MULTINUCLEAR NUCLEAR-MAGNETIC-RESONANCE STUDIES OF NA CATION-STABILIZED COMPLEX FORMED BY D(G-G-T-T-T-T-C-G-G) IN SOLUTION - IMPLICATIONS FOR G-TETRAD STRUCTURES

There has been much recent interest in the self-association of short deoxyguanosine-rich motifs within single-stranded DNAs to generate monovalent cation modulated four-stranded helical segments called G-quadruplexes stabilized by hydrogen-bonded G-tetrad alignments. We have addressed structural aspects of this novel alignment and report on multinuclear 1H, 31P and 13C nuclear magnetic resonance studies on the d(G2T4CG2) deoxynonanucleotide with Na cation as counterion in aqueous solution at low temperature. This sequence forms stable structures even though it cannot align by Watson-Crick hydrogen bond formation (see the paper on d(G2T5G2) describing optical and calorimetric measurements by Jin, R., Breslauer, K. J., Jones, R. A. & Gaffney, B. L. (1990), Science, 250, 543-546). The four narrow exchangeable protons detected between 11.5 and 12.0 parts per million (p.p.m.), which are common to the d(G2T4CG2) deoxynonanucleotide and the d(G2TCG2) deoxyhexanucleotide sequences, are assigned to deoxyguanosine imino protons hydrogen-bonded to carbonyl acceptor groups. These narrow imino protons are not detected for d(IGN5IG) and d(I2N5G2), where two deoxyguanosine residues are replaced by two deoxyinosine residues in the deoxynonanucleotide sequences. This implies that the 2-amino protons of deoxyguanosine must also participate in hydrogen bond formation and stabilize the structured conformation of d(G2T4CG2) in Na cation-containing solution. We have completely assigned the base and sugar H1′, H2′,2, {combining double acute accent} H3′, and H4′ protons of the d(G2T4CG2) oligomer following analysis of two-dimensional nuclear Overhauser enhancement spectroscopy and two-dimensional correlated spectroscopy data sets in 0.1 m-NaCl, 10 mm-sodium phosphate, 2H2O solution at 0 °C. The relative magnitude of the nuclear Overhauser enhancements (NOEs) between the base H8 and its own sugar H1′ protons of individual deoxyguanosine residues establishes that G1 and G8 adopt syn orientations while G2 and G9 adopt anti orientations about the glycosidic bond in the d(G1-G2-T3-T4-T5-T6-C7-G8-G9) sequence in both Na and K cation-containing aqueous solution. Consequently, any structure proposed for the tetramolecular complex of d(G2T4CG2) must exhibit alternating G(syn) and G(anti) glycosidic torsion angles within each strand. The directionality and magnitude of the observed NOEs are consistent with the G(syn)-G(anti) steps adopting right-handed helical conformations in solution. We also note that the H8 protons of G1 and G8 (7.35 to 7·45 p.p.m.) in a syn alignment are shifted significantly upfield from the H8 protons of G2 and G9 (8·0 to 8·3 p.p.m.) in an anti alignment. Two-dimensional proton-detected heteronuclear (1H31P and 1H13C) spectra have been recorded at natural abundance on the d(G2T4CG2) sequence in aqueous solution. The phosphorus resonances are dispersed over 1·5 p.p.m. with the G2-T3 and T6-C7 phosphates shifted to low field and the T4-T5 phosphate shifted to high field of the unperturbed spectral region. These results suggest that the pyrimidine-rich T4C segment in d(G2T4CG2) is involved either in hairpin loop or in hairpin bulge formation. The base and sugar carbon resonances have been assigned and we note that the base, C-1′, C-3′ and C-4′ carbons of G1 and G8 in a syn alignment resonate to low field of the corresponding carbon atoms of G2 and G9, which adopt an anti alignment. The nuclear magnetic resonance results are discussed in relation to proposed models for G-tetrad alignments in G-quadruplex structures. © 1991.