| Project/Area Number |
14580609
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bioorganic chemistry
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| Research Institution | KYOTO INSTITUTE OF TECHNOLOGY |
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Principal Investigator |
MURAKAMI Akira KYOTO INSTITUTE OF TECHNOLOGY, FACULTY OF ENGINEERING AND DESIGN, PROFESSOR, 繊維学部, 教授 (60210001)
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| Co-Investigator(Kenkyū-buntansha) |
IWASE Reiko KYOTO INSTITUTE OF TECHNOLOGY, FACULTY OF ENGINEERING AND DESIGN, ASSISTANT PROFESSOR, 繊維学部, 助手 (90283697)
YAMAOKA Tetsuji KYOTO INSTITUTE OF TECHNOLOGY, FACULTY OF ENGINEERING AND DESIGN, ASSOCIATE PROFESSOR, 繊維学部, 助教授 (50243126)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2003: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2002: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Keywords | antisense / fluorescence probe / pyrene / gene diagnosis / transcriptome / gene destruction / analytical protocol for gene function / 蛍光修飾RNAプローブ / アンチセンス法 |
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| Research Abstract |
The antisense strategy, which can destruct the target gene by oligonucleotides in a sequence specific manner, has been established as the novel tool for the gene therapy and the gene analysis. To pursue the strategy toward the gene therapy, it is crucial and inevitable to decide the sequence of the antisense oligonucleotides. The information of the tertiary structure of the RNA is essential for the purpose, and, however, the diversity of the structure prevents us from viewing the whole figure. In this study, we focused in the establishment of the method to analyze the tertiary structure of native folded RNAs by fluorescence-conjugated oligonucleotides. We developed two types of antisense oligonucleotides. The one is the 2'-0-pyrene-conjugated 2'-0-Methl type oligonucleotide (OMUpy). By addition of OMUpy to the native folded RNA, the fluorescence intensity drastically increased 100 times compared with OMUpy alone. As the addition of OMUpy to the RNA with the mismatch sequence scarcely affected the intensity, it was concluded that OMUpy can be an excellent probe for the analysis of the binding site on the target RNA. The other is the Ru-complex-labeled oligonucleotide (Ru-probe). From the fluorescence anisotropy assay, the rotational motion of Ru-probe was remarkably changed upon addition to the native folded RNA. The change in rotational motion is tightly correlated with the apparent molecular size and, accordingly, the restriction of the motion suggests that Ru-probe hybridized with the target RNA. The advantage of the method is that the B/F separation is not necessary for the evaluation of the hybrid formation. Using these two fluorescent oligonucleotide probes, we demonstrated that the accessible sites on the target RNA could be decided in the homogeneous media without B/F separation. It was also demonstrated that these methods be used to monitor the genetic flow in living cells.
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