Most cited article - PubMed ID 8720835
Through-bond correlation of imino and aromatic resonances in 13C-, 15N-labeled RNA via heteronuclear TOCSY
The paper presents a set of triple-resonance two-dimensional experiments for correlating all quaternary carbons in RNA bases to one or more of the base protons. The experiments make use of either three-bond proton-carbon couplings and one selective INEPT step (the long-range selective HSQC experiment) to transfer the magnetization between a proton and the carbon of interest and back, or they rely on one- and/or two-bond heteronuclear (the H(CN)C and H(N)C experiments) or carbon-carbon (the H(C)C experiment) couplings and multiple INEPT transfer steps. The effect of the large one-bond carbon-carbon coupling in t(1) is removed by a constant time evolution or by a selective refocusing. The performance of the proposed approach is demonstrated on a 0.5 mM 25-mer RNA. The results show that the experiments are applicable to samples containing agents for weak molecular alignment. The design of the correlation experiments has been supported by ab initio calculations of scalar spin-spin couplings in the free bases and the AU and GC base pairs. The ab initio data reveal surprisingly high values of guanine (2)J(N1C5) and uracil (2)J(N3C5) couplings that are in a qualitative agreement with the experimental data. The sensitivity of the spin-spin couplings to base pairing as well as the agreement with the experiment depend strongly on the type of nuclei involved and the number of bonds separating them.
Triple resonance HCN and HCNCH experiments are reliable methods of establishing sugar-to-base connectivity in the NMR spectra of isotopicaly labeled oligonucleotides. However, with larger molecules the sensitivity of the experiments is drastically reduced due to relaxation processes. Since the polarization transfer between 13C and 15N nuclei relies on rather small heteronuclear coupling constants (11-12 Hz), the long evolution periods (up to 30-40 ms) in the pulse sequences cannot be avoided. Therefore any effort to enhance sensitivity has to concentrate on manipulating the spin system in such a way that the spin-spin relaxation rates would be minimized. In the present paper we analyze the efficiency of the two known approaches of relaxation rate control, namely the use of multiple-quantum coherence (MQ) and of the relaxation interference between chemical shift anisotropy and dipolar relaxation - TROSY. Both theoretical calculations and experimental results suggest that for the sugar moiety (H1'-C1'-N1/9) the MQ approach is clearly preferable. For the base moiety (H6/8-C6/8-N1/9), however, the TROSY shows results superior to the MQ suppression of the dipole-dipole relaxation at moderate magnetic fields (500 MHz) and the sensitivity improvement becomes dramatically more pronounced at very high fields (800 MHz). The pulse schemes of the triple-resonance HCN experiments with sensitivity optimized performance for unambiguous assignments of intra-residual sugar-to-base connectivities combining both approaches are presented.
- MeSH
- Quantum Theory MeSH
- Magnetics MeSH
- Nuclear Magnetic Resonance, Biomolecular methods MeSH
- Nucleosides chemistry MeSH
- RNA chemical synthesis chemistry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Nucleosides MeSH
- RNA MeSH
