Development of the stable isotope aided multidimensional NMR for the structure determination of nucleic acids

开发用于核酸结构测定的稳定同位素辅助多维核磁共振

基本信息

批准号：
05101004
负责人：
KYOGOKU Yoshimasa
金额：
$ 166.4万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Specially Promoted Research
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1996
项目状态：
已结题

项目摘要

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.

已经建立了通过化学合成对核苷稳定的同位素标记的方法，在该方法中，整个氮/碳核或特定位点的核苷方法被 ^<13> C/ ^<15> N核代替。除他们外，还开发了在2'或5'位置上进行立体特定剥离的方法。从这些核苷的单体单元中制备了用于聚合物合成的单体单位，并用于合成标记的寡核苷酸。通过利用 ^1H， ^1H， ^<13> c， ^> c， ^<15> n和 ^p之间的直接自旋旋转耦合，用于顺序信号分配的几个脉冲序列。这些技术对应于蛋白质多维NMR。基于分配的蛋白质技术，已提出了用于确定二面角的J的方法Alpha-XI，CHI。通过将有限数量的NOE与J值相结合，可以确定低分子量寡核苷酸的结构。为了确定较高的分子量寡核苷酸的结构，NOE的强度应该是准确的，并且应在不同条件下进行充分使用NOE。一种新方法用于通过利用偶极 - 偶极相互作用的距离距离距离的距离距离的距离，从而允许最短的距离距离距离距离距离距离，从而开发了多个位点的样品。此外，提出了测量多个标记位点之间距离和二面角的方法。在不久的将来，可以通过使用稳定的同位素标记的样品来确定RNA发夹的整个三级结构。一些样品（例如Holliday结和CRO/DNA复合物）被部分标记，并获得了结构信息。