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靶标之一。几年前，发现了灯笼离子的显着催化剂，并且在pH 7时水解了DNA中高度稳定的磷酸二键链链接。CE（IV）离子特别活跃。但是，CE（IV）很容易形成金属氢氧化物的凝胶，并且此特征不可能限制其应用范围。更广泛的应用需要均质和催化活性CE（IV）复合物。在这项研究中，我们对（1）催化活性CE（IV）复合物的发展进行了四次，以及（2）核酸中靶磷酸剂的激活方法。（1）EDTA的CE（IV）复合物在物理条件下迅速水解寡核苷酸。相反，二核苷酸未水解为可测量的范围。 DNA底物的聚合程度对于目前的催化剂至关重要。通过与选定的寡胺的合作，该复合物的活性显着增加。 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.相应的磷酸二键连接显着通过与DNA（（i）和（ii））的非共价相互作用激活，因此金属离子在生理条件下立即进行了位点选择性科学。通过非共价组合（III）离子和一个在其实习位置（二进制系统）中携带acridine的DNA的非共价组合也可以实现序列选择性的RNA科学。靶磷酸二酯键也被修饰的DNA激活，并优选水解。与三元系统相比，DNA-ACR/LU（III）二元系统的活性（约20％）。