A more unified picture for the thermodynamics of nucleic acid duplex melting: a characterization by calorimetric and volumetric techniques

Abstract

We use a combination of calorimetric and volumetric techniques to detect and to characterize the thermodynamic changes that accompany helix-to-coil transitions for five polymeric nucleic acid duplexes. Our calorimetric measurements reveal that melting of the duplexes is accompanied by positive changes in heat capacity (DeltaCP) of similar magnitude, with an average DeltaCP value of 64.6 +/- 21.4 cal deg-1 mol-1. When this heat capacity value is used to compare significantly different transition enthalpies (DeltaHo) at a common reference temperature, Tref, we find DeltaHTref for duplex melting to be far less dependent on duplex type, base composition, or base sequence than previously believed on the basis of the conventional assumption of a near-zero value for DeltaCP. Similarly, our densimetric and acoustic measurements reveal that, at a given temperature, all the AT- and AU-containing duplexes studied here melt with nearly the same volume and compressibility changes. In the aggregate, our results, in conjunction with literature data, suggest a more unified picture for the thermodynamics of nucleic acid duplex melting. Specifically, when compared at a common temperature, the apparent large differences present in the literature for the transition enthalpies of different duplexes become much more compressed, and the melting of all-AT- and all-AU-containing duplexes exhibits similar volume and compressibility changes despite differences in sequence and conformation. Thus, insofar as thermodynamic properties are concerned, when comparing duplexes, the temperature under consideration is as important as, if not more important than, the duplex type, the base composition, or the base sequence. This general behavior has significant implications for our basic understanding of the forces that stabilize nucleic acid duplexes. This behavior also is of practical significance in connection with the use of thermodynamic databases for designing probes and for assessing the affinity and specificity associated with hybridization-based protocols used in a wide range of sequencing, diagnostic, and therapeutic applications.

Figure 1

(A) Excess heat capacity profiles for the poly[d(AT)]⋅poly[d(AT)] duplex at different salt concentrations ranging from 32 mM Na⁺ (curve 1) to 200 mM Na⁺ (curve 6). The difference in the pre- and posttransition baselines reflects a positive heat capacity change, ΔC_P, for the denaturation of the polynucleotide duplex. (B) Expanded scale representation of the excess heat capacity profiles of poly[d(AT)]⋅poly[d(AT)] at three of the salt concentrations to facilitate visualization of the heat capacity change. The average ΔC_P measured for all polynucleotides [ΔC_P = 64.6 ± 21.4 cal⋅K⁻¹⋅(mol of base pair)⁻¹] is indicated by the dotted lines.

Figure 2

Comparison of the published calorimetric enthalpy values for all RNA/RNA, RNA/DNA, and DNA/DNA polynucleotide duplexes plotted against their melting temperatures. The experimental data in Fig. 2 (■) are taken from the extensive collection of polynucleotide thermodynamic data compiled by Klump (3, 9). The solid line corresponds to the best fit straight line to these values. This line has a slope, ΔH/T_m (=ΔC_P), equal to 47 cal⋅ K⁻¹⋅(mol of base pair)⁻¹.

Figure 3

The dependence of volume change (ΔV) on temperature for the poly[d/r(A)]⋅poly[d/r(T/U)] system. Solid squares (■) correspond to the values listed in Table 2 determined by thermal denaturation of the polynucleotides; solid circles (●) correspond to values derived from the titration of poly[rA] with poly[rU] (Table 3); triangles correspond to published ΔV values reported by Wu and Macgregor (39) for poly[d(AT)]⋅poly[d(AT)] (▴) and poly[d(A)]⋅poly[d(T)] (Δ).

Figure 4

The change in adiabatic compressibility (ΔK_s) with temperature for the poly[d/r(A)]⋅poly[d/r(T/U)] system. Solid squares (■) correspond to the values listed in Table 2 determined by thermal denaturation of the polynucleotides; solid circles (●) correspond to data derived from the titration poly[r(A)] with poly[r(U)] at various temperatures (Table 3).