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Many factors that change the temperature position and interval of the DNA helix-coil transition often also alter the shape of multi-peak differential melting curves (DMCs). For DNAs with a multi-peak DMC, there is no agreement on the most useful definition for the melting temperature, Tm, and temperature melting width, ΔT, of the entire DNA transition. Changes in Tm and ΔT can reflect unstable variation of the shape of the DMC as well as alterations in DNA thermal stability and heterogeneity. Here, experiments and computer modeling for DNA multi-peak DMCs varying under different factors allowed testing of several methods of defining Tm and ΔT. Indeed, some of the methods give unreasonable "jagged" Tm and ΔT dependences on varying relative concentration of DNA chemical modifications (rb), [Na(+)], and GC content. At the same time, Tm determined as the helix-coil transition average temperature, and ΔT, which is proportional to the average absolute temperature deviation from this temperature, are suitable to characterize multi-peak DMCs. They give smoothly varying theoretical and experimental dependences of Tm and ΔT on rb, [Na(+)], and GC content. For multi-peak DMCs, Tm value determined in this way is the closest to the thermodynamic melting temperature (the helix-coil transition enthalpy/entropy ratio).

Many experiments demonstrate that regions with higher GC-content in natural DNAs unwind at higher temperatures adsorbing more heat than equivalently sized regions with lower GC-content. This simple observation implies that normalized calorimetric melting profiles (calorimetric cDMCs) will not be equivalent differential melting curves (DMCs). We propose simple expressions for long natural and random DNA sequences to reciprocally convert DMCs and corresponding calorimetric cDMCs. The expressions are confirmed by the Poland–Fixman–Freire method and an approach based upon mixtures of homopolymeric duplexes. Using these expressions and experimental calorimetric data, we demonstrate that the average relative deviation between DMC and cDMC is proportional to the temperature melting range of the helix-coil transition Δ T . Corresponding difference between melting temperatures is proportional to Δ T 2 . In general, sequence and ionic conditions influence the deviation through their effect on Δ T . On the basis of the developed approach, we propose a method to determine the thermodynamic melting temperature (ratio of calorimetric enthalpy and entropy of the helix-coil transition) for natural DNAs from optical DMCs without calorimetric experiments.

The Poland-Fixman-Freire formalism was adapted for modeling of calorimetric DNA melting profiles, and applied to plasmid pBR 322 and long random sequences. We studied the influence of the difference (HGC -HAT ) between the helix-coil transition enthalpies of AT and GC base pairs on the calorimetric melting profile and on normalized calorimetric melting profile. A strong alteration of DNA calorimetrical profile with HGC -HAT was demonstrated. In contrast, there is a relatively slight change in the normalized profiles and in corresponding ordinary (optical) normalized differential melting curves (DMCs). For fixed HGC -HAT , the average relative deviation (S) between DMC and normalized calorimetric profile, and the difference between their melting temperatures (Tcal -Tm ) are weakly dependent on peculiarities of the multipeak fine structure of DMCs. At the same time, both the deviation S and difference (Tcal -Tm ) enlarge with the temperature melting range of the helix-coil transition. It is shown that the local deviation between DMC and normalized calorimetric profile increases in regions of narrow peaks distant from the melting temperature.

It is generally accepted that DNA conserves its secondary structure after a freeze-thaw cycle. A negligible amount of degradation occurs after this procedure. Degradation becomes appreciable only after multiple cycles of freezing and thawing. In this study, we have found that a single freeze-thaw cycle in alkaline medium (pH>or=10.8) gives rise to denaturation of calf thymus DNA, although the melting temperature of intact DNA in the solution used for the freeze-thaw experiments is higher than 60 degrees C. The degree of denaturation is almost independent of the regime of freezing. The melting curve obtained after DNA is frozen at -2 degrees C and then thawed is almost the same as after a freezing carried out in liquid nitrogen (-196 degrees C). However, incubation in the same solution at 0 degrees C for 24 hours without freezing does not give rise to any denaturation. The degree of denaturation caused by freezing increases with pH (if pH>or=10.8) and decreases with Na2CO3 concentration at fixed pH and [Na+], although Na2CO3 decreases the melting temperature of intact DNA. A preliminary treatment of DNA with cisplatin or transplatin (0.01 Pt atoms per nucleotide) gives rise to a full recovery of the DNA secondary structure after freezing and thawing similar to what occurs after heating DNA to 100 degrees C and quick cooling. Possible mechanisms that may cause DNA denaturation during a freeze-thaw cycle in alkaline medium are discussed.

A computer modeling of thermodynamic properties of a long DNA of N base pairs that includes omega interstrand crosslinks (ICLs), or omega chemical modifications involving one strand (monofunctional adducts, intrastrand crosslinks) has been carried out. It is supposed in our calculation that both types of chemical modifications change the free energy of the helix-coil transition at sites of their location by deltaF. The value deltaF0 corresponds to stabilization, i.e., to the increase in melting temperature. It is also taken into account that ICLs form additional loops in melted regions and prohibit strand dissociation after full DNA melting. It is shown that the main effect of interstrand crosslinks on the stability of long DNA's is caused by the formation of additional loops in melted regions. This formation increases DNA melting temperature (Tm) much stronger than replacing omega base pairs of AT type with GC. A prohibition of strand dissociation after crosslinking, which strongly elevates the melting temperature of oligonucleotide duplexes, does not influence melting behavior of long DNA's (Nor=1000 bp). As was demonstrated earlier for the modifications involving one or the other strand, the dependence of the shift of melting temperature deltaTm on the relative number of modifications r=omega/(2N) is a linear function for any deltaF, and deltaTm(r) identical with 0 for the ideal modifications (deltaF=0). We have shown that deltaTm(r) is the same for periodical and random distribution if the absolute value of deltaF is less 2 kcal. The absolute value of deltaTm(r) at deltaF2 kcal and deltaF-2 kcal is higher for periodical distribution. For interstrand crosslinks, the character of the dependence deltaTm(r) is quite different. It is nonlinear, and the shape of the corresponding curve is strongly dependent on deltaF. For "ideal" interstrand crosslinks (deltaF=0), the function deltaTm(r) is not zero. It is monotone positive nonlinear, and its slope decreases with r. If r0.004, then the entropy stabilizing effect of interstrand crosslinking itself exceeds the influence of a distortion of the double helix at sites of their location. The resulting deltaTm(r) is positive even in the case of the infinite destabilization at sites of the ICLs (deltaF---infinity). In general, stabilizing influence of interstrand crosslinks is almost fully compensated for by local structural distortions caused by them if 0r0.01 and -infinitydeltaFapproximately -5 kcal per mole of ICLs. The absolute value of deltaTm(r) in this case is approximately 10 times lower than for ideal ICLs as well as for local destabilization without crosslinking. Interstrand crosslinks formed by cisplatin do not change the melting temperature of long DNA's because of this compensation effect.

Addition of Na(2)CO(3) to almost salt-free DNA solution (5.10(-5)M EDTA, pH=5.7, T(m)=26.5 degrees C) elevates both pH and the DNA melting temperature (T(m)) if Na(2)CO(3) concentration is less than 0.004 M. For 0.004 M Na(2)CO(3), T(m)=58 degrees C is maximal and pH=10.56. Further increase in concentration gives rise to a monotonous decrease in T(m) to 37 degrees C for 1M Na(2)CO(3) (pH=10.57). Increase in pH is also not monotonous. The highest pH=10.87 is reached at 0.04 M Na(2)CO(3) (T(m)=48.3 degrees C). To reveal the cause of this DNA destabilization, which happens in a narrow pH interval (10.56/10.87) and a wide Na(2)CO(3) concentration interval (0.004/1M), a procedure has been developed for determining the separate influences on T(m) of Na(+), pH, and anions formed by Na(2)CO(3) (HCO(3)(-) and CO(3)(2-)). Comparison of influence of anions formed by Na(2)CO(3) on DNA stability with Cl(-) (anion inert to DNA stability), ClO(4)(-) (strong DNA destabilizing "chaotropic" anion) and OH(-) has been carried out. It has been shown that only Na(+) and pH influence T(m) in Na(2)CO(3) solution at concentrations lower than 0.001 M. However, the T(m) decrease with concentration for [Na(2)CO(3)]>/=0.004 M is only partly caused by high pH=10.7. Na(2)CO(3) anions also exert a strong destabilizing influence at these concentrations. For 0.1M Na(2)CO(3) (pH=10.84, [Na(+)]=0.2M, T(m)=42.7 degrees C), the anion destabilizing effect is higher 20 degrees C. For NaClO(4) (ClO(4)(-) is a strong "chaotropic" anion), an equal anion effect occurs at much higher concentrations approximately 3M. This means that Na(2)CO(3) gives rise to a much stronger anion effect than other salts. The effect is pH dependent. It decreases fivefold at neutral pH after addition of HCl to 0.1M Na(2)CO(3) as well as after addition of NaOH for pH greater than 11.2.

DNA chemical modifications caused by the binding of some antitumor drugs give rise to a very strong local stabilization of the double helix. These sites melt at a temperature that is well above the melting temperatures of ordinary AT and GC base pairs. In this work we have examined the melting behavior of DNA containing very stable sites. Analytical expressions were derived and used to evaluate the thermodynamic properties of homopolymer DNA with several different distributions of stable sites. The results were extended to DNA with a heterogeneous sequence of AT and GC base pairs. The results were compared to the melting properties of DNA with ordinary covalent interstrand cross-links. It was found that, as with an ordinary interstrand cross-link, a single strongly stabilized site makes a DNA's melting temperature (T(m)) independent of strand concentration. However in contrast to a DNA with an interstrand cross-link, a strongly stabilized site makes the DNA's T(m) independent of DNA length and equal to T(infinity), the melting temperature of an infinite length DNA with the same GC-content and without a stabilized site. Moreover, at a temperature where more than 80% of base pairs are melted, the number of ordinary (non-modified) helical base pairs (n) is independent of both the DNA length and the location of the stabilized sites. For this condition, n(T) = (2 omega-a)S/(1-S) and S = exp[DeltaS(T(infinity)-T)/(RT)] where omega is the number of strongly stabilized sites in the DNA chain, a is the number of DNA ends that contain a stabilized site, and DeltaS, T, and R are the base pair entropy change, the temperature, and the universal gas constant per mole. The above expression is valid for a temperature interval that corresponds to n0.2N for omega=1, and n0.1N for omega1, where N is the number of ordinary base pairs in the DNA chain.

Covalent and strong coordination binding to DNA of a large number of antitumour drugs and other compounds leads to interstrand cross-link formation. To investigate cross-link influence on double helix stability, two methods are developed for the calculation of melting curves. The first method is based on Poland's approach. It requires computer time proportional to u.N, where u is the average distance (in base pairs) between neighboring cross-links and N is the number of base pairs in the DNA chain. The method is more suitable when u is not large, and small loops formed by interstrand cross-links in melted regions strongly affect DNA melting. The computer time for the second method, based on the Fixman-Freire approach, does not depend on the number of cross-links and is proportional to I.N (I is the number of exponential functions used for a decomposition of the loop entropy factor). It is more appropriate when N and u are large, and therefore particular values of the entropy factors of small loops do not influence DNA melting behavior.

Binding of the antitumor compound cisplatin to DNA locally distorts the double helix. These distortions correlate with a decrease in DNA melting temperature (Tm). However, the influence of cisplatin on DNA stability is more complex because it decreases the DNA charge density. In this way, cisplatin increases the melting temperature and partially compensates for the destabilizing influence of structural distortions. The stabilization is stronger at low Na+ ion concentration. Due to this compensation, the total decrease in the DNA melting temperature after cisplatin binding is much lower than the decrease caused by the distortions themselves, especially at low [Na+]. It is shown in this study that, besides Na+ concentration, pH also strongly influences the value of a change in the melting temperature caused by cisplatin. In alkaline medium (pH=10.5-10.8), a fall in the melting temperature caused by platination is enhanced several times with respect to neutral medium. Such a stronger drop in Tm is explained by a decrease in pK values of base pairs caused by lowering the charge density under platination that facilitates proton release. At neutral pH, the proton release is low for both control and platinated DNA and does not influence the melting behavior. Therefore, lowering in the charge density under platination, besides stabilization, gives additional destabilization just in alkaline medium. Destabilization caused by structural distortions due to this pH induced compensation of stabilizing effect is more pronounced. In the presence of carbonate ion, destabilization caused by high pH value is strengthened. As a decrease in DNA charge density, interstrand crosslinking caused by cisplatin also increases the DNA stability due to loss in the entropy of the melted state. However, computer modeling of DNA stability demonstrates that interstrand crosslinks formed by cisplatin do not stabilize long DNA. It is shown that the increase in Tm caused by interstrand crosslinking itself is compensated for by a local destabilization of the double helix at the sites of location of interstrand crosslinks formed by cisplatin.

DNA interstrand cross-links are usually formed due to bidentate covalent or coordination binding of a cross-linking agent to nucleotides of different strands. However interstrand linkages can be also caused by any type of chemical modification that gives rise to a strong local stabilization of the double helix. These stabilized sites conserve their helical structure and prevent local and total strand separation at temperatures above the melting of ordinary AT and GC base pairs. This local stabilization makes DNA melting fully reversible and independent of strand concentration like ordinary covalent interstrand cross-links. The stabilization can be caused by all the types of chemical modifications (interstrand cross-links, intrastrand cross-links or monofunctional adducts) if they give rise to a strong enough local stabilization of the double helix. Our calculation demonstrates that an increase in stability by 25 to 30 kcal in the free energy of a single base pair of the double helix is sufficient for this "cross-linking effect" (i.e. conserving the helicity of this base pair and preventing strand separation after melting of ordinary base pairs). For the situation where there is more then one stabilized site in a DNA duplex (e.g., 1 stabilized site per 1000 bp), a lower stabilization per site is sufficient for the "cross-linking effect" (18 - 20 kcal). A substantial increase in DNA stability was found in various experimental studies for some metal-based anti-tumor compounds. These compounds may give rise to the effect described above. If ligand induced stabilization is distributed among several neighboring base pairs, a much lower minimum increase per stabilized base pair is sufficient to produce the cross-linking effect (1 bp- 24.4 kcal; 5 bp- 5.3 kcal; 10 bp- 2.9 kcal, 25 bp- 1.4 kcal; 50 bp- 1.0 kcal). The relatively weak non-covalent binding of histones or protamines that cover long regions of DNA (20- 40 bp) can also cause this effect if the salt concentration of the solution is sufficiently low to cause strong local stabilization of the double helix. Stretches of GC pairs more than 25 bp in length inserted into poly(AT) DNA also exhibit properties of stabilizing interstrand cross-links.

A theoretical method is developed for calculation of melting curves of covalent complexes of DNA with antitumor drugs. The method takes into account all the types of chemical modifications of the double helix caused by platinum compounds and DNA alkylating agents: 1) monofunctional adducts bound to one nucleotide; 2) intrastrand cross-links which appear due to bidentate binding of a drug molecule to two nucleotides that are included into the same DNA strand; 3) interstrand cross-links caused by bidentate binding of a molecule to two nucleotides of different strands. The developed calculation method takes into account the following double helix alterations at sites of chemical modifications: 1) a change in stability of chemically modified base pairs and neighboring ones, that is caused by all the types of chemical modifications; 2) a change in the energy of boundaries between helical and melted regions at sites of chemical modification (local alteration of the factor of cooperativity of DNA melting), that is caused by all the types of chemical modifications, too; 3) a change in the loop entropy factor of melted regions that include interstrand cross-links; 4) the prohibition of divergence of DNA strands in completely melted DNA molecules, which is caused by interstrand cross-links only. General equations are derived, and three calculation methods are proposed to calculate DNA melting curves and the parameters that characterize the helix-coil transition.

In the previous paper (D.Y. Lando, J. Biomol. Struct. Dynam, 15, 129-140 (1997)) the melting of cross-linked DNA with N base pairs and omega interstrand cross-links has been considered theoretically. In the present study on the basis of these results, two simple schemes are developed for the computation of melting curves of cross-linked DNA. The investigation of influence of interstrand linking on DNA stability has been carried out by computer simulation. It is shown that the relative concentration of cross-links, CCT = omega/N, their distribution along a DNA molecule, and particular values of the entropy factors of small loops formed by cross-links in melted regions strongly affect the DNA melting temperature, Tm. On the contrary, for DNA without cross-links, a ten-fold increase or decrease in the entropy factors of small loops does not cause the Tm variation. The comparison of the results of calculation with experimental data suggests that the majority of types of cross-link neither maintain ordered parallel orientation of bases in melted regions nor increase considerably the thermostability of cross-linked base pairs. Four different ways of influence of interstrand cross-linking on the DNA double helix stability are considered. It is shown that cross-linking significantly enhances the influence of single strand stiffness in melted regions on DNA melting behavior.