Investigator(Kenkyū-buntansha) |
NOJIMA Takahiko Kyusyu Institute of Technology, School of Engineering, Department of Applied Chemistry, Research Associate, 大学院・工学研究院, 助手 (00291930)
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| Project Period (FY) |
2004 – 2005
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| Project Status |
Completed (Fiscal Year 2005)
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| Budget Amount *help |
¥16,100,000 (Direct Cost: ¥16,100,000)
Fiscal Year 2005: ¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2004: ¥12,300,000 (Direct Cost: ¥12,300,000)
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| Keywords | telomere DNA / tetraplex / extracellular potassium ion / sensing / thrombin-binding aptamer (TBA) / oligonucelotide / fluorescence resonance energy transfer (FRET) / Pyrene excimer / 蛍光共鳴エネルギー移動(FRET) / カリウムイオン / テロメアDNA配列 / 四本鎖DNA構造 / 蛍光励起共鳴エネルギー移動 / ピレン / エキシマー発光 / 神経細胞 |
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| Research Abstract |
Potassium ion plays an important role in biological systems. Recently, it became known that deficiency or anomaly of the potassium ion channel binding protein brings about an irregular heartbeat. It is hence important to monitor the concentration change of potassium ion in organisms and it is required to quantitate potassium ion in the presence of a very high concentration of sodium ion. However, no one succeeded in quantitating potassium ion under such "harsh" conditions. In this project, we studied a new reagent by using DNA tetraplex formation which is one form of complex structures of DNA. Firstly, we have been studying a fluorescent probe having two dyes at the termini of an oligonucleotide (oligo) used as the backbone for visualization of potassium ion in the living cells. The detection mechanism is based on the formation of a tetraplex DNA structure in the oligo induced by potassium ion, which enhances fluorescence resonance energy transfer (FRET) tremendously. Oligo derivatives
… More
, F-TBA-T and F-7TBA-T carrying thrombin-binding aptamer (TBA) sequence and FAM and TAMRA dyes at their each end were designed to achieve the fluorometric monitoring of potassium ion concentrations in a living organisms. These oligos could form a tetraplex structure upon addition of potassium ion. Spectrophotometric experiments revealed that the fluorescence enhancement on the FRET was larger in the case of F-7TBA-T having 7-nucleotides at 5'-end of TBA sequence than in the case of F-TBA-T due to the elimination of the quenching by dye-dye interaction. As a second step, we devised a thrombin-binding aptamer oligo carrying pyrenes at both termini to improve the performance of this system. Pyrene excimer fluorescence was generated effectively in the presence of potassium ion and the performance of this new system was better than that of the FRET system reported previously. The fluorescence imaging of extracellular potassium ion surrounding the taste cells was also attempted. This was the first fluorescent probe, which can be potentially exploited for the real time monitoring of potassium ion in extracellular milieu. Less
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