DNA fragment conformations. 1H-NMR comparative studies of helix-coil transition and conformation of d(C-A-C-G-T-G) and d(G-T-G-C-A-C). Influence of helix formation on proton chemical shifts.

Abstract

The conformation and the helix‐coil transition of two complementary hexamers, d(C‐A‐C‐G‐T‐G) and d(G‐T‐G‐C‐A‐C), were investigated as a function of temperature by 1H‐NMR spectroscopy at 500 MHz and 90 MHz. At high temperature (t > 70–80°C), in the coil form, the proton chemical shifts depend strongly on the nature and position in the sequence of the residue. Purine and pyrimidine residues can be distinguished not only by the difference in chemical shift of the H 2′ and H 2” resonances but also by that of the H 3′ or H 4′ protons. For equivalent positions, the dA proton resonances are always located at lower fields than those of other residues. For residues of the same nature the chemical shift of the H 3′ or H 4′ protons decreases in the following order: δ(pdNp) > δ(dNp) > δ(pdN), where dN = dA, dG, dC or dT; moreover the terminal residue can easily be identified by either the H 1′ resonance [δ(pdN) > δ(pdNp, dNp)] or the H 2” resonance for the purine family [δ(pdN) < δ(pdNp, dNp) ≤δ(pdNp, dNp, pdN), where dN = dA, dG] and the H 2′ resonance for the pyrimidine family [δ(pdN) > δ(pdNp, dNp), where dN = dC or dT].The chemical shift difference between the coil and helix forms is generally greater for the pyrimidine than for the purine residues and the proton resonances of the external residues often exhibit different temperature‐dependent profiles. The results suggest that, as in the helical form the bases of adjacent residues are close to each other, the pyrimidine base proton resonances are shifted not only by the change in environment but also by the ring‐current effects from neighbouring purine bases. In the intercalated purine‐pyrimidine sequences, the protons of an internal pyrimidine residue are statistically subjected to opposite ring‐current effects from the base of the preceding and following residues. The difference in temperature‐dependent behaviour of the H 2” resonances between the initial or internal purine residues (where the phosphate group is located at the 3′ position) on the one hand and the terminal residue on the other can be explained by the change in conformation of the phosphate groups from the coil to the helix form.From measurements of proton coupling constants and observation of the NH resonances, it was concluded that the duplexes of both hexamers, d(C‐A‐C‐G‐T‐G) and d(G‐T‐G‐C‐A‐C), adopt the B‐helical conformation and open sequentially from the exterior with increasing temperature. The midpoint temperatures, t1/2, coil proportions and helix‐coil dissociation constants Kd, were determined from the melting curves. The dissociation rate constant, kd, was deduced versus temperature from the line width of the H 1′ and H 5 resonances. The midpoint temperature of each residue and the difference between the t1/2 external and central residues depend strongly on the number of the dG · dC base pairs and their positions in the sequence, whereas the dissociation enthalpy appears to be independent for sequences of the same size.