2000 Fiscal Year Final Research Report Summary
Development of amide-linked RNA and 2', 4'-aminomethyl-linked DNA for targeting RNA.
| Project/Area Number |
11680589
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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 |
IWASE Reiko Kyoto Inst. of Technology Faculty of Textile Science Research Associate, 繊維学部, 助手 (90283697)
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| Co-Investigator(Kenkyū-buntansha) |
MURAKAMI Akira Kyoto Inst. of Technology Faculty of Textile Science Professor, 繊維学部, 教授 (60210001)
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| Project Period (FY) |
1999 – 2000
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| Keywords | RNA derivatives / RNA binding / antisense / 2', 4'-aminomethyl linkage / amide linkage / oligonucleotide / A -conformation / nuclease registance |
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| Research Abstract |
Amide-linked RNA derivatives and 2', 4'-aminomethyl-linked oligonucleotides were synthesized. Properties of these modified oligonucleotides were examined for the binding to the complimentary RNA in addition to the nuclease resistance.
1. Synthesis and properties of amide-linked RNA derivatives
The amide-linked uridylyl-uridine dinucleoside derivative was synthesized using 5'-N-monomethoxytrityl-5'-amino-3'-carboxymethyl-3', 5'-dideoxyuridine derivative and 5'-amino-5'deoxyuridine derivative. A phosphonium type of condensing reagent, PyBOP, was found to be effective for the rapid amide-bond formation. By means of 1H-NMR analysis, the ribose conformation of the upstream uridine unit was found to be fixed in the C3'-endo conformation. This condensing method was applied to the solid-phase synthesis of amide-linked RNA oligomers, U^*U^*U^*U^*U^*U (1), and CCCUUUU^*U^*U^*U (2)(^* : amide linkage). The UV-melting temperature of 2-RNA duplex was slightly lower to that of the unmodified duplex (Δ
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Tm-2℃). CD spectra analysis indicated that the 2-RNA duplex has A-form structure. These results suggest that the amide-linked RNA derivatives have RNA binding affinity in addition to the nuclease resistance.
2. Synthesis and properties of 2', 4'-aminomethyl-linked oligonucleotides
A 2', 4'-aminomethyl-linked uridylyl-thymidine dinucleoside phosphoramidite was synthesized in 11 steps from 1- (β-D-arabinofuranosyl) uracil with 5'-amino-5'-deoxythymidine. Oligonucleotides containing 2', 4'-aminomethyl linkages, dUaTUaTT (3) and dCCCTUaTTTTT (4) (a : 2', 4'-aminomethyl linkage), were synthesized using the UaT phosphoramidite by solid-phase synthesis. The oligomer 3 was digested by a 3'-exonuclease only at the 3'-terminal phosphodiester linkage. This result suggests that the 3'-terminal 2', 4'- aminomethyl internucleoside residue can prevent degradation of the oligonucleotide by 3-exonucleases. The oligomer 4 containing of a 2', 4'-aminomethyl linkage at the center of the strand showed relatively lower affinity to DNA and RNA.However, the CD spectra of 4-RNA duplex indicated A-form structure. This result suggests that the 2', 4'-aminomethyl linkage could incorporate into A-form structure without the global distortion. Less
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