The NMR Core includes investigators at 6 institutions who are developing isotopically labeled ribonucleoside triphosphate (rNTPs) reagents and RNA labeling methodologies (Dayie, Williamson), and NMR data acquisition/assignment methodologies (Al-Hashimi, Summers, Marchant, Tolbert, Dayie, Johnson, Bax), to facilitate NMR studies of large RNAs. The NMR Core also interacts extensively with the Case and Johnson labs of the Computational Core, and with the Chiu, Zheng, and Pornillos labs of the cryoEM Core, to facilitate development of hybrid methodologies and force fields that enable structural and dynamical studies of larger HIV RNAs. Hybrid NMR/SAXS methods are also being developed and evaluated (Tolbert, Case, Dayie). The NMR Core also provides screening services to assist CRNA members in the identification of viable new RNA targets for structural analysis, and to support efforts by Hargrove to identify RNA-targeting antivirals. Over the past four years, the NMR Core has contributed to several technical innovations and discoveries including: (1) development of a novel fragmentation/annealing-based approach (fr-RNA) for detection and signal assignment of long-range secondary structures; (2) development of improved nucleotide-specific partial deuteration strategies (2H-editing) for NMR signal assignment; (3) development of improved methods detecting and preventing self-templated run-on during T7 RNA polymerase dependent synthesis; (4) development of a novel NMR method for unambiguous identification of intermolecular base pairing in large RNAs; (5) development of a novel NMR/mutagenesis method for measuring the rate of formation of intermolecular base pairs; (6) development of new tools for predicting and validating 1H NMR signal assignments by analysis of depositions in the BioMagResBank database; (7) contributed to the development of a new software package for rapid NMR data processing, and semi-automated RNA signal assignment; (8) development of new pulse sequences for rapid collection of isotopically edited RNA NMR spectra; and for the measurement and characterization of micro-to-millisecond conformational exchange in RNA; (9) development of new 2H, 13C, and 15N-labeled reagents to facilitate structural and dynamical studies of large RNAs; and (10) conducted service work for members of the CRNA (Beemon, Bieniasz, Hargrove, Boris-Lawrie, Parent, Heng), as well as for other NIH funded centers (James Hurley, HARC, Berkeley), several of which led to new structural projects. The NMR Core will continue to push the size limits of NMR and will support studies of new protein and RNA targets identified by the CRNA and other NIH funded groups. The methodological innovations that have been developed are generally applicable to the rapidly expanding field of RNA structural biology.
