Automated NMR resonance assignment strategy for RNA via the phosphodiester backbone based on high-dimensional through-bond APSY experiments
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 H1â?²C1â?²CH 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 98Â*% assignment completeness for all 13Câ??1H, 15N1/9 and 31P resonances of a stem-loop with 14 nucleotides.
Source: Journal of Biomolecular NMR