| Project/Area Number |
13680669
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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 |
IWASAE Reiko Kyoto Institute of Technology, Department of Polymer Science and Engineering, Research Associate, 繊維学部, 助手 (90283697)
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| Co-Investigator(Kenkyū-buntansha) |
MURAKAMI Akira Kyoto Institute of Technology, Department of Polymer Science and Engineering, Professor, 繊維学部, 教授 (60210001)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2002: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2001: ¥1,600,000 (Direct Cost: ¥1,600,000)
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| Keywords | amide-linked RNA / RNA binding sffinity / C3'-endo conformatiaon / rigidity / fluoresccein / cellular uptake / pyrene / fluorescent probe / 機能性修飾核酸 / 3'-endo構造 / 細胞膜透過性 / 蛍光標識 / 二重鎖形成能 / RNA選択的結合能 / PyAOP |
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| Research Abstract |
Amidelinked RNA was synthesized by solidphase method using 5'Nmonomethoxytrityl5'amino3'carboxymethy1 3', 5'dideoxyuridine derivative (1) with a phosphonium type of condensing reagent, PyA OP.The solidphase method is compatible with the synthesis of natural type of RNA, Chimera RNAs containing amidelinked RNA and natural RNA sgments, (amide-4: rCCCUUUUaUaUaU, amide6: rCCCUUaUaUaUaUaU, a:amidelinkage), were synthesized The UV melting temperature (Tm) of the amide 4/RNA duplex was almost same as that of the unmodified RNA/RNA duplex. CD spectral analysis indicated that the amide4/RNA duplex has A form structure. The amide 6/RNA duplex also formed A typcduplex. However, decreased RNA binding affinity of amide 6 was observed compared with unmodified RNA. CD spectral analysis indicated that the backbone conformation of amide 6 was more rigid than that of unmodified RNA. These results suggest that the RNA binding affinity of amidelinked RNA is influenced by the two characteristics, the fixed
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C3'endo conformation of the furanose ring and the rigid backbone conformation. These findings suggest that tetramer of amidelinked RNA segment is appropriate length for sufficient RNA binding affinity of the chimera RNAs.
Hexamer of amidelinked RNA was labeled with fluorescein to evaluate the cellular uptake of the amidelinked RNA. The labeled amidelinked RNA was found to permeate across HeLa cell membrane, It was also observed that the efficiency of cellular uptake of amidelinked RNA was less than that of phosphorothioate DNA. The localization of the amidelinked RNA in cells was shown in the cytoplasmrather than nucleus.
5' 0 Dimethoxytrityl 3' carboxymethyl 2' 0 pyrenylmethy1 3' deoxyuridine( 2) was synthesized in order to fix the conformation of the furanow ring of pyrenemodified uridine. NMR analysis indicated that the conformation of the uridine derivative was fixed to C3' endo conformation. Synthesis of pyrenemodifiedoligonucleotide consisting of 2 and the fluorescent properties will be invcstigated to improve the sensitivity of RNA detection of conventional pyrenemodified oligonucleotides. Less
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