Relationship between calorimetric profiles and differential melting curves for natural DNAs.

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 ΔT. Corresponding difference between melting temperatures is proportional to ΔT2. In general, sequence and ionic conditions influence the deviation through their effect on ΔT. 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.

Estimation of the diversity between DNA calorimetric profiles, differential melting curves and corresponding melting temperatures.

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.

Determination of melting temperature and temperature melting range for DNA with multi-peak differential melting curves.

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).

Comparative thermal and thermodynamic study of DNA chemically modified with antitumor drug cisplatin and its inactive analog transplatin.

Antitumor activity of cisplatin is exerted by covalent binding to DNA. For comparison, studies of cisplatin-DNA complexes often employ the very similar but inactive transplatin. In this work, thermal and thermodynamic properties of DNA complexes with these compounds were studied using differential scanning calorimetry (DSC) and computer modeling. DSC demonstrates that cisplatin decreases thermal stability (melting temperature, Tm) of long DNA, and transplatin increases it. At the same time, both compounds decrease the enthalpy and entropy of the helix-coil transition, and the impact of transplatin is much higher. From Pt/nucleotide molar ratio rb=0.001, both compounds destroy the fine structure of DSC profile and increase the temperature melting range (ΔT). For cisplatin and transplatin, the dependences δTm vs rb differ in sign, while δΔT vs rb are positive for both compounds. The change in the parameter δΔT vs rb demonstrates the GC specificity in the location of DNA distortions. Our experimental results and calculations show that 1) in contrast to [Pt(dien)Cl]Cl, monofunctional adducts formed by transplatin decrease the thermal stability of long DNA at [Na(+)]>30mM; 2) interstrand crosslinks of cisplatin and transplatin only slightly increase Tm; 3) the difference in thermal stability of DNA complexes with cisplatin vs DNA complexes with transplatin mainly arises from the different thermodynamic properties of their intrastrand crosslinks. This type of crosslink appears to be responsible for the antitumor activity of cisplatin. At any [Na(+)] from interval 10-210mM, cisplatin and transplatin intrastrand crosslinks give rise to destabilization and stabilization, respectively.

Temporal behavior of DNA thermal stability in the presence of platinum compounds. Role of monofunctional and bifunctional adducts.

Penetrating into cell nuclei, antitumor drug cisplatin sequentially forms various intermediate and final adducts destroying local DNA structure. The demonstrated disappearance of the fine structure of melting curve of long DNAs along with a strong decrease in melting enthalpy conforms to the structural impact. However, the negative thermal effect (δT(m)) caused by cisplatin is relatively small if neutral medium is used in melting experiments. Cisplatin's inactive analogs transplatin and diethylenetriaminechloroplatinum {Pt[(dien)Cl]Cl} also distort DNA structure but their thermal effect is even positive. We have found that the use of alkaline medium in melting experiments strengthens the negative thermal effect for cisplatin. For transplatin and Pt[(dien)Cl]Cl, the thermal effect becomes negative that makes it qualitatively consistent with structural distortions. Those changes are explained by elimination of nonspecific electrostatic stabilization of DNA under platination. Additionally, alkaline medium fixes intermediate states of DNA platination and makes them stable against heating. These results allowed us to monitor δT(m) under binding of platinum compounds to DNA and their further transformation. The kinetic and thermal characteristics of monofunctional and bifunctional adducts were evaluated. It has been demonstrated that monofunctional adducts of cisplatin, transplatin and Pt[(dien)Cl]Cl produce approximately the same thermal destabilization. Cisplatin intrastrand crosslinks cause a two-fold stronger thermal destabilization than its monofunctional adducts. The value of δT(m) for cisplatin's final adducts is ten times larger than for transplatin. This difference mainly comes from the much stronger thermal destabilizing power of cisplatin's intrastrand crosslinks, which are responsible for antitumor activity of this compound.

Thermal stability of DNA with interstrand crosslinks.

Although many anticancer drugs exert their biological activity by forming DNA interstrand crosslinks (ICLs), the thermodynamics of biologically relevant long crosslinked DNAs has not been intensively studied in contrast to short duplexes. Here, we carry out computer modeling of the shift of melting temperature of long DNAs caused by ICLs taking into account crosslinking effect in itself and concomitant local alterations in the free energy (δG) of the helix-coil transition at sites of ICLs. Depending on δG, DNA interstrand crosslinks at per nucleotide concentration r = 0.05 can change the melting temperature by value from -17 to +47°C, and the influence weakly depends on DNA sequence and GC content. A change in melting temperature caused by introduction of interstrand crosslinking in modified DNA at sites of modifications also depends on δG and varies from 0 to +12°C. Comparison with experiment for the three platinum crosslinking compounds demonstrates utility of the theoretical method for understanding how crosslinking compounds can influence the melting behavior. On the basis of the method, interdependence of local distortions and crosslinking in itself was studied for thermal effect of ICLs. A method for evaluating the nature of the structural alteration that produces a change in thermal stability for short crosslinked DNA is also proposed. The methods can be used for comparative thermodynamic characterization of various DNA crosslinking agents.

DNA denaturation under freezing in alkaline medium.

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.

Melting of crosslinked DNA: VI. Comparison of influence of interstrand crosslinks and other chemical modifications formed by antitumor compounds on DNA stability.

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 deltaF>0 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 (N>or=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 deltaF>2 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 r<0.004, 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 0

Distribution of unselectively bound ligands along DNA.

Unselective and reversible adsorption of ligands on DNA for a model of binding proposed by Zasedatelev, Gursky, and Volkenshtein is considered. In this model, the interaction between neighboring ligands located at the distance of i binding centers is characterized by the statistical weight ai. Each ligand covers L binding centers. For this model, expressions for binding averages are represented in a new simple form. This representation is convenient for the calculation of the fraction of inter-ligand distances of i binding centers fd(i) and the fraction of binding centers included in the distances of i binding centers fbc(i) for various types of interaction between bound ligands. It is shown that, for non-cooperative binding, contact cooperativity and long-range cooperativity, the fraction of the zero inter-ligand distance fd(0) is maximal at any relative concentration of bound ligands (r). Calculations demonstrate that, at low r, fd(0) approximately r.ao, and fd(i) approximately r at 11/r-L, then fd(i) rapidly decreases with i at any r for all types of inter-ligand interaction. At high ligand concentration (r is close to rmax=L(-1)), fd(0) is close to unity and fd(i) rapidly decreases with i for any type of inter-ligand interaction. For strong contact cooperativity, fd(0) is close to unity in a much lager r interval ((0.5-1).rmax), and fd(1) approximately ao(-1) at r approximately 0.5.rmax. In the case of long-range interaction between bound ligands, the dependence fd(i) is more complex and has a maximum at i approximately (1/r-L)1/2 for anti-cooperative binding. fbc(i) is maximal at i approximately 1/r-L for all types of binding except the contact cooperativity. A strong asymmetry in the influence of contact cooperativity and anticooperativity on the ligand distribution along DNA is demonstrated.

Matrix formalism for site-specific binding of unstructured proteins to multicomponent lipid membranes.

We describe a new approach to calculate the binding of flexible peptides and unfolded proteins to multicomponent lipid membranes. The method is based on the transfer matrix formalism of statistical mechanics recently described as a systematic tool to study DNA-protein-drug binding in gene regulation. Using the energies of interaction of the individual polymer segments with different membrane lipid species and the scaling corrections due to polymer looping, we calculate polymer adsorption characteristics and the degree of sequestration of specific membrane lipids. The method is applied to the effector domain of the MARCKS (myristoylated alanine rich C kinase substrate) protein known to be involved in signal transduction through membrane binding. The calculated binding constants of the MARCKS(151-175) peptide and a series of related peptides to mixed PC/PS/PIP2 membranes are in satisfactory agreement with in vitro experiments.

Compensation of DNA stabilization and destabilization effects caused by cisplatin is partially disturbed in alkaline medium.

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.

Na2CO3 influence on DNA double helix stability: strong anion destabilizing 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.

Thermodynamics of DNA containing very stable chemically modified base pairs.

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 n<0.2N for omega=1, and n<0.1N for omega>1, where N is the number of ordinary base pairs in the DNA chain.

Melting of cross-linked DNA v. cross-linking effect caused by local stabilization of the double helix.

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.

Modeling of DNA condensation and decondensation caused by ligand binding.

A theoretical method for computer modeling of DNA condensation caused by ligand binding is developed. In the method, starting (s) and condensed (c) states are characterized by different free energies for ligand free DNA (F(s) and F(c) respectively), ligand binding constants (K(s) and K(c)) and stoichiometry dependent parameters (c(sm) and c(cm) - maximum relative concentration of bound ligands (per base pair) for starting and condensed state respectively). The method allows computation of the dependence of the degree of condensation (the fraction of condensed DNA molecules) on ligand concentration. Calculations demonstrate that condensation transition occurs under an increase in ligand concentration if F(s) < F(c) (i.e. S(sc) = exp [- (F(c) - F(s)) / (RT)], the equilibrium constant of the s-c transition, is low (S(sc) << 1)) and K(s) < K(c). It was also found that condensation is followed by decondensation at high ligand concentration if the condensed DNA state provides the number of sites for ligand binding less than the starting state (c(sm) > c(cm)). A similar condensation-decondensation effect was found in recent experimental studies. We propose its simple explanation.

Short-range interactions and size of ligands bound to DNA strongly influence adsorptive phase transition caused by long-range interactions.

Long-range interaction between all the ligands bound to DNA molecule may give rise to adsorption with the character of phase transition of the first kind (D. Y. Lando, V. B. Teif, J. Biomol. Struct & Dynam. 18, 903-911 (2000)). In this case, the binding curve, c(c(o)), is characterized by a sudden change of the relative concentration of bound ligands ((c)) at a critical concentration of free (unbound) ligands, c(o)=c(ocr), from a low c value to a high one where c(o) is molar concentration of free ligands. Such a transition might be caused by some types of DNA condensation or changes in DNA topology. For the study of the conditions necessary for adsorption with the character of phase transition, a calculation procedure based on the method of the free energy minimum is developed. The ligand size and two types of interactions between ligands adsorbed on DNA molecule are taken into consideration: long-range interaction between all the ligands bound to DNA and contact interactions between neighboring ligands. It was found that a) Stronger long-range interaction is required for longer ligands to induce phase transition that is occurred at greater c(ocr) values; b) Pure contact interaction between neighboring ligands can not itself initiate phase transition. However contact cooperativity strongly decreases the threshold value of energy of long-range interaction necessary to give rise to the transition.