PULSE SEQUENCE DEVELOPMENT

到目前为止，二维、三维和四维的应用 NMRFAM的多核核磁共振实验几乎完全集中在 关于蛋白质的分析，这些蛋白质构成了可用蛋白质的大部分 样品中富含15N、13C，在某些情况下还富含2H。目前， 生产标记RNA的技术已经变得相当成熟 直截了当(想必在不久的将来，DNA也是如此)， 而且很可能会有越来越多的研究人员希望 使用核磁共振方法进行RNA结构测定项目。至 促进RNA脉冲序列的快速实现 分析，我们正在将这一努力与一个协作项目相结合，以 确定35 N.T.的结构。均一为15N的RNA 15N，13C标记。在这一点上，一些双倍和 三重共振的2D、3D和4D实验已经在 布鲁克DMX-600和DMX-750控制台。PULSE计划将是 NMRFAM的用户和通过脉冲序列均可使用 在经过充分测试和记录后，可以在BioMagResBank上找到该库。 目前实施的一些实验包括：2D 15N HSQC (用于亚胺和氨基)；2D 13C恒定时间HSQC(用于核糖和 芳香族)；核糖的3DHCCH-TOCSY；3DHCN和选择性HCN 对于碱基H8/H6与核糖H1的相关性？通过N9/N1共振；3D 15N编辑的NOESY，带H2O翻转(亚氨基和氨基)；3D 13C编辑 NOESY(核糖/H5)；3D 13C编辑的NOESY(芳香族)；4D 13C/15N 适用于15N T1、T2的HMQC-NOESY-HSQC[ribose/aromatic(13C)-imino(15N)]；HSSC 她也不知道。这些实验中的大多数都已在 文献，但尚未放入BMRB图书馆。其他 已经尝试了基于文献描述的实验， 却没能在我们手中发挥作用。这可能是由于 增加了35个N.T.的线宽。正在研究的RNA相对于 大多数这些报告中使用的较小的RNA。

Until now, application of two-, three- and four-dimensional multinuclear NMR experiments at NMRFAM has focused almost exclusively on analysis of proteins, which have comprised the bulk of available samples enriched in 15N, 13C, and, in some cases, 2H. Currently, the techniques for production of labeled RNAs have become fairly straightforward (likewise for DNA, presumably, in the near future), and it is likely that increasing numbers of researchers will want to pursue RNA structure determination projects using NMR methods. To facilitate the rapid implementation of pulse sequences for RNA analysis, we are combining this effort with a collaborative project to determine the structure of a 35 n.t. RNA which has been uniformly 15N and 15N, 13C labeled. At this point, a number of double- and triple-resonance 2D, 3D, and 4D experiments have been implemented on the Bruker DMX-600 and DMX-750 consoles. The pulse programs will be made available both to users at NMRFAM and via the pulse sequence library at BioMagResBank after being fully tested and documented. Some of the currently implemented experiments include: 2D 15N HSQC (for iminos and aminos); 2D 13C constant-time HSQC (for ribose and aromatics); 3D HCCH-TOCSY for ribose sugars; 3D HCN and HCN-selective for correlation of base H8/H6 to ribose H1? via N9/N1 resonances; 3D 15N-edited NOESY with H2O flip-back (imino and amino); 3D 13C-edited NOESY(ribose/H5); 3D 13C-edited NOESY(aromatic); 4D 13C/15N HMQC-NOESY-HSQC[ribose/aromatic(13C)-imino(15N)]; HSQCs for 15N T1, T2 and hetNOE. Most of these experiments have been described in the literature but have not been put in the BMRB library. Other experiments have been attempted based upon literature descriptions, but have failed to work in our hands. This may be a consequence of the increase linewidths of the 35 n.t. RNA under study relative to smaller RNAs used in most of these reports.