Two-dimensional 1H and 31P NMR spectra and restrained molecular dynamics structure of a mismatched GA decamer oligodeoxyribonucleotide duplex.

Assignment of the 1H and 31P NMR spectra of a tandem G.A mismatched base pair decamer oligodeoxyribonucleotide duplex, d(CCAAGATTGG)2, has been made by two-dimensional 1H-1H and heteronuclear 31P-1H correlated spectroscopy. Unusual downfield 31P resonances have been assigned by a pure absorption phase constant-time heteronuclear 31P-1H correlated spectrum to be associated with the phosphates on the 5'- and 3'-sides of the mismatched guanosine residue. JH3'-P coupling constants for each of the phosphates of the decamer were obtained from the 1H-31P J-resolved selective proton-flip 2D spectrum. The two most downfield-shifted 31P resonances each appear to consist of two overlapping signals that can be resolved into two distinct doublets with different coupling constants in the J-resolved spectrum. This as well as the temperature dependence of the 31P spectra demonstrates that two distinct conformations exist at lower temperatures. By use of a modified Karplus relationship, the C4'-C3'-O3'-P torsional angles (epsilon) were obtained. A linear correlation between 31P chemical shifts and the measured coupling constants is quite good (only when the larger set of coupling constants of the two most downfield 31P signals is included). The 31P chemical shifts as well as the measured coupling constants tend to follow the positional variation seen in other duplexes of interior phosphates resonating more upfield than terminal residues and of interior phosphates exhibiting smaller coupling constants; however, this pattern is disrupted at the site of the mismatch. Modeling and initial NOESY distance restrained molecular mechanics energy minimization and restrained molecular dynamics support previous observations that the mismatched guanine and adenine bases are both in anti conformations. Most significantly, the epsilon backbone torsional angle variaions calculated from the NOESY distance restrained structures are in agreement with both the crystal structure values and the measured JH3'-P coupling constants.