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Preparation of Artificial nuclease and creation of new-biotechnology

人工核酸酶的制备及新生物技术的创造

基本信息

批准号：
11308023
负责人：
KOMIYAMA Makoto
金额：
$ 20.61万
依托单位：
the University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A).
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11308023/
关键词：
Artificial nuclease Hydrolysis Nucleic Acid RNA DNA Lanthanide Intercalator Cerium アミン制限酵素リン酸ジエステル

项目摘要

Non-enzymatic hydrolysis of DNA has been one of the most significant targets for chemists. Several years ago, the remarkable catalysis by the lanthanide ions was found, and highly stable phosphodiester linkages in DNA were hydrolyzed at pH 7. The Ce (IV) ion is especially active. However, the Ce (IV) easily forms a gel of metal hydroxide, and this feature imposes limitations to the scope of its application. Homogeneous and catalytically active Ce (IV) complexes are required for more versatile applications. In this research, we fourcused on the development of (1) catalytically active Ce (IV) complexes, and (2) the methods of the activation of the target phosphodiesters in nucleic acids.(1) Homogeneous Ce (IV) complex of EDTA promptly hydrolyzes oligonucleotides under physiological conditions. In contrast, dinucleotides are not hydrolyzed to measurable extents. The degree of polymerization of DNA substrate is crucial for the present catalysis. The activity of this complex is significantly increased by the cooperation with selected oligoamines. The reaction in the presence of spermine (10 μmol dm^<-3>) is about 5O times as fast as that in its absence.(2) Non-covalent systems (ternary system), composed of (i) DNA oligomer bearing an acridine, (ii) unmodified DNA, and (iii) free lanthanide (III) ion, selectively and efficiently hydrolyse RNA at the target pohosphodiester. The corresponding phosphodiester linkage is notably activated by non-covalent interactions with the DNAs ((i) and (ii)), and thus the site-selective scission by the metal ion promptly proceeds under physiological conditions. The sequence-selective RNA scission is also achieved by non-covalent combinations of lanthanide (III) ion and one DNA bearing an acridine in its intern al position (binary system). The target phosphodiester linkage is also activated by the modified DNA, and preferentially hydrolyzed. The DNA-Acr/Lu (III) binary system is still more (about 20%) active than the ternary system.

DNA 的非酶水解一直是化学家最重要的目标之一。几年前，人们发现了镧系离子的显着催化作用，DNA 中高度稳定的磷酸二酯键在 pH 7 时被水解。Ce (IV) 离子尤其活跃。然而，Ce(IV)容易形成金属氢氧化物凝胶，这一特性限制了其应用范围。更广泛的应用需要均质且具有催化活性的 Ce (IV) 配合物。在本研究中，我们重点开发了（1）催化活性Ce（IV）配合物，以及（2）核酸中目标磷酸二酯的活化方法。（1）EDTA的均质Ce（IV）配合物在生理条件下迅速水解寡核苷酸。相反，二核苷酸没有水解到可测量的程度。 DNA 底物的聚合度对于目前的催化至关重要。通过与选定的寡胺合作，该复合物的活性显着增加。精胺(10μmol dm^-3)存在下的反应速度比精胺不存在时的反应快约5O倍。(2)非共价系统(三元系统)，由(i)带有吖啶的DNA寡聚物、(ii)未修饰的DNA和(iii)游离的镧系元素(III)离子组成，选择性且有效地在目标磷酸二酯处水解RNA。相应的磷酸二酯键明显被与DNA的非共价相互作用激活((i)和(ii))，因此金属离子的位点选择性断裂在生理条件下迅速进行。序列选择性 RNA 断裂也可以通过镧系元素 (III) 离子和内部位置带有吖啶的 DNA（二元系统）的非共价组合来实现。目标磷酸二酯键也被修饰的 DNA 激活，并优先水解。 DNA-Acr/Lu (III) 二元系统仍然比三元系统更活跃（约 20%）。