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

建立了化学合成法标记核苷稳定同位素的方法，即用~(13)&gt；C/~(-lt；15)~(gt；N)核取代全氮/碳或特定位置的氮/碳原子核。此外，还开发了在2‘或5’位置上进行立体专位氢化的方法。由这些核苷制备了用于合成聚合物的单体单元，并用于合成标记的寡核苷酸。开发了几个脉冲序列，用于利用^1H，^&lt；13&gt；C，^&lt；N和^&lt；31&gt；P之间的直接自旋-自旋耦合进行序列信号分配。这些技术对应于蛋白质多维核磁共振的技术。在此基础上，提出了用于确定二面角α-xi，chi的J的获取方法。通过结合有限数量的NOE和J值，可以确定低分子量寡核苷酸的结构。为了确定高分子量寡核苷酸的结构，要求NOE的强度准确，并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，允许最短距离的自旋对交叉峰，发展了一种新的多位固相标记样品的信号指认方法。提出了多个标记点间距离和二面角的测量方法。在不久的将来，有可能用这种方法来确定固态核苷的构象。用稳定的同位素标记的样品确定了RNA发夹的整个三级结构。对Holliday结和Cro/DNA复合体等样品进行了部分~(13)&gt；C标记，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.