DUPLEX AND TRIPLE-HELIX FORMATION WITH DA19 AND DT19 - THERMODYNAMIC PARAMETERS FROM CALORIMETRIC, NMR, AND CIRCULAR-DICHROISM STUDIES

Differential scanning calorimetric (DSC), NMR, and circular dichroism studies (CD) have been performed on solutions with dA19 and dT19 at 1:1 and 1:2 mole ratios at different [NaCl] in PIPES buffer (pH=7.0). An asymmetry exists in the transition regions for the excess heat capacity curves for the dA19/dT19 mole ratio of 1:1 that increases with increasing [NaCl]. In a 1:2 mixture of dA19 and dT19 with [NaCl] > 1.0 M the transition region is much narrower, and the total DELTAH is about 50% larger. The CD and NMR spectra for the 1:1 mixture at 0-degrees-C have the same features reported for poly(dA)*poly(dT), confirming the existence of a tight duplex in 1:1 mixtures of dA19 and dT19 at low temperatures. Also for the 1:2 mixture, the negative ellipticity in the CD spectra below 230 nm is characteristic for formation of triple helix in dA/dT systems. Integration of the DSC curves, which is independent of any model, provides DELTAH(AT) = 106 +/- 4 kcal/mol and DELTAH(AT2) = 153 +/- 3 kcal/mol for duplex and triple helix dissociation, respectively. A model employing coupled equilibria between duplex and triple helix fits the excess heat capacity curves extremely well for both the 1:1 and 1:2 mixtures with DELTAH(AT) and DELTAH(AT2), within experimental error, the same at each [NaCl] and equal to the values derived from integration. The average DELTAH for dissociation of a dA*dT base pair in dA19*dT19 is nearly 0.8 kcal/mol less than reported for poly(dAdT) and 3.0 kcal/mol less than that for poly(dA)*poly(dT). The smaller than expected DELTAH for dA19*dT19 could conceivably be due to decreased base-pair interactions at the 5'-end of dA*dT tracts, as has been proposed (Nadeau, J.; Crothers, D. H. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 2622-2626) from imino proton NMR studies. Also, in the triple helix complex, the average DELTAH for the dA19*dT19 Hoogsteen base pair is nearly one-half less than that reported for the corresponding polymer complex; thus, caution must be used when employing the homopolymer systems as models for the dA*dT tracts in DNA.