DEVELOPMENT OF ARTIFICIAL RIBONUCLEASE BEARING NOVEL FUNCTIONALIZED ANTISENSE OLIGONUCLEOTIDES
| Project/Area Number |
05808051
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Bioorganic chemistry
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| Research Institution | GUNMA UNIVERSITY |
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Principal Investigator |
SHINOZUKA Kazuo GUNMA UNIVERSITY FACULTY OF ENGINEERING ASSOCIATE PROFESSOR, 工学部, 助教授 (20206105)
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥1,800,000 (Direct Cost: ¥1,800,000)
Fiscal Year 1994: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1993: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | RNase. / mimic of RNase. / rRNA. / hydorolytic mecanism. / C-5 modified 2'-deoxyuracil. / acid-base catalytic sites. |
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| Research Abstract |
Novel polyamine derivatives (I-IV) were prepard from tris (2-aminoethyl) amine as a mimic of naturally occurring RNase. These compounds bear imidazole and/or primary amine groups as acid-base catalytic sites along with or without intercalating agent. Among the derivatives, the compound (III) which has a combination of imidazole and primary amine moiety along with the intercalator exhibited most potent RNA cleaving activity. The compound (I) in which an imidazole moiety of (III) was substituted by a primary amine moiety also exhibited remarkably high activity. The compound which lacks the intercalator (II) as well as the compound (IV) which has a longer linker arm for the primary amine compared to (III) were inactive.
The mechanism of the RNA cleaving reation by (I) and (III) was investigated by several different experiments. The results suggest that the compounds cleave RNA by hydrolytic mechanism.
The active sites of the compound (III), two primary amine groups, wereincorporated in C-5 position of 2'-deoxyuracil. The obtained novel nucleoside was converted to fully protected form of 3'-phosphoramidite and used for the synthesis of oligomer.
Two types of oligonucleotides (DNA1 and DNA2) were prepared using the above phosphoramidite. The DNA1 is complementary to 5S rRNA loop region and DNA2 is complementary to the stem region. Both oligomers are 11-merlong and have C-5 modified 2'-deoxyuracil at their 5'-terminus. These oligomers were mixed with 5S rRNA for prolonged period and the results were assayd by denaturing polyacrilamide gel electrophoresis. As the result, DNA1 gave two new fragements which were supposed to be generated by the strand scission of the substrate. On the other hand, DNA2 gave no new bands at all. These results indicate that the hybrid compound consisted of RNA cleaving agent and oligonucleotide will be a useful candidate as a new functionallized antisense agent.
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Report
(3 results)
Research Products
(6 results)
