Calorimetric and spectroscopic investigation of the helix-to-coil transition of a ribo-oligonucleotide: rA 7U 7

Abstract

The double-strand to single-strand transition of the self-complementary ribo-oligonucleotide A 7 U 7 is investigated by means of differential scanning calorimetry, equilibrium u.v. melting and temperature-jump spectroscopy. It is found that these different physical techniques provide equivalent means for determining the melting temperature of the transition. The calorimetric experiments provide a value of 99·3 kcal (mol double strand) −1 for the helix-to-coil transition of A 7 U 7 . This represents the first direct determination of the enthalpy change accompanying such a transition in a ribo-oligonucleotide. Assuming all fourteen base-pairs to be equivalent, this corresponds to 7·1 kcal (mol A·U base-pair) −1 . Comparison with data obtained from polymeric systems permits the conclusion that an A·U base-pair in an oligomer is energetically equivalent to an A·U base-pair in a polymer. The data also appear to indicate that there is no significant variation of the ΔH with salt concentration. Values are also calculated for ΔG and ΔS . Values of ΔH V.H. (van't Hoff enthalpy change) are derived from the spectroscopic and calorimetric data and compared with the ΔH obtained directly from the calorimetric experiment. This comparison reveals that the thermally induced helix-to-coil transition of A 7 U 7 is not a two-state process. Calculations presented in the Appendix indicate that the transition is better represented by a model in which terminal base-pair fraying and intermediate states are taken into account. The relative reliability of the various optical techniques is discussed. It is noted that ΔH cal. is in better agreement with the ΔH V.H. derived from a log c versus l/ t m plot ( c is concentration) than from the slope at the midpoint of an α versus t melting curve, where α is the fraction in single strand. In addition, it is better to use a sloping rather than a flat low-temperature baseline to evaluate absorbance versus temperature profiles. Further analysis of the data permits the determination of values for s and β8 (or κ ). 8 is the equilibrium constant for formation of an A·U base-pair in an already existing helix and β8 (or κ ) is the equilibrium constant for bimolecular helix nucleation by an A·U base-pair.