| Project/Area Number |
05101004
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| Research Category |
Grant-in-Aid for Specially Promoted Research
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| Allocation Type | Single-year Grants |
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| Review Section |
Chemistry
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| Research Institution | Osaka University |
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Principal Investigator |
KYOGOKU Yoshimasa Osaka University, Insisute for Protein Research, Professor, 蛋白質研究所, 教授 (90012632)
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| Co-Investigator(Kenkyū-buntansha) |
AKUTSU Hideo Yokohama National University, Faculty of Engineering, Professor, 工学部, 教授 (60029965)
KAINOSHO Masatsune Tokyo Metropolitan University, Faculty of Science, Professor, 理学部, 教授 (20137029)
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| Project Period (FY) |
1993 – 1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥260,000,000 (Direct Cost: ¥260,000,000)
Fiscal Year 1996: ¥14,000,000 (Direct Cost: ¥14,000,000)
Fiscal Year 1995: ¥71,000,000 (Direct Cost: ¥71,000,000)
Fiscal Year 1994: ¥87,000,000 (Direct Cost: ¥87,000,000)
Fiscal Year 1993: ¥88,000,000 (Direct Cost: ¥88,000,000)
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| Keywords | stable isotope / multidimensional NMR / solid state NMR / nucleic acids / protein-nucleic acid complex / NOE / dihedral angle / stereospecific labeling / 異種核多極子2次元相関 / 立体特異的重水素化 / ホリディ構造 / 内部回転角 / 多次元核磁気共鳴 / 異種核双極子2次元相関 / 選択的標識 / 蛋白質-核酸複合体 / 分子認識 / 核酸構造 / 多次NMR / 高分解能NMR / 糖の二面角 / NOEの精度 / 固体HOHAHA / 動的構造 / プリンヌクレオシド醗酵法 / T7RNAポリメラーゼ / 2次元相関スペクトル / 固相合成 |
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| Research Abstract |
The methods for stable isotope labeling of nucleosides by chemical synthesis has been established, where whole nitrogen/carbon nuclei or those at specific sites were replaced by ^<13>C/^<15>N nuclei. In addition to them the methods for stereospecifically deuterating at the 2' or 5' positions were developed. From these nucleosides monomer units for the polymer synthesis were prepared and used for the synthesis of labeled oligonucleotides.
Several pulse sequences for the sequential signal assignment by utilizing direct spin-spin coupling among ^1H,^<13>C,^<15>N and ^<31>P were developed. These techniques correspond to those of the protein multidimensional NMR.Based on the assignments the methods for obtaining J used for determing dihedral angles, alpha-xi, chi, have been proposed. By combining the limited number of NOEs with the J values, the structure of low molecular weight oligonucleotides could be determined. For the structure determination of higher molecular weight oligonucleotides, the intensity of NOE should be accurate and also NOEs at different conditions should be fully used.
A new method for signal assignments of the solid state labeled samples at multiple sites was developed by utilizing distance dependency of the dipole-dipole interaction which allows cross peaks for the shortest distance spin pairs. Moreover the methods for measuring distances and dihedral angles among multiple labeled sites were proposed. In near future it may be possible to determine the conformation of nucleosides in the solid state by this method.
The whole tertiary structure of an RNA hairpin was determined by using the stable isotope labeled sample. Some samples like the Holliday junction and Cro/DNA complexes were partially ^<13>C labeled and structural informations were obtained.
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