Effect of distortions in the phosphate backbone conformation of six related octanucleotide duplexes on CD and 31P NMR spectra.

Abstract

We examined the structural properties of six octanucleotide duplexes d(TGACGTCA), d(ACTGCAGT), d(CTTCGAAG), d(CATCGATG), d(GTACGTAC), and d(CATGCATG). Circular dichroism (CD) and 2D 31P and 1H NMR spectroscopies were used in conjunction. Although of the B-DNA type, it was possible to arrange CD spectra into two families, A and B. Family A resembled poly(dG-dC) with a positive signal at approximately 280 nm and a negative one at approximately 260 nm, while family B resembled poly(dA-dT) with a positive signal at approximately 270 nm and a negative one at approximately 250 nm. All 31P resonances were assigned through constant-time heteronuclear 31P-1H correlated spectra. J(H3'-P) coupling constants related to dihedral angeles epsilon (C4'-C3'-O3'-P) were determined from 1H-31P J-resolved selective proton-flip 2D experiments. A good correlation was observed between 31P chemical shifts and coupling constants for all oligonucleotides. The patterns of these two parameters vs the base position along the sequences were almost similar. They were confronted with CD spectra. The results indicated that the position and magnitude of the signals were mainly affected by the CpG and ApT steps whose 31P chemical shifts were the farthest away from the mean 31P chemical shift value. This is in keeping with greater rigidity at these steps and should explain the influence of the local order on the shape of the CD spectra. Lastly, both UV absorption and 31P chemical shifts vs temperature provided normal temperature melting (Tm) values for all of the octanucleotide duplexes except for d(CTTCGAAG), for which the Tm was approximately 10 degrees C lower compared to its counterpart d(CATCGATG). The decrease in the thermal stability of this octanucleotide duplex was imputed to its contained TT and AA repeats, which might be able to induce correlated base destacking and phosphate group distortion in the oligonucleotide and especially on the intermediate CpG. We demonstrate that the CpG step displayed 31P NMR properties similar to those found in mismatched nucleotides exclusively in the d(CTTCGAAG) duplex.