NMR aided structural analysis of Holiday Junction

Holiday Junction 的核磁共振辅助结构分析

基本信息

批准号：
09044098
负责人：
ONO Akira
金额：
$ 5.5万
依托单位：
TOKYO METOROPOLITAN UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09044098/
关键词：
Heteronuclear Mutidimensional NMR Stable isotope labeling Chemical Synthesis of DNA Holliday Junction Holliday Junction Holliday junction NMR 安定同位体標識核酸安定同位体利用NMR 安定同位体 DNA 化学合成

项目摘要

The synthetic method for site and stereoselectively ィイD12ィエD1H/ィイD113ィエD1C-labeled glucose had been investigated. After examination of the reaction conditions for introducing deuteron at 5-positions of ィイD113ィエD1C-labeled ribose by the deuteride transfer from deuterated reagent to the 5-oxopentose derivatives synthesized from ィイD113ィエD1C-labeled glucose, the procedure introducing deuteron at the 5-position of ribose with desired R/S ratios. Finally, the synthetic route for site and stereoselectively ィイD12ィエD1H/ィイD113ィエD1C-labeled nucleosides had been established.We examined the reaction conditions for synthesizing labeled dNMP by phosphorylating the 5'OH of labeled nucleosides. The yields for the phosphrylation of 2'-deoxynucleosides are lower than those for the phosphrylation of ribo-nucleosides. However, the yields have been improved by adding protonsponge as a base.The reaction conditions have been investigated for in vitro replication using labeled dNTP and exo-nuclease activity free Klenow polymerase. We found that the commercially available enzyme gave good results.Through NMR studies using ィイD113ィエD1C/ィイD115ィエD1N-fully labeled DNA duplexes, NMR scalar coupling across Watson-Crick base pair hydrogen bonds were observed. This phenomenon can be used for robust determination of base pairs in large complexes such as the Holliday Junction.We tried to express Ruv C, a Holliday Junction specific endonuclease, and its mutants using high expression vectors in order to study Ruv C-Holliday Junction complexes by NMR, however, the purpose has not achieved because of the low solubility of the enzyme.

研究了通过氘代试剂在 D113D1C 标记核糖 5 位上引入氘核的反应条件，研究了立体选择性 D12D1H/D113D1C 标记葡萄糖的合成方法。 D113D1C-标记最后，建立了位点和立体选择性D12D1H/D113D1C标记核苷的合成路线。我们考察了磷酸化合成标记dNMP的反应条件。标记核苷的 5'OH 磷酸化的产率。 2'-脱氧核苷的磷酸化反应低于核糖核苷的磷酸化反应，但通过添加质子海绵作为碱基，产率得到了提高。使用标记的 dNTP 和无核酸外切酶活性的 Klenow 对反应条件进行了体外复制研究。我们发现市售的酶效果良好。通过使用NMR研究。 D113D1C/D115D1N 完全标记的 DNA 双链体，观察到跨 Watson-Crick 碱基对氢键的 NMR 标量耦合，这种现象可用于稳健测定大型复合物（例如霍利迪连接点）中的碱基对。我们尝试表达 Ruv C， Holliday Junction 特异性核酸内切酶及其突变体，使用高表达载体研究 Ruv C-Holliday Junction然而，由于酶的溶解度较低，因此未能达到目的。