PULSE SEQUENCE DEVELOPMENT

脉冲序列开发

• 批准号: 6309181
• 负责人: BRIAN F VOLKMAN
• 金额: $ 0.75万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-15 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6309181
• 关键词: biomedical equipment development; biomedical resource; nuclear magnetic resonance spectroscopy; technology /technique

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.

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 分析蛋白质，蛋白质构成了大部分可用的蛋白质 富含15 N、13 C，在某些情况下还富含2 H的样品。 目前 生产标记RNA的技术已经变得相当 简单明了（DNA也是如此，大概在不久的将来）， 而且很可能越来越多的研究人员想要 利用核磁共振方法进行RNA结构测定项目。 到 促进RNA的脉冲序列的快速实施， 分析，我们正在将这一努力与一个合作项目相结合， 确定一个35 n.t. RNA的结构，该RNA已被均匀地15 N 和15 N、13 C标记。 在这一点上，一些双重和 三重共振2D，3D和4D实验已经实施， 布鲁克DMX-600和DMX-750控制台。 脉冲程序将是 提供给NMRFAM的用户和通过脉冲序列 库在BioMagResBank后进行全面测试和记录。 目前实施的一些实验包括：2D 15 N HSQC (for亚氨基和氨基）; 2D 13 C恒定时间HSQC（对于核糖和 3D HCH-TOCSY用于核糖; 3D HCN和HCN选择性 碱基H8/H6与核糖H1的相关性？ 通过N9/N1共振; 3D 15 N-编辑的NOESY与H2O翻转（亚氨基和氨基）; 3D 13 C-编辑 NOESY（核糖/H5）; 3D 13 C编辑的NOESY（芳香族）; 4D 13 C/15 N HMQC-NOESY-HSQC[核糖/芳族（13 C）-亚氨基（15 N）]; 15 N T1、T2的HSQC 和HetNOE。 这些实验中的大多数已经在 文献，但没有放在BMRB图书馆。 其他 已经基于文献描述尝试了实验， 但在我们手中却失败了 这可能是由于 所研究的35 n.t. RNA的线宽相对于 大多数这些报告中都使用了较小的RNA。