Sequential assignments in uniformly 13C- and 15N-labelled RNAs: The HC(N,P) and HC(N,P)-CCH-TOCSY experiments

Abstract

An approach for the simultaneous acquisition of HCN and HCP as well as HCN-CCH-TOCSY and HCP-CCH-TOCSY triple resonance data sets for 13C-/15N-labelled RNAs is presented. The new HCN-CCH-TOCSY scheme unambiguously links all sugar resonances to the base nitrogen. In addition, simultaneous acquisition of HCN-CCH-TOCSY and HCP-CCH-TOCSY data sets provides sequential and base-type information in a single experiment, thereby saving data acquisition time as well as providing complementary data sets that are useful in clarifying ambiguous assignments. Virtually complete sequence-specific phosphate-ribose 1H, 31P, and base 15N1,9, assignments as well as partial 13C assignments could be obtained in a single experiment for a 0.5-mM sample of a 19-mer ribonucleotide.

Structure of an RNA Hairpin Loop with a 5‘-CGUUUCG-3‘ Loop Motif by Heteronuclear NMR Spectroscopy and Distance Geometry

“…Here we used a new experiment, HC(N/P)-CCH-TOCSY (Ramachandran et al, 1996), which provides valuable information on base types via the 15 N chemical shifts that can be used for the clarification of ambiguous sequential assignments in the HCP-CCH-TOCSY subspectrum. For a detailed description of this approach as applied to the 19mer studied here, see Ramachandran et al (1996).…”

Automated NMR resonance assignment strategy for RNA via the phosphodiester backbone based on high-dimensional through-bond APSY experiments

“…It serves to sequentially connect resonances via the 31 P frequencies to achieve sequence-specific resonance assignment. Experiments with the same magnetization transfer pathway were published as a 3D HCP-CCH COSY (Marino et al 1995;Ramachandran et al 1996;Tate et al 1995), which provided frequencies for 31 P and for the 13 C-1 H group on which the magnetization transfer ends. We modified the experiment to a 5D pulse sequence by including a constant-time evolution of the first 13 C dimension during the second 13 C-31 P transfer period, and a semi-constant-time evolution on the proton where the magnetization transfer starts.…”

Identification of Ribose-Base Sequential NOEs According to Base Types in Uniformly13C-Labeled RNAs

“…Under the typical 13 C acquisisignments based on through-bond coherence transfer along tion times normally employed in the HMQC-NOESY exthe phosphate-ribose backbone (4)(5)(6)(7)(8)(9)(10). Although these periment, it may be difficult to resolve such carbon-carbon methods are contributing to the determination of detailed couplings and hence to achieve purine/pyrimidine distinc-NMR solution structures for RNA molecules of up to about tion.…”