Two- and three-dimensional 31P-driven NMR procedures for complete assignment of backbone resonances in oligodeoxyribonucleotides

Abstract

We describe a strategy for sequential assignment of 31P and deoxyribose 1H NMR resonances in oligode-oxyribonucleotides. The approach is based on 31P-1H J-cross-polarization (hetero TOCSY) experiments, recently demonstrated for the assignment of resonances in RNA [Kellogg, G.W. (1992) J. Magn. Reson., 98, 176; Kellogg, G.W. et al. (1992) J. Am. Chem. Soc., 114, 2727]. Two-dimensional heteroTOCSY and heteroTOCSY-NOESY experiments are used to connect proton spin systems from adjacent nucleotides in the dodecamer d(CGCGAATTCGCG)2 entirely on the basis of through-bond scalar connectivities. All phosphorus resonances of the dodecamer are assigned by this method, and many deoxyribose 1H resonances can be assigned as well. A new three-dimensional heteroTOCSY-NOESY experiment is used for backbone proton 4', 5' and 5" resonance assignments, completing assignments begun on this molecule in 1983 [Hare, D.R. et al. (1983) J. Mol. Biol., 171, 319]. Numerical simulations of the time dependence of coherence transfer aid in the interpretation of heteroTOCSY spectra of oligonucleotides and address the dependence of heteroTOCSY and related spectra on structural features of nucleic acids. The possibility of a generalized backbone-driven 1H and 31P resonance-assignment strategy for oligonucleotides is discussed.

Analysis of Phosphorylated Metabolites in Crayfish Extracts by Two-Dimensional1H-31P NMR Heteronuclear Total Correlation Spectroscopy (heteroTOCSY)

“…The 31 P-1 H heteroTOCSY experiment has the advantages of a single mixing period, greater sensitivity from 1 H detection, in-phase cross-peaks, and a fully phase-sensitive representation of the data. For example, 1 H-detected 2D 31 P-1 H heteroTOCSY has been used to correlate the backbone phosphorus with the ribose protons of DNA at low millimolar concentrations (15)(16)(17). With sufficiently long mixing times, magnetization was relayed out as far as the anomeric H1Ј.…”

Mutational and Structural Analysis of Stem-loop IIIc of the Hepatitis C Virus and GB Virus B Internal Ribosome Entry Sites

“…Backbone 31 P resonances were assigned based on the H3 0 shifts and the 1 H-31 P hetero-TOCSY correlation. 31 Coupling constants among the sugar protons were obtained from the DQF-COSY spectra. The absence of H1 0 -H2 0 cross-peaks in the TOCSY and DQF-COSY spectra for all sugar moiety except for the two apical sugar moiety in the loop indicates that most of the bases adopt predominantly C 3 0 -endo sugar puckering as found in A-form helices.…”

“…31 All NMR data were processed using Felix version 97 (MSI). The number of t 1 data points was increased by twofold, using mirrorimage linear prediction algorithm provided in the Felix program, before Fourier transformation.…”

Solution-State NMR Experiments Based on Heteronuclear Cross-Polarization

“…As an important aspect to be pointed out, the transfer mechanism in all proposed HCP experiments always involved in-phase to in-phase (IPto-IP) coherence transfer in which only magnetization components that are parallel to the phase of the HCP process were efficiently transferred. Examples can be found in 1D HCP directed to small molecules such as the measurement of carbon-carbon coupling constants at natural abundance [15], 1 H- 29 Si NMR spectroscopy to be applied on organosilicon compounds [16][17], X-excited 1 H-detected experiments to measure relaxation 31 P data [18][19][20][21][22] or for structural 31 P studies [23][24] in nucleic acids, 117/119 Cd NMR studies in metalloproteins [25][26], proton-carbon transfers in 2D, 3D and 4D experiments to be applied in proteins and nucleic acids [27][28][29][30][31], and other experiments in which band-selective HCP transfer was usually performed on specific spectral regions [32][33].…”