Experiments for correlating quaternary carbons in RNA bases.
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.
Fiala
Radovan
R
National Centre for Biomolecular Research, Masaryk University, Kotlárská 2, 611 37 Brno, Czech Republic. fiala@ncbr.chemi.muni.cz
Munzarová
Markéta L
ML
Sklenár
Vladimír
V
D002244:Carbon / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D011687:Purines / Q000737:chemistry*; D011743:Pyrimidines / Q000737:chemistry*; D012313:RNA / Q000737:chemistry*
D011687:Purines; D011743:Pyrimidines; D012313:RNA; D002244:Carbon