Towards structural genomics of RNA: rapid NMR resonance assignment and simultaneous RNA tertiary structure determination using residual dipolar couplings.

We report a new residual dipolar couplings (RDCs) based NMR procedure for rapidly determining RNA tertiary structure demonstrated on a uniformly (15)N/(13)C-labeled 27 nt variant of the trans-activation response element (TAR) RNA from HIV-I. In this procedure, the time-consuming nuclear Overhauser enhancement (NOE)-based sequential assignment step is replaced by a fully automated RDC-based assignment strategy. This approach involves examination of all allowed sequence-specific resonance assignment permutations for best-fit agreement between measured RDCs and coordinates for sub-structures in a target RNA. Using idealized A-form geometries to model Watson-Crick helices and coordinates from a previous X-ray structure to model a hairpin loop in TAR, the best-fit RDC assignment solutions are determined very rapidly (<five minutes of computational time) and are in complete agreement with corresponding NOE-based assignments. Orientational constraints derived from RDCs are used simultaneously to assemble sub-structures into an RNA tertiary conformation. Through enhanced speeds of application and reduced reliance on chemical shift dispersion, this RDC-based approach lays the foundation for rapidly determining RNA conformations in a structural genomics context, and may increase the size limit of RNAs that can be examined by NMR. c) 2002 Elsevier Science Ltd.