Abstract
A fast, robust and reliable strategy for automated sequential resonance assignment for uniformly [13C, 15N]-labeled RNA via its phosphodiester backbone is presented. It is based on a series of high-dimensional through-bond APSY experiments: a 5D HCP-CCH COSY, a 4D H1′C1′CH TOCSY for ribose resonances, a 5D HCNCH for ribose-to-base connection, a 4D H6C6C5H5 TOCSY for pyrimidine resonances, and a 4D H8C8(C)C2H2 TOCSY for adenine resonances. The utilized pulse sequences are partially novel, and optimized to enable long evolution times in all dimensions. The highly precise APSY peak lists derived with these experiments could be used directly for reliable automated resonance assignment with the FLYA algorithm. This approach resulted in 98 % assignment completeness for all 13C–1H, 15N1/9 and 31P resonances of a stem-loop with 14 nucleotides.
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Acknowledgments
The Swiss National Science Foundation (SNSF project 200021_120048) and the Institute for Molecular Biology and Biophysics (ETH Zurich) are gratefully acknowledged for financial support. P.G. gratefully acknowledges financial support by the Lichtenberg program of the Volkswagen Foundation and the Japan Society for the Promotion of Science (JSPS).
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Krähenbühl, B., El Bakkali, I., Schmidt, E. et al. Automated NMR resonance assignment strategy for RNA via the phosphodiester backbone based on high-dimensional through-bond APSY experiments. J Biomol NMR 59, 87–93 (2014). https://doi.org/10.1007/s10858-014-9829-z
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DOI: https://doi.org/10.1007/s10858-014-9829-z