Correlation of Tm and sequence of DNA duplexes with delta H computed by an improved empirical potential method

T_m values for 20 DNA duplexes with different repeating base sequences provided the data base for developing a rational and relatively simple methodology for computing apparent enthalpies for the helix → coil transitions of DNA helices, ΔH _calc. The computational variables and their range of acceptable values were selected on the basis of physically plausible arguments. Over 350,000 different combinations of the variables were tested for degree of fit. It was therby possible to find a combination giving a high degree of linear fit between T_m and ΔH _calc (correlation coefficient, 0.99), with T_m values deviating (on average) from the regression line by only ±2.17°C. Most of this uncertainty is attributed to experimental limitations, although computational approximations also contribute. With ΔH _calc for the melting of each of the unique complementary dinucleotide fragments computed by the method developed, it is possible to estimate T_m and (relative) ΔH _calc reliable for the melting of any particular DNA [with base pairs G(I)·C and A·T] given only its base sequence. The ΔH_calc values for the complementary dinucleotide fragments, together with statistical considerations, make it apparent that T_m of DNAs with repeating base sequence show only an approximate linear dependence on G·C content because A·T and G·G pairs do not contribute to helix stability independently of the base-pair sequence in which they occur. In fact, the nearestneighbor stacking interactions are so significant that certain complementary dinucleotide fragment sequences with 0,50, and 100% G·C content have the same stability.     

Polynucleotides. 13. Stoichiometric and thermodynamic studies of polynucleotide helices with non-complementary residues

Stoichiometry and thermodynamic properties of polyadenylate–polyuridylate helices containing varying proportions of near-randomly distributed non-complementary uridine residues were charactrized from an analysis of their mixing curves and melting profiles measured at 259 nm and at appropriate longer wavelength isochromic points. The noncomplementary residues in this homopolymer–copolymer system (in which the homopolymer has the capacity to readjust with respect to the residues with which it is in opposition) show absolute preference for an extrahelical conformation even when situated in … AAUAA … sequences and must occur therefore as single loops. As the frequency of extrahelical residues in creases, the electrostatic energy of these complexes becomes greater, and is particularly severe for the three-stranded helices. Thus, an adenyl-ate-uridylate copolymer containing 35.2 mole percent uridine residues does not form a three-stranded complex with polyuridylate even in 0.7M Na⁺at O°C. The imperfections introduced into the helix lattice by extrahelical residues decrease the cooperativity of thermal denaturation as well as T_m. However, for the helices with extrahelical residues in low frequency (～1 per helix turn) only small increases in concentration of charge-neutralizing ions are required to bring T_m to the level of their perfect analogs. Two-stranded helices with a higher density of extra helical residues (～5 per helix turn) show [Na⁺] dependence of T_m characteristic of perfect three-stranded helices. These findings together with the absence of an effect of these imperfections on the hypochromicity per base-pair suggest only minimal disruption of helix continuity or distortion of stacking interactions that normally in volve the base pairs or triplets.     

Position-dependent effects on stability in tricyclo-DNA modified oligonucleotide duplexes

A series of oligodeoxyribonucleotides and oligoribonucleotides containing single and multiple tricyclo(tc)-nucleosides in various arrangements were prepared and the thermal and thermodynamic transition profiles of duplexes with complementary DNA and RNA evaluated. Tc-residues aligned in a non-continuous fashion in an RNA strand significantly decrease affinity to complementary RNA and DNA, mostly as a consequence of a loss of pairing enthalpy ΔH. Arranging the tc-residues in a continuous fashion rescues T_m and leads to higher DNA and RNA affinity. Substitution of oligodeoxyribonucleotides in the same way causes much less differences in T_m when paired to complementary DNA and leads to substantial increases in T_m when paired to complementary RNA. CD-spectroscopic investigations in combination with molecular dynamics simulations of duplexes with single modifications show that tc-residues in the RNA backbone distinctly influence the conformation of the neighboring nucleotides forcing them into higher energy conformations, while tc-residues in the DNA backbone seem to have negligible influence on the nearest neighbor conformations. These results rationalize the observed affinity differences and are of relevance for the design of tc-DNA containing oligonucleotides for applications in antisense or RNAi therapy.     

Free energy of imperfect nucleic acid helices. 3. Small internal loops resulting from mismatches

Physical studies of enzymioally synthesized oligoribonucleotides of defined sequence are used to evaluate quantitatively the destabilizing influence of mismatched bases in a double helix. The series (A-)₄G(-C)_n(-U)₄, N = 1 to 6, exist as imperfect dimer helices when N is equal to or less than 4, and as monomolecular hairpin helices when N is 5 and 6. Internal loops become progressively more destabilizing as their size increases from 2 to 4 to 6 nucleotides resulting from 1, 2 and 3 consecutive mismatched base pairs. However, the stability of a helix will generally be greater if a given number of mismatched pairs occur consecutively rather than in isolation from one another.These data may be used for improved calculations of stability of RNA secondary structure, to estimate the frequency of structural fluctuations in a double helix and to assess the stability of modified polynucleotide helices. An unmodified double helix of one million randomly arranged base pairs should contain on the time average approximately 10 G.C and 500 A.U pairs in non-hydrogen bonded, unstacked conformations at 25 °C. Our estimate of the effect of mismatching on T_m values of high polymers is less precise because of the long temperature extrapolation required. However, we estimate that DNA or RNA treated with mutagens which interrupt up to 20% of the nucleotide pairs will show a drop of about 1.2 deg. C in melting temperature with each unit per cent of modification.     

The structure of triple-stranded G-2C polynucleotide helices.

The thermal stability of a new polynucleotide complex has been used to establish the hydrogen-bonding structure of three-stranded C-G·CH⁺ helices. In the Hoogsteen structure, the 8NH₂ group of 8NH₂GMP can form a third hydrogen bond to the CH⁺ strand, but in the alternative structure, the 8NH₂ group can form no interbase hydrogen bonds. For the new complex, 8NH₂GMP·2 poly(C), a transition temperature of 80°C is observed under conditions in which the corresponding complex formed with 5′-GMP has a T_m of 20°C. We conclude from this 60° elevation of transition temperature that a third hydrogen bond is formed by the 8NH₂ group and that the structure must have Hoogsteen bonding. In order to be compatible with this structure in regular helices formed by U,C copolymers, A·2U bonding would also have to have a Hoogsteen structure.     

The formation of catalytically competent enzyme–substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase

Human alkyladenine DNA glycosylase (AAG) protects DNA from alkylated and deaminated purine lesions. AAG flips out the damaged nucleotide from the double helix of DNA and catalyzes the hydrolysis of the N-glycosidic bond to release the damaged base. To understand better, how the step of nucleotide eversion influences the overall catalytic process, we performed a pre-steady-state kinetic analysis of AAG interaction with specific DNA-substrates, 13-base pair duplexes containing in the 7th position 1-N6-ethenoadenine (εA), hypoxanthine (Hx), and the stable product analogue tetrahydrofuran (F). The combination of the fluorescence of tryptophan, 2-aminopurine, and 1-N6-ethenoadenine was used to record conformational changes of the enzyme and DNA during the processes of DNA lesion recognition, damaged base eversion, excision of the N-glycosidic bond, and product release. The thermal stability of the duplexes characterized by the temperature of melting, T_m, and the rates of spontaneous opening of individual nucleotide base pairs were determined by NMR spectroscopy. The data show that the relative thermal stability of duplexes containing a particular base pair in position 7, (T_m(F/T)?T_m(εA/T)?T_m(Hx/T)?T_m(A/T)) correlates with the rate of reversible spontaneous opening of the base pair. However, in contrast to that, the catalytic lesion excision rate is two orders of magnitude higher for Hx-containing substrates than for substrates containing εA, proving that catalytic activity is not correlated with the stability of the damaged base pair. Our study reveals that the formation of the catalytically competent enzyme–substrate complex is not the bottleneck controlling the catalytic activity of AAG.     

Relative stabilities of triple helices composed of combinations of DNA, RNA and 2'-O-methyl-RNA backbones: chimeric circular oligonucleotides as probes.

Described is a systematic study of the effects of varied backbone structure on the stabilities of pyr.pur.pyr triple helices. The effects were measured using six circular 34 base oligonucleotides containing DNA (D), RNA (R) and/or 2'-O-methyl-RNA (M) residues designed to bind a complementary single-stranded purine target strand by triple helix formation. Eighteen different backbone combinations were studied at pH 5.5 and 7.0 by optical melting experiments and the results compared with the stabilities of the corresponding Watson-Crick duplexes. When the target purine strand is DNA, all circles form pH-dependent triple helical complexes which are considerably stronger than the duplexes alone. When RNA is the target, five of the nine complexes studied are of the pH-dependent triplex type and the other four complexes are not significantly stronger than the corresponding duplexes. The results are useful in the design of the highest affinity ligands for single- and double-stranded DNAs and RNAs and also point out novel ways to engender DNA- or RNA-selective binding.     

Parallel nucleic acid helices with hoogsteen base pairing: Symmetry and structure

Molecular structures for parallel DNA and RNA double helices with Hoogsteen pairing are proposed for the first time. The DNA helices have sugars in the C2′-endo region and the phosphodiester conformations are (trans, gauche^?), and the RNA helices have sugars in the C3′-endo region and the phosphodiester conformations are (gauche^?, gauche^?). A pseudorotational symmetry relates the two parallel strands of DNA helices and a screw symmetry relates the two strands of RNA helices, which have an associated tilt of the The conformational space of parallel helices with Hoogsteen base pairing, unlike the Watson-Crick duplex, is highly restricted due to the unique positioning of the symmetry axis in the former case. The features of the parallel double helix with Hoogsteen pairing are compared with the Watson-Crick duplex and the corresponding triple helix. © 1994 John Wiley & Sons, Inc.     

Conformational constraints of cyclopentane peptide nucleic acids facilitate tunable binding to DNA

We report a series of synthetic, nucleic acid mimics with highly customizable thermodynamic binding to DNA. Incorporation of helix-promoting cyclopentanes into peptide nucleic acids (PNAs) increases the melting temperatures (T_m) of PNA+DNA duplexes by approximately +5°C per cyclopentane. Sequential addition of cyclopentanes allows the T_m of PNA + DNA duplexes to be systematically fine-tuned from +5 to +50°C compared with the unmodified PNA. Containing only nine nucleobases and an equal number of cyclopentanes, cpPNA-9 binds to complementary DNA with a T_m around 90°C. Additional experiments reveal that the cpPNA-9 sequence specifically binds to DNA duplexes containing its complementary sequence and functions as a PCR clamp. An X-ray crystal structure of the cpPNA-9–DNA duplex revealed that cyclopentanes likely induce a right-handed helix in the PNA with conformations that promote DNA binding.     

A Parallel Stranded Linear DNA Duplex Incorporating dG · dC Base Pairs

Abstract

DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA · dT base pairs. We have substituted four dA · dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1 · D2) with dG · dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG · dC base pairs (ps-D5 · D6) is 10-16°C lower and the van't Hoff enthalpy difference Δ_vH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl₂) compared to that of ps-Dl · D2. Based on energy minimizations of a ps-[d(T₅GA₅) · d(A₅CT₅)] duplex using force field calculations we propose a model for the conformation of a trans dG · dC base pair in a ps helix.     

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 (H_GC?H_AT) 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 H_GC?H_AT 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 H_GC?H_AT, the average relative deviation (S) between DMC and normalized calorimetric profile, and the difference between their melting temperatures (T_cal?T_m) are weakly dependent on peculiarities of the multipeak fine structure of DMCs. At the same time, both the deviation S and difference (T_cal?T_m) 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 DNA base compositions from melting profiles in dilute buffers

Equations were determined for the dependency of the melting temperature (T_m) of DNA upon the logarithm of the sodium ion concentration, for four DNA samples of widely different base compositions (θ_GC). The slopes of these T_m versus log M equations wore found to decrease with increasing θ_{G C}of the samples. An empirical equation relating T_m, log M (Na⁺) and θ_{G C} was derived, which also accounts for differences in T_m versus log M slopes. Data from the literature for some synthetic polynucleotides and for the crab(Cancer pagarus) satellite poly AT are discussed in relation to the above finding. The changes in T_m versus log M slopes with θ_{G C} are interpreted in terms of changes in the thermodynamic parameters ΔS and ΔH with base composition.     

Raman studies of nucleic acids. VII. Poly A-poly U and poly G-poly C

Laser-excited Raman spectra of the double-helical complexes poly A·poly U and poly G·poly C are reported for ²H₂O and H₂O solutions. The spectra are discussed in relation to their use as quantitative reference spectra for determining the dependence of the Raman scattering of RNA on secondary structure. The Raman line at 815 cm^?1, due to the phosphodiester group, exhibits the same intrinsic intensity in spectra of poly A·poly U and poly G·poly C and is thus dependent only upon the amount of ordering of the helix and not on the kinds of nucleotides involved. The hypochromic Raman lines in spectra of poly A·poly U are identified and their intensity changes are determined quantitatively over the temperature range 32–85°C. Comparison of the spectra in the 1500–1750 cm^?1 region reveals that the Raman lines from carbonyl group vibrations of uracil are about sevenfold more intense than those of guanine and cytosine for both paired and unpaired states and will thus dominate the spectra of RNA. The Raman frequencies in this region are also compared with previously reported infrared frequencies and give evidence of being strongly perturbed by base-stacking interactions in the helices.     

Sequence and conformational effects on imino proton exchange in A.T- and A.U-containing DNA and RNA duplexes

¹H-nmr relaxation has been used to study the effect of sequence and conformation on imino proton exchange in adenine–thymine (A · T) and adenine–uracil (A · U) containing DNA and RNA duplexes. At low temperature, relaxation is caused by dipolar interactions between the imino and the adenine amino and AH2 protons, and at higher temperature, by exchange with the solvent protons. Although room temperature exchange rates vary between 3 and 12s^?1, the exchange activation energies (E_α) are insensitive to changes in the duplex sequence (alternating vs homopolymer duplexes), the conformation (B-form DNA vs A-form RNA), and the identity of the pyrimidine base (thymine vs uracil). The average value of the activation energy for the five duplexes studied, poly[d(A-T)], poly[d(A) · d(T)], poly[d(A-U)], Poly[d(A) · d(U)], and poly[r(A) · r(U)], was 16.8 ± 1.3 kcal/mol. In addition, we find that the average E_α for the A.T base pairs in a 43-base-pair restriction fragment is 16.4 ± 1.0 kcal/mol. This result is to be contrasted with the observation that the E_α of cytosine-containing duplexes depends on the sequence, conformation, and substituent groups on the purine and pyrimidine bases. Taken together, the data indicate that there is a common low-energy pathway for the escape of the thymine (uracil) imino protons from the double helix. The absolute values of the exchange rates in the simple sequence polymers are typically 3–10 times faster than in DNAs containing both A · T and G · C base pairs.     

Structural Studies of DNA Fragments: The G·T Wobble Base Pair in A,B and Z DNA; The G·A Base Pair in B-DNA

Abstract

The crystal structures of five double helical DNA fragments containing non-Watson-Crick complementary base pairs are reviewed. They comprise four fragments containing G·T base pairs: two deoxyoctamers d(GGGGCTCC) and d(GGGGTCCC) which crystallise as A type helices; a deoxydodecamer d(CGCGAATTTGCG) which crystallises in the B-DNA conformation; and the deoxyhexamer d(TGCGCG), which crystallises as a Z-DNA helix. In all four duplexes the G and T bases form wobble base pairs, with bases in the major tautomer forms and hydrogen bonds linking N1 of G with 02 of T and 06 of G with N3 of T. The X-ray analyses establish that the G·T wobble base pair can be accommodated in the A, B or Z double helix with minimal distortion of the global conformation. There are, however, changes in base stacking in the neighbourhood of the mismatched bases. The fifth structure, d(CGCGAATTAGCG), contains the purine purine mismatch G·A where G is in the anti and A in the syn conformation. The results represent the first direct structure determinations of base pair mismatches in DNA fragments and are discussed in relation to the fidelity of replication and mismatch recognition.     

Cooperative nonenzymic base recognition II. thermodynamics of the helix-coil transition of oligoadenylic + oligouridylic acids

The properties of oligonucleotide helices of adeuylic- and uridylic acid oligomers have been investigated by measurements of hypo-and hyperchromieity. High ionic strengths favor the formation of triple helices. Thus, the double helix-coil transition can be studied (without interference by triple helices) only at low ionic-strength. A “phase diagram” is given representing the T_m-values of the various transitions at different ionic strengths for the system A(pA)₁₇ + U(pU)₁₇. Oligonucleolides of chain lengths <8 always form both double and triple helices at the nucleotide concentrations required for base pairing. For this reason the double helix-coil transition without coupling of the triple helix equilibrium can only be measured for chain lengths higher than 7. Melting curves corresponding to this transition have been determined for chain lengths 8, 9, 10, 11, 14 and 18 at different concentrations. An increase in nucleotide concentration leads to an increase in melting temperature. The shorter the chain length the lower the T_m-value and the broader the helix-coil transition. The experimental transition curves have been analysed according to a staggering zipper model with consideration of the stacking of the adeuylic acid single strands and the electrostatic repulsion of tlip phosphate charges on opposite strands. The temperature dependence of the nucleation parameter has been accounted for by a slacking factor x. The stacking factor expresses the magnitude of the stacking enthalpy. By curve fitting xwas computed to be 0.7, corresponding to a stacking enthalpy of about S kcal/mole. The model described allows the reproduction of the experimental transition curves with relatively high accuracy. In an appendix the thermodynamic parameters of the stacking equilibrium of poly A and of the helix-coil equilibria of poly A + poly U at neutral pH are calculated (ΔH_A = ?7.9 kcal/mole for the poly A stacking and ΔH₁₂ = ?10.9 kcal/mole for the formation of the double helix from the randomly coiled single strands). A formula for the configurational entropy of polymers derived by Flory on the basis of a liquid lattice model is adapted to calculate the stacking entropies of adenylic oligomers.     

The intranuclear helices of Euplotes: Their substructure,localization, and apparent migration to the cytoplasm

Study of the fine structure of the macronucleus in Euplotes eurystomus, a ciliate protozoon, during various stages of the cell division cycle has yielded new information about intranuclear helices. They are frequently observed at the periphery of chromatin bodies or next to the nuclear envelope, and they appear to be a constituent of nucleoli. The fibril that forms a helix is about 11–15 nm thick, and torus profiles of helices cut in cross section are about 35 nm in diameter. In substructure the helix is composed of a thin strand 3–5 nm thick which is coiled to form the 11–15 nm fibril; so the helix is a super-coiled structure. The intranuclear helices are present in the macronucleus throughout the cell cycle. They do not show obvious changes of relative abundance nor changes of relative localization in the nucleus, with one exception: they were never observed in the diffuse zone of replication bands. Evidence is presented indicating that nuclear helices migrate to the cytoplasm through nuclear pores. Although the chemical composition of the Euplotes intranuclear helices is unknown, information in the literature on similar helices in Amoeba indicates that they contain RNA and not DNA. The observations on Euplotes helices are consistent with a concept of “packaged” RNA for transport to the cytoplasm.     

Chain length and oligonucleotide stability at high pressure

The effect of hydrostatic pressure on the helix-coil transition temperature (T_m) was measured for the DNA oligomers (dA)_n(dT)_n, where n = 11, 15, and 19, in 50 mM NaCl. The data were analyzed in light of previously published data for the polymer, poly(dA)·poly(dT) under the same conditions. As has been observed for DNA polymers, increasing the hydrostatic pressure led to an increase in the T_m of the oligomers; however, the effect of pressure diminished with decreasing chain length. The value of dT_m/dP decreased linearly with the inverse of the chain length varying from 3.15 × 10⁻²°C MPa⁻¹ for the polymer to 0.7 × 10⁻²°C MPa⁻¹ for the 11-mer. The two-state or van't Hoff enthalpy (ΔH_vH) of the helix-coil transition was obtained by analysis of the half-width of the thermal transition. As expected, ΔH_vH decreases with decreasing chain length. In contrast to the behavior of the polymer, poly(dA)·poly(dT), and (dA)₁₉(dT)₁₉, the ΔH_vH of the two shorter duplex oligonucleotides displayed a small pressure dependence dΔH_vH/dP≃−0.4 kJ MPa⁻¹ in both cases. The changes observed in the T_m and ΔH_vH were not sufficient to explain the magnitude of the chain-length dependence of the pressure effect. To interpret the large chain-length dependence of dT_m/dP, we propose that the terminal base pairs contribute a negative volume change to the helix-coil transition. Base pairs distant from the ends exhibit behavior characterized by the polymer where end effects are assumed to be negligible, i.e., a positive volume change for the helix-coil transition. The negative volume change of separating terminal bases may originate from the imperfect interactions these base pairs form with water due to the existence of several energetically equivalent conformations. This is reminiscent of one of the mechanisms proposed to be important in the pressure-induced dissociation of multimeric proteins into their constituent subunits. © 1996 John Wiley & Sons, Inc.     

Thermodynamic characterization of ethidium bromide binding to a unique site on yeast tRNAphe.

A self-consistent thermodynamic characterization of the binding of ethidium to yeast phenylalanine-specific tRNA at 25°C, pH 7.0, in 11 nM MgCl₂, 375 nM NaCl, and 25 mM sodium phosphate has been obtained. Two ethidium molecules bind per tRNA under these conditions. The stronger site has a dissociation constant equal to 1.9 ± 0.5 μM and ΔH_dis°′ = 12 ± 1 Kcal/mol, and the weaker sites has a dissociation constant equal to 24 ± 9 μM and ΔH_dis°′ = 8.9 ± 1.5 Kcal/mol. The average calorimetric ΔH_dis°′ for the to sites 10.6 ± 0.4 kcal/mol. The thermodynamics of binding to the stranger sites are most probably the thermodynamics of interaction between A·U (6) and A·U (7), the unique site identified by Jones and Kearns. The binding is enthalpically driven and classical hydrophobic interactions do not appear to be important in the binding reaction.     

Microcalorimetric Investigation of DNA,poly(dA)poly(dT) and poly[d(A-C)]poly[d(G-T)] Melting in the Presence of Water Soluble (Meso tetra (4 N oxyethylpyridyl) Porphyrin) and its Zn Complex

Abstract

Thermodynamic parameters of melting process (δH_m, T_m, δT_m) of calf thymus DNA, poly(dA)poly(dT) and poly(d(A-C))·poly(d(G-T)) were determined in the presence of various concentrations of TOEPyP(4) and its Zn complex. The investigated porphyrins caused serious stabilization of calf thymus DNA and poly poly(dA)poly(dT), but not poly(d(A-C))poly(d(G-T)). It was shown that TOEpyp(4) revealed GC specificity, it increased T_m of satellite fraction by 24°C, but ZnTOEpyp(4), on the contrary, predominately bound with AT-rich sites and increased DNA main stage T_m by 18°C, and T_m of poly(dA)poly(dT) increased by 40 °C, in comparison with the same polymers without porphyrin. ZnTOEpyp(4) binds with DNA and poly(dA)poly(dT) in two modes—strong and weak ones. In the range of r from 0.005 to 0.08 both modes were fulfilled, and in the range of r from 0.165 to 0.25 only one mode—strong binding—took place. The weak binding is characterized with shifting of T_m by some grades, and for the strong binding T_m shifts by ～ 30–40°C. Invariability of ΔH_m of DNA and poly(dA)poly(dT), and sharp increase of T_m in the range of r from 0.08 to 0.25 for thymus DNA and 0.01–0.2 for poly(dA)poly(dT) we interpret as entropic character of these complexes melting. It was suggested that this entropic character of melting is connected with forcing out of H₂O molecules from AT sites by ZnTOEpyp(4) and with formation of outside stacking at the sites of binding. Four-fold decrease of calf thymus DNA melting range width ΔT_m caused by increase of added ZnTO- Epyp(4) concentration is explained by rapprochement of AT and GC pairs thermal stability, and it is in agreement with a well-known dependence, according to which ΔT～T_GC-T_AT for DNA obtained from higher organisms (L. V. Berestetskaya, M. D. Frank-Kamenetskii, and Yu. S. Lazurkin. Biopolymers 13, 193–205 (1974)). Poly (d(A-C))poly(d(G-T)) in the presence of ZnTOEpyp(4) gives only one mode of weak binding. The conclusion is that binding of ZnTOEpyp(4) with DNA depends on its nucleotide sequence.