Theory of helix-coil transition on DNA-ligand complexes: the effect to two types of interaction of ligand on the parameters of transition

The effect of ligand interacting with native DNA by two types on the parameters of helix-coil transition in homopolymers is considered using the most probable distribution method (Yu.S. Lazurkin et al., Biopolymers 1970). It is shown that at a small relative concentration of ligand the melting enthalpy (delta H) of DNA may be obtained from the universal formula which contains only values directly known from the experiments. It is shown that the formula for the change of melting temperature and width of melting range depending on the total ligand concentration in solution is converted into the corresponding formulae which are defined for the case when only one type of interaction of ligand and DNA is considered.     

Theoretical and Experimental Study of DNA Helix-Coil Transition in Acidic and Alkaline Medium

Abstract

The theoretical approach to the calculation of the influence of selective binding of small ligands on DNA helix-coil transition has been described in the previous paper (Lando D.Yu., J. Biomol. Struct. Dyrt., (1994)). In the present paper that method is used for the study of DNA protonation and deprotonation in acidic and alkaline medium by theoretical analysis of pH effect on DNA heat denaturation.

The mechanism of DNA protonation in acidic medium and pK values of nucleotides are well known. It gave us an opportunity to check the theory without any fitting of pK values. A good agreement between experimental and calculated functions T_m(pH) and ΔT(pH) (melting temperature and melting range width) obtained for acidic medium proved the validity of the theory. However, for alkaline medium there was not even qualitative agreement when the agreed-upon mechanism of deprotonation was considered. Looking into the cause of the discrepancy, we have studied the DNA melting for different mechanisms of deprotonation by calculation of T_m(pH) and ΔT(pH). As a result, it has been established that the discrepancy is due to deprotonation of bonded GC base pairs of helical DNA regions (pK= 11).

It was shown that the early known protonation and newly found deprotonation of helical DNA essentially stabilised double helix in alkaline and acidic medium.     

A Theoretical Consideration of the Influence of Selective Binding of Small Ligands on DNA Helix-Coil Transition

Abstract

The influence of small ligands on DNA helix-coil transition is considered theoretically. The ligands are supposed to interact with DNA base pairs reversibly and selectively. The long-range ligand-ligand interactions are taken into account. Simple formulae are derived for calculation of melting curve alteration induced by ligand binding. The proposed theoretical approach requires neither knowledge of nucleotide sequence nor long computation time.     

Transition Metal Ligands as Novel DNA-Base Substitutes

Abstract

The base modified nucleoside dBP, carrying a non-hydrogen-bonding non-shape complementary base was incorporated into oligonucleotides (Brotschi, C.; Häberli, A.; Leumann C.J. Angew. Chem. Int. Ed. 2001, 40, 3012–3014). This base was designed to coordinate transition metal ions into well defined positions within a DNA double helix. Melting experiments revealed that the stability of a dBP: dBP base couple in a DNA duplex is similar to a dG: dC base pair even in the absence of transition metal ions. In the presence of transition metal ions, melting experiments revealed a decrease in duplex stability which is on a similar order for all metal ions (Mn²⁺, Cu²⁺, Zn²⁺, Ni²⁺) tested.     

Magnesium ion effect on the helix-coil transition of DNA

The effect of magnesium ions on the parameters of the DNA helix-coil transition has been studied for the concentration range 10^?6–10^?1M at the ionic strengths of 10^?3M Na⁺. Special attention has been given to the region of low ion concentrations and to the effect of polyvalent metallic impurities present in DNA. It has been shown that binding with Mg⁺⁺ increases the DNA stability, the effect being observed mainly in the concentration range 10^?6–10^?4M. At[Mg⁺⁺]>10^?2M the thermal stability of DNA starts to decrease. The melting range extends to concentrations ～10^?5M and then decreases to 7–8°C at the ion content of 10^?3M. Asymmetry of the melting curves is observed at low ionic strengths ([Na⁺] = 10^?3M) and [Mg⁺⁺] ? 10^?5M. The results, analyzed in terms of the statistical thermodynamic theory of double-stranded homopolymers melting in the presence of ligands, suggest that the effects observed might be due to the ion redistribution from denatured to native DNA. An experimental DNA–Mg⁺⁺ phase diagram has been obtained which is in good agreement with the theory. It has been shown that thermal denaturation of the system may be an efficient method for determining the ion-binding constants for both native and denatured DNA.     

Mixed Mode of Ligand-DNA Binding Results in S-Shaped Binding Curves

Abstract

S-shaped binding curves often characterize interactions of ligands with nucleic acid molecules as analyzed by different physicochemical and biophysical techniques. S-shaped experimental binding curves are usually interpreted as indicative of the positive cooperative interactions between the bound ligand molecules. This paper demonstrates that S-shaped binding curves may occur as a result of the “mixed mode” of DNA binding by the same ligand molecule. Mixed mode of the ligand-DNA binding can occur, for example, due to 1) isomerization or dimerization of the ligands in solution or on the DNA lattice, 2) their ability to intercalate the DNA and to bind it within the minor groove in different orientations. DNA- ligand complexes are characterized by the length of the ligand binding site on the DNA lattice (so-called “multiple-contact” model). We show here that if two or more complexes with different lengths of the ligand binding sites could be produced by the same ligand, the dependence of the concentration of the complex with the shorter length of binding site on the total concentration of ligand should be S-shaped. Our theoretical model is confirmed by comparison of the calculated and experimental CD binding curves for bis-netropsin binding to poly(dA-dT) poly(dA-dT). Bis-netropsin forms two types of DNA complexes due to its ability to interact with the DNA as monomers and trimers. Experimental S-shaped bis-netropsin-DNA binding curve is shown to be in good correlation with those calculated on the basis of our theoretical model. The present work provides new insight into the analysis of ligand-DNA binding curves.     

Theoretical calculations of the helix–coil transition of DNA in the presence of large,cooperatively binding ligands

Theoretical calculations are conducted on the helix–coil transition of DNA, in the presence of large, cooperatively binding ligands modeled after the DNA-binding proteins of current biological interest. The ligands are allowed to bind both to helx and to coil, to cover up any number of bases or base pairs in the complex, and to interact cooperatively with their nearest neighbors. The DNA is treated in the infinite homogeneous Ising model approximation, and all calculations are done by Lifson's method of sequence-generating functions. DNA melting curves are calculated by computer in order to expolore the effects on the transition of ligand size, binding constant, free activity, and ligand–ligand cooperativity. The calculations indicate that (1) at the same intrinsic free energy change per base pair of the complexes, small ligands, for purely entropic reasons, are more effective than are large ligands in shifting the DNA melting temperature; (2) the response of the DNA melting temperature to increased ligand binding constant K and/or free ligand activity L is adequately represented at high values of KL (but not at low KL) by a simple independent site model; (3) if curves are calculated with the total amount of added ligand remaining constant and the free ligand activity allowed to vary throughout the transition, biphasic melting curves can be obtained in the complete absence of ligand–ligand cooperativity. In an Appendix, the denaturation of poly[d(A-T)] in the presence of the drug, netropsin, is used to verify some features of the theory and to illustrate how the theory can be used to obtain numerical estimates of the ligand binding parameters from the experimental melting curves.     

Thermodynamic description of oligonucleotide self-association in DNA concatamer structures

A scheme of equilibrium formation of concatamers by two different oligonucleotides has been considered. It is shown that in the general case, the dependence of the concentration of oligonucleotide components on temperature cannot be found in analytical form. Therefore, it is impossible to find the thermodynamic parameters of concatamer formation (ΔH ⁰, ΔS ⁰) and melting temperatures by analyzing the profiles of thermal denaturation of oligonucleotide complexes. An algorithm for numerical solution of implicit dependences has been developed. A number of approaches are considered that simplify the analysis of heat denaturation curves for concatamer complexes. It is shown that the dependence of the efficiency of concatamerization on temperature can be described analytically when duplex fragments have close stability and there is no cooperativity at the oligonucleotide junction. In this case, the dependence of melting temperature on thermodynamic parameters and oligonucleotide concentration has the same form as in the case of the duplex structure formed by a pair of non-self-complementary oligonucleotides. The ability of various model approaches to describe the experimental curves of concatamer heat denaturation is evaluated. For concatamer structures used as signal amplifiers in DNA hybridization analysis, a function is introduced that shows the relative contribution of a concatamer of given length to the magnitude of signal amplification. The dependence of the maximum of this function on the concentration of oligonucleotides, the thermodynamic characteristics of their complexes, and temperature has been determined. It is shown by the gel retardation assay that the function of the length distribution of concatamers qualitatively correlates with the experimental dependences.     

Determination of Melting Sequences in DNA and DNA-Protein Complexes by Difference Spectra

A graphical formula is presented for determining the base ratio of melted DNA. By use of this formula, the composition of sequences which melt in different portions of the melting curves of Clostridium DNA, Escherichia coli DNA, and mouse DNA were determined. As the DNA melts, the per cent of adenine and thymine (AT) in the melted sequences decreases linearly with temperature. The average composition of sequences which melt in a given part of the melting curve is proportional to the base ratio of the DNA. The concentration and average composition of sequences were determined for three parts of the melting curves of the DNA samples, and a frequency distribution curve was constructed. The curve is symmetrical and has a maximum at about 56% AT. The distribution of GC-rich sequences on the E. coli chromosome was estimated by shearing, partially melting, and fractionating the DNA on hydroxylapatite. GC-rich sequences appear to occur every thousand base pairs, and have a maximum length of about 180 base pairs. The graphical formula was applied to the determination of the composition of sequences which melt in different parts of the melting curve of chromatin. Throughout the melting curve, the composition of the melting sequences is about 60% AT, which appears to suggest that relatively long sequences are melting simultaneously. Their melting temperature may be a function of the composition of the protein on different parts of the DNA. The problem of light scattering in DNA-protein and DNA was also investigated. A formula is presented which corrects for light scattering by relating the intensity of the scattered light to the rate of change of absorbance of DNA with wavelength.     

Correlation between percentage guanine-cytosine content and melting temperature of deoxyribonucleic acid

An explicit relation between the percentage (G + C)-content X_GC, and the melting temperature (T_m) of a natural DNA, is derived froma a statistical mechanics calculation with due consideration given to the base pair bonding and stacking interactions. The well-known Marmur-Doty empirical formula, linearly relating these two quantities, can now be understood in terms of the fundamental processes. We also propose a simpler experimental procedure for the determination of X_GC. It is similar to the melting temperature method, but we take only one reading at a prescribed temperature of any natural DNA in addition to the two normalization readings. It should be more sensitive for DNAs having X_GC ranging from 35 to 65%.     

Effect of ionic strength on the pKa of ligands bound to DNA

The pK_a of 3,8-diamino-6-phenyl-phenanthridine (DAPP), a nonquaternary analog of ethidium bromide, has been determined spectrophotometrically as a function of sodium ion concentration both free in solution and complexed to DNA. Unwinding angle determinations with this compound were determined with Col El DNA using ethidium bromide as a standard. The unwinding angle for DAPP was 24 ± 2° relative to 26° for ethidium, and this suggests that DAPP binds in a manner quite similar to ethidium and with no significant outside bound DAPP under these experimental conditions. Isobestic behavior was obtained on spectrophotometric pH titration above pH 5 as long as the ratio of DNA-phosphate to ligand was between 100 and 300 and the DNA phosphate concentration was approximately 0.01M or greater. The loss of isosbestic behavior which occurred below pH 5 is probably due to titration of the 8 amino group of the ligand complexed to DNA. To circumvent this problem, pK_a values and the extinction coefficient of the acidic species were both determined by a computer program using experimental data obtained above pH 5. The pK_a of the free compound has only a minor dependence on ionic strength, while the pK_a of the ligand bound to DNA in an intercalated complex depends strongly on the sodium ion concentration. The pK_a of the DAPP-DNA complex is a linear function of –log[Na⁺] as predicted by the ion-condensation theory of polyelectrolytes. It was determined that DAPP is essentially completely bound to DNA under the conditions of these experiments by (1) determination of apparent pK_a values as a function of total DNA concentration, (2) calculation of binding constants for the neutral species of DAPP, and (3) spectral analysis of the protonated and neutral species of DAPP bound to DNA relative to DAPP free in solution. These results support the ion-condensation theory; provide an independent method for measuring ψ*, the average number of counterions associated per phosphate of DNA in the intercalated conformation; and illustrate that there are no specific pH effects or absolute pK_a values for ligands bound to DNA, but only ionic-strength-dependent results.     

Fine structure of DNA melting curves.

Theoretical calculations predict that the differential melting curves for random polynucleotide sequences having lengths up to several tens of thousands of base pairs have a clear-cut fine structure. This structure appears in the form of multiple narrow peaks 0.3–0.4°C wide on the bell shaped main curve. The differential melting curves have different shapes for different specific sequences. The theory also predicts the disappearance of the fine structure when the length of the sequence increases and when circular, covalently closed DNA is considered instead of the open structure. The predictions of the theory were confirmed by the measurements of differential melting curves for open and covalently closed circular forms of DNA for PM2 phage (N = 10⁴ base pairs) and also for other phage DNA's of different length: T7 (N = 3.8 × 10⁴); S_D (N = 9.2 × 10⁴); T2 (N = 17 × 10⁴). It was shown that the effect of fine structure results mainly from the cooperative melting out of DNA regions 300–500 base pairs long.     

Long-Range Interactions between Ligands Bound to a DNA Molecule Give Rise to Adsorption with the Character of Phase Transition of the First Kind

Abstract

Influence of long-range interactions between ligands bound to DNA molecule on the character of their adsorption is studied using computer modeling. For this investigation, two calculation procedures are developed. They are based upon the method of the free energy minimum and on the partition function method. The both procedures demonstrate that in the case of a strong enough attraction between all the bound ligands their binding to DNA has the character of phase transition of the first kind. There is a break in the binding curve c(c₀) where c—relative concentration of bound ligands, c ₀—molar concentration of free ligands. The break occurs because there is an interval of central degrees of binding (～50% of the maximum c value) that are prohibited for individual DNA molecules. Such a transition might be caused by some types of DNA condensation. Attraction between the neighboring ligands only, adjacent or/and separated by double helix regions, does not cause this effect.     

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.     

Slow relaxational processes in the melting of linear biopolymers: A theory and its application to nucleic acids

We treat the problem of the mean time of complete separation of complementary chains of a duplex containing N base pairs. A combination of analytical and computer methods is used to obtain the exact solution in the form of a compact expression. This expression is used to analyze the limits of application of the equilibrium theory of helix–coil transition in oligo- and polynucleotides. It also allows the melting behavior of a biopolymer to be predicted when its melting is nonequilibrium. In the case of oligonucleotides for which the equilibrium melting takes place at a high value of the stability constant s, the general expression turns into the equation of Craig, Crothers, and Doty, used by them to determine the rate constant k_f of the growth of a helical region from temperature-jump experiments. For the case of fragmented DNA with N ～ 10², the melting process is shown to be completely nonequilibrium, and as a result, the observed melting temperature should be higher than that for the equilibrium. A simple equation is obtained that makes possible calculation of the real, “kinetic” melting temperature T_k. As N increases, the observed melting temperature should approach the equilibrium value, T_m. This analysis has explained quantitatively the peculiar chain-length dependence of the experimentally observed shift in the DNA melting temperature during fragmentation. A thorough analysis is given of the nonequilibrium effects in the melting process of long DNA molecules (N ? 10³). The main conclusion is that even in the presence of profound hysteresis phenomena, the melting profile observed on heating may differ only slightly from the equilibrium profile.     

Statistical Mechanical Approach for Predicting the Transition to Non-B DNA Structures in Supercoiled DNA

Abstract

Supercoiling causes global twist of DNA structure and the supercoiled state has wide influence on conformational transition. A statistical mechanical approach was made for prediction of the transition probability to non-B DNA structures under torsional stress. A conditional partition function was defined as the sum over all possible states of the DNA sequence with basepair 1 and basepair n being in B-form helix and a recurrence formula was developed which expressed the partition function for basepair n with those for less number of pairs. This new definition permits a quick enumeration of every configuration of secondary structures. Energetic parameters of all conformations concerned, involving B-form, interior loop, cruciform and Z-form, were included in the equation. The probability of transition to each non-B conformation could be derived from these conditional partition functions. For treatment of effects of superhelicity, supercoiling energy was considered, and a twist of each conformation was determined to minimize the supercoiling energy. As the twist itself affects the transition probability, the whole scheme of equations was solved by renormalization technique. The present method permits a simultaneous treatment of serveral types of conformations under a common torsional stress.

A set of energetic parameters of DNA secondary structures has been chosen for calculation. Some DNA sequences were submitted to the calculation, and all the sequences that we submitted gave stable convergence. Some of them have been investigated the critical supercoil density for the transition to non-B DNA structures. Even though the reliability of the set of parameters was not enough, the prediction of secondary structure transition showed good agreement with reported observation. Hence, the present algorithm can estimate the probability of local conformational change of DNA under a given supercoil density, and also be employed to predict some specific sequences in which conformational change is sensitive to superhelicity.     

Thermal stability and renaturation of DNA in dimethyl sulfoxide solutions: Acceleration of the renaturation rate

The thermal stability and renaturation kinetics of DNA have been studied as a function of dimethyl sulfoxide (DMSO) concentration. Increasing the concentration of DMSO lowers the melting temperature of DNA but results in an increased second-order renaturation rate. For example, in a DNA solution containing 0.20M NaCl, 0.01M Tris (pH 8.0), and 0.001M EDTA, the addition of 40% DMSO lowers the melting temperature of the DNA by 27°C and approximately doubles the optimal renaturation rate. The effect of DMSO on the renaturation rate is shown to be at least partially due to its effect on the solution dielectric constant and to be consistent with the polyelectrolyte counterion condensation theory of Manning [(1976) Biopolymers 15 , 1333–1343].     

Thermodynamics of DNA Containing Very Stable Chemically Modified Base Pairs

Abstract

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 homopolymers 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 _∞, 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) = (2ω-a) S (1- S ) and S = exp[ΔS(T∞-T)/(RT)] where ω is the number of strongly stabilized sites in the DNA chain, a is the number of DNA ends that contain a stabilized site, and ΔS, 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 ω=1, and n<0.1N for ω>1, where N is the number of ordinary base pairs in the DNA chain.     

Helix-coil transition in nucleoprotein. Effect of ionic strength on thermal denaturation of polylysine-DNA complexes

Thermal denaturation of direct-mixed and reconstituted polylysine–DNA complexes in 2.5 × 10^?4 M EDTA, pH 8.0 and various concentrations of NaCl has been studied. For both complexes, increasing ionic strength of the solution raises T_m, the melting temperature of free base pairs. The linear dependence of T_m on log Na⁺ indicates that the concept of electrostatic shielding on phosphate lattice of an infinitely long pure DNA by Na⁺ can be applied to short free DNA segments in a nucleoprotein. For a direct-mixed polylysine–DNA complex, the melting temperature of bound base pairs T_m′ remains constant at various ionic strengths. On the other hand, the T_m′ in a reconstituted polylysine–DNA complex is shifted to lower temperature at higher ionic strength. This phenomenon occurs for reconstituted complex with long polylysine of one thousand residues or short polylysine of one hundred residues. It is shown that such a decrease of T_m′ is not due to a reduction of coupling melting between free and bound regions in a complex when the ionic strength is raised. It is also not due to intermolecular or intramolecular change from a reconstituted to a direct-mixed complex. It is suggested that this phenomenon is due to structural change on polylysine-bound regions by ionic strength. It is suggested further that Na⁺ may replace water molecules and bind polylysine-bound regions in a reconstituted complex. Such a dehydration effect destabilizes these regions and lowers T_m′. This explanation is supported by circular dichroism (CD) results.     

87 The melting of DNA in the presence of meso-tetra-pyridyl porphyrins with different peripheral substituents

The influence of water-soluble cationic 3N- and 4N-pyridyl porphyrins with different peripheral substituents (oxyethyl, buthyl, allyl, and metallyl) on melting parameters of DNA has been studied. Results indicate that the presence of porphyrin changes the shape and parameters of DNA melting curve. The increase of porphyrins concentration results in the increase of the melting temperature (Tm) and the melting interval (ΔT) of DNA. At the porphyrin-DNA concentration ratio r?=?0.01, changes in the melting temperature have not been observed. The melting intervals almost do not change upon adding of the 4N-porphyrins, while the decrease of ΔT, in the presence of 3N-porphyrins, is observed. Because the intercalation binding mechanism occurs in GC-rich regions of DNA, we assume that 3N-porphyrins, intercalated in GC-rich regions, reduce the thermal stability of these sites, bringing them closer to the thermal stability of the AT-sites, which is the reason for the decrease in the melting interval. While at the relative concentration r?=?0.01 for 4-N porphyrins, already the external binding mechanism “turns on” and the destabilizing effect of porphyrins on GC-pairs compensates stabilizing effect on AT-pairs, as a result of which change in the melting of DNA upon complexation with these porphyrins is not observed. The decrease of the hypochromic effect also indicates the intercalation of investigated porphyrins in the DNA structure, which weakens the staking interaction of base pairs of DNA. The increase of the hypochromic effect of DNA upon binding with porphyrin depends on the type of peripheral substituents of the porphyrin. The results show that porphyrins with butyl and allyl substituents weaken staking interaction of base pairs less than porphyrins with other substituents. The largest change was observed for metallyl porphyrins. It can be the result of bulky peripheral substituents, which make significant local changes in DNA structure.