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 (⌬C P ) of similar magnitude, with an average ⌬C P value of 64.6 ؎ 21.4 cal deg ؊1 mol ؊1 . When this heat capacity value is used to compare significantly different transition enthalpies (⌬H o ) at a common reference temperature, T ref , we find ⌬H T ref 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 ⌬C P . Similarly, our densimetric and acoustic measurements reveal that, at a given temperature, all the ATand 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.Thermodynamic studies of nucleic acids have produced data of both fundamental and practical importance. On the fundamental side, such studies have provided insight into the nature and strength of the forces that stabilize nucleic acids in the myriad of structural states they can assume (1-7). On the practical side, such studies have produced databases that are used to predict the stability and selectivity of hybridization events required in a broad range of nucleic acid-based diagnostic and therapeutic protocols (3, 8-11, 19, 42-44).Three aspects of the current nucleic acid thermodynamic library that are conspicuously deficient are values for the heat capacity change(s), ⌬C P , the volume changes, ⌬V, and the compressibility changes, ⌬K s , which accompany nucleic acid conformational transitions. These deficiencies are of particular concern because ⌬C P , ⌬V, and ⌬K s provide unique insights into the role of solvent i...

We report a complete thermodynamic characterization of the stability and the melting behavior of an oligomeric DNA triplex. The triplex chosen for study forms by way of major-groove Hoogsteen association of an all-pyrimidine 15-mer single strand (termed y15) with a Watson-Crick 21-mer duplex composed ofone purine-rich strand (termed u21) and one pyrimidine-rich strand (termed y2l). We find that the near-UV CD spectrum of the triplex can be duplicated by the addition of the B-like CD spectrum ofthe isolated 21-mer duplex and the CD spectrum of the 15-mer single strand. Spectroscopic and calorimetric measurements show that the triplex (y15u21y21) melts by two well-resolved sequential transitions. The first transition (melting temperature, Tm, =300C) is pH-dependent and involves the thermal expulsion of the 15-mer strand to form the free duplex u21y21 and the free single strand y15. The second transition (Tm 650C) is pH-independent between pH 6 and 7 and reflects the thermal disruption ofthe u21-y21 Watson-Crick duplex to form the component single strands. The thermal stability of the y15-u21y21 triplex increases with increasing Na' concentration but is nearly independent of DNA strand concentration. Differential scanning calorimetric measurements at pH 6.5 show the triplex to be enthalpically stabilized by only 2.0 ± 0.1 kcal/mol of base triplets (1 cal = 4.184 J), whereas the duplex is stabilized by 6.3 ± 0.3 kcal/mol of base pairs. From the calorimetric data, we calculate that at 250C the y15-u21-y21 triplex is stabilized by a free energy of only 1.3 ± 0.1 kcal/mol relative to its component u21-y21 duplex and y15 single strand, whereas the 21-mer duplex is stabilized by a free energy of 17.2 ± 1.2 kcal/mol relative to its component single strands. The y15 single strand modified by methylation of cytosine at the C-S position forms a triplex with the u21y2I duplex, which exhibits enhanced thermal stability. The spectroscopic and calorimetric data reported here provide a quantitative measure of the influence of salt, temperature, pH, strand concentration, and base modification on the stability and the melting behavior of a DNA triplex. Such information should prove useful in designing third-strand oligonucleotides and in defining solution conditions for the effective use of triplex structure formation as a tool for modulating biochemical events.More than three decades have passed since the first description of polynucleotide triple helices (1). In the ensuing years a small number of investigators interested in the fundamental properties of nucleic acids have studied the structure (2) and physical properties (3-6) of triple-helical nucleic acids. Most of the work in this area has focused on triple helices composed of one polypurine strand and two polypyrimidine strands; however, triplexes of (polypurine)2 polypyrimidine also are known (7-10).The widely accepted structural model for polypurine (polypyrimidine)2 triple helices is based on x-ray fiber diffraction studies on poly(A) poly(U)2 (2, 11) and poly(dA)...

The abasic site in DNA may arise spontaneously, as a result of nucleotide base damage, or as an intermediate in glycosylase-mediated DNA-repair pathways. It is the most common damage found in DNA. We have examined the consequences of this lesion and its sequence context on DNA duplex structure, as well as the thermal and thermodynamic stability of the duplex, including the energetic origins of that stability. To this end, we incorporated a tetrahydrofuran abasic site analogue into a family of 13-mer DNA duplexes, wherein the base opposite the lesion (A, C, G, or T) and the base pairs neighboring the lesion (C.G or G.C) were systematically varied and characterized by a combination of spectroscopic and calorimetric techniques. The resulting data allowed us to reach the following conclusions: (i) the presence of the lesion in all sequence contexts studied does not alter the global B-form conformation characteristic of the parent undamaged duplex; (ii) the presence of the lesion induces a significant enthalpic destabilization of the duplex, with the magnitude of this effect being dependent on the sequence context; (iii) the thermodynamic impact of the lesion is dominated by the identity of the neighboring base pairs, with the cross strand partner base exerting only a secondary thermodynamic effect on duplex properties. In the aggregate, our data reveal that even in the absence of lesion-induced alterations in global structure, the abasic lesion can significantly alter the thermodynamic properties of the host duplex, with the magnitude of this impact being strongly dependent on sequence context.