We present a protocol for determining the relative orientation and dynamics of A-form helices in 13C/15N isotopically enriched RNA samples using NMR residual dipolar couplings (RDCs). Non-terminal Watson–Crick base pairs in helical stems are experimentally identified using NOE and trans-hydrogen bond connectivity and modeled using the idealized A-form helix geometry. RDCs measured in the partially aligned RNA are used to compute order tensors describing average alignment of each helix relative to the applied magnetic field. The order tensors are translated into Euler angles defining the average relative orientation of helices and order parameters describing the amplitude and asymmetry of interhelix motions. The protocol does not require complete resonance assignments and therefore can be implemented rapidly to RNAs much larger than those for which complete high-resolution NMR structure determination is feasible. The protocol is particularly valuable for exploring adaptive changes in RNA conformation that occur in response to biologically relevant signals. Following resonance assignments, the procedure is expected to take no more than 2 weeks of acquisition and data analysis time.
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We thank members of the Al-Hashimi laboratory for insightful comments and Dr. Alex Kurochkin for his expertise and for maintenance of the NMR instruments. H.M.A. acknowledges fruitful collaborations with the groups of Carol Fierke (The University of Michigan) and Ioan Andricioaei (The University of Michigan). We gratefully acknowledge the Michigan Economic Development Cooperation and the Michigan Technology Tri-Corridor for the support in the purchase 600 MHz spectrometer. This work was supported by funding from the NIH (RO1 AI066975-01).
The authors declare no competing financial interests.
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Bailor, M., Musselman, C., Hansen, A. et al. Characterizing the relative orientation and dynamics of RNA A-form helices using NMR residual dipolar couplings. Nat Protoc 2, 1536–1546 (2007). https://doi.org/10.1038/nprot.2007.221
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