| Project/Area Number |
13132204
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| Research Category |
Grant-in-Aid for Scientific Research on Priority Areas
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| Allocation Type | Single-year Grants |
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| Review Section |
Science and Engineering
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| Research Institution | The University of Tokyo |
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Principal Investigator |
KOMIYAMA Makoto The University of Tokyo, Research Center for Advanced Science and Technology, Professor, 先端科学技術研究センター, 教授 (50133096)
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| Co-Investigator(Kenkyū-buntansha) |
ASANUMA Hiroyuki The University of Tokyo, Research Center for Advanced Science and Technology, Associate Professor, 先端科学技術研究センター, 助教授 (20282577)
SUMAOKA Jun The University of Tokyo, Graduate School of Engineering, Assistant Professor, 大学院工学系研究科, 助手 (10280934)
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| Project Period (FY) |
2001 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥115,700,000 (Direct Cost: ¥115,700,000)
Fiscal Year 2004: ¥17,300,000 (Direct Cost: ¥17,300,000)
Fiscal Year 2003: ¥18,100,000 (Direct Cost: ¥18,100,000)
Fiscal Year 2002: ¥38,300,000 (Direct Cost: ¥38,300,000)
Fiscal Year 2001: ¥42,000,000 (Direct Cost: ¥42,000,000)
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| Keywords | DNA / Artificial Restriction Enzyme / Phosphodiester / RNA / Peptide Nucleic Acid / Cerium / Hydrolysis / Acridine / PNA / リン酸エステル / 制限酵素 / 機能性核酸 / バイオテクノロジー / 遺伝子操作 |
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| Research Abstract |
We have achieved the sequence selective scissions of both DNA and RNA by using our artificial systems.
1. DNA cleavage
We found that the phosphodiester linkages in gap sites were preferentially hydrolyzed by a Ce(IV)/EDTA complex. Even though the complex was not covalently bound to any sequence-recognizing moiety, the DNA scission selectively occurs at the gap sites, since the linkages therein is more susceptible to catalysis by the Ce(IV) complex than are those in double-stranded portions. Furthermore, this gap-selective DNA hydrolysis was greatly promoted by introducing monophosphate groups at the gap sites and recruiting the Ce(IV) complex to the gap site.
By combining Ce(IV)/EDTA with, two pseudocomplementary peptide nucleic acids (pcPNAs), both strands in double-stranded DNA were selectively hydrolyzed at the target site. When two pcPNAs invaded into the double-stranded DNA, only the designated portion in each of the two strands was single-stranded like structure. On the treatment of this invasion complex with Ce(IV)/EDTA, both of the single-stranded portions are selectively hydrolyzed. With the use of two pcPNAs bearing either aminocarboxylates or phosphates, the DNA scission at target site was greatly promoted. Furthermore, the hydrolytic scission products were successfully connected with foreign double-stranded DNA by using T4 ligase.
2. RNA cleavage,
When oligonucleotides bearing two acridine groups form heteroduplexes with substrate RNA, the two phosphodiester linkages in front of the acridines were selectively activated and preferentially hydrolyzed by lanthanide ion. By using these systems, RNA fragments of predetermined length were obtained from long RNA substrates and analyzed by MALDI-TOF MS. Single nucleotide polymorphisms in homozygous and heterozygous samples were accurately and easily detected in terms of difference in mass number.
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