UV Resonance Raman and Circular Dichroism Studies of a DNA Duplex Containing an A₃T₃ Tract:  Evidence for a Premelting Transition and Three-Centered H-Bonds

Abstract

The presence of A n and A n T n tracts in double-helical sequences perturbs the structural properties of DNA molecules, resulting in the formation of an alternate conformation to standard B-DNA known as B‘-DNA. Evidence for a transition occurring prior to duplex melting in molecules containing A n tracts was previously detected by circular dichroism (CD) and calorimetric studies. This premelting transition was attributed to a conformational change from B‘- to B-DNA. Structural features of A n and A n T n tracts revealed by X-ray crystallography include a large degree of propeller twisting of adenine bases, narrowed minor grooves, and the formation of three-centered H-bonds between dA and dT bases. We report UV resonance Raman (UVRR) and CD spectroscopic studies of two related DNA dodecamer duplexes, d(CGCAAATTTGCG)2 (A3T3) and d(CGCATATATGCG)2 [(AT)3]. These studies address the presence of three-centered H-bonds in the B‘ conformation and gauge the impact of these putative H-bonds on the structural and thermodynamic properties of the A3T3 duplex. UVRR and CD spectra reveal that the premelting transition is only observed for the A3T3 duplex, is primarily localized to the dA and dT bases, and is associated with base stacking interactions. Spectroscopic changes associated with the premelting transition are not readily detectable for the sugar−phosphate backbone or the cytosine and guanosine bases. The temperature-dependent concerted frequency shifts of dA exocyclic NH2 and dT C4O vibrational modes suggest that the A3T3 duplex forms three-centered hydrogen bonds at low temperatures, while the (AT)3 duplex does not. The enthalpy of this H-bond, estimated from the thermally induced frequency shift of the dT C4O vibrational mode, is approximately 1.9 kJ/mol or 0.46 kcal/mol.

Molecular dynamics simulations of DNA curvature and flexibility: Helix phasing and premelting

“…110,115,116 Hunter's analysis 114 suggests that a steric clash between the T-CH 3 group in the major groove and the neighboring 5Ј sugar ring is at least partly responsible. Crystallography 117 and resonance Raman spectroscopy 118,119 suggest that an additional "bifurcated" H-bond between cross-strand A-N6 and T-O4 may be a source of the stability. Whatever the reason, ApA steps in crystal structures of uncomplexed DNA turn out to be essentially straight and relatively rigid 113 and support the non-A-tract model as opposed to the ApA wedge model.…”

“…O4 distances of Յ2.8 Å . 167 However, NMR 168 and resonance Raman studies, 118,119 explicitly support an interaction, and the issue may reduce to how much free energy of stabilization is contributed by this interaction. The net contribution from each instance would be expected to be small at best, but could, of course, be cumulative.…”

Discrimination between UV radiation‐induced and thermally induced spectral changes in AT‐paired DNA oligomers using UV resonance Raman spectroscopy

“…Figure 3 illustrates typical UVRR spectra acquired on 12-mer oligo(dA)Ðoligo(dT) DNA samples heated to different temperatures and showing a recovery from Raman hypochromicity consistent with the literature. 4,8 In combination with Table 2, it can be seen that UVRRS is a powerful tool for selectively enhancing the base-pair vibrations while minimizing the vibrations from the DNA backbone and solvent. In fact, only one backbone vibration is readily observed in the spectrum of Fig.…”

Effect of Premelting on Conductivity of DNA-Lipid Films

“…The existence of "bubbles" [6,7] as well as the temperature induced local perturbations at T < T dn , termed "premelting," [8][9][10][11][12] has been confirmed experimentally, and the biological aspects of these local denaturations were discussed in a number of studies (see, e.g., [6]). Local deformations should cause breaking of a long-range order in the DNA structure (i.e., interruption of the parallel base-pair (bp) stackings) similar to an order breaking in solid bodies due to the dislocation introduction.…”

“…Hence, we cannot conclude the hypochromicity observed here to the effect of DNA premelting without further investigation. Figure 4: Temperature-dependent premelting effect in Raman frequencies of (a) the admixture of complex vibration of the B-DNA phosphodiester backbone conformation and thymine and cytosine ring modes [18], (b) dT C2 -endo sugar pucker and anti glycosyl torsion mode [8], and (c) dG and dA ring stretching vibrations sensitive to hydrogen bonding [18,19] as well as dT [10]. The bands shift toward lower frequencies starting near 35…”