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The nature of stacking equilibria in polynucleotides

Temperature melting profiles of adenylyladenosine and cytidylylcytidine were examined by ultraviolet absorption, circular dichroism, and optical rotatory dispersion. They showed the following characteristics: profiles for different absorption wavelengths are identical as defined by least‐squares computed optical and thermodynamic parameters; melting curves obtained by the three spectroscopic methods are likewise in all cases identical by a series of criteria; in terms of the operational equilibrium constant for base‐stacking all van't Hoff plots are linear; absorption spectra and circular dichroism curves at a series of temperatures for both dinucleoside phosphates give excellent isosbestic points; a matrix‐rank analysis of the family of complete spectra at different temperatures gives a value of two for the numbers of components in both cases. Four criteria are thus fulfilled of a two‐state system for the stacking equilibrium. The results are not compatible with a multi‐state scheme in which the substates are optically distinguishable. There is no measurable effect of ionic strength up to 1.0. In a presence of high concentrations of lithium chloride the criteria for two‐state behavior are no longer fulfilled, and examination of infrared spectra gives evidence of complex formation by this salt, though not by sodium chloride. A destacking profile, similar to that observed on heating, is obtained by progressive addition of ethylene glycol to the solution. It is suggested that in dimers, though not in higher oligomers or polymers, the stacking equilibrium can be operationally regarded as a two‐state system, and that the meaningful thermodynamic parameters can be extracted by analytical treatment, of the type here developed, of the melting profiles.