PULSE SEQUENCE DEVELOPMENT

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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.