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

用化学合成法建立了核苷稳定同位素标记方法，用^<13>C/^<15>N核代替完整的氮/碳核或特定位置的氮/碳核。此外，还开发了在2′或5′位置进行立体特异氘化的方法。从这些核苷中制备了用于聚合物合成的单体单元，并用于标记寡核苷酸的合成。利用^1H，^<13>C,^<15>N和^<31>P之间的直接自旋耦合，开发了几种脉冲序列用于顺序信号分配。这些技术对应于蛋白质多维核磁共振。在此基础上，提出了确定二面角J的方法。将有限数目的noe与J值结合，可以确定低分子量寡核苷酸的结构。对于高分子量寡核苷酸的结构测定，既要保证NOE强度的准确性，又要充分利用不同条件下的NOE。利用偶极-偶极相互作用的距离依赖性，提出了一种固体标记样品多位点信号赋值的新方法，该方法允许最短距离自旋对的交叉峰。此外，还提出了多个标记位点之间距离和二面角的测量方法。在不久的将来，用这种方法测定固态核苷的构象可能成为可能。用稳定同位素标记样品测定了RNA发夹的三级结构。一些样品如Holliday结和Cro/DNA复合物被部分^<13>C标记，并获得了结构信息。

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.