Three-State Diagram for DNA

Abstract

Experimental phase diagrams (A form, B form, Coil) were built in the coordinates (a, alcohol fraction: T, temperature) for the natural DNAs and poly d(A-T). The main parameter of the B-A transition,—cooperativity length, v _o, was estimated by the slopes of the branches A-B, A-Coil, B-Coil in the vicinity of the triple point: v _o +AD0- 10-20 base pairs, which corresponds to the energy for the B/A junction of 1.2–1.8 kcal/mol.

We discovered two new effects which are due to the coexistence of the three different conformations in one polymeric molecule: an increase in the melting temperature above that for the ‘ideal’ triple point (i.e. for the case of the ideal phase transitions); a widening of the melting curve within the B-A transition range.

The physics of these phenomena is discussed.     

A Study of DNA Melting in Concentrated Water-Alcohol Solutions

Abstract

The DNA melting profiles with high resolution have been studied for conditions corresponding to the B and A conformations of DNA in water-alcohol solutions. The melting profiles of the A-form and B-form DNA, their mean melting temperatures and melting range width were found to differ. DNA was shown to be heterogeneous in respect of the B-A transition, the GC-rich regions more readily converting into the A form than AT-rich ones. The presence of boundaries between the A and B sections within the transition zone did not smooth off the fine structure of melting profiles.     

A Study of the B-A Transition in DNA by Gel Electrophoresis

Abstract

A procedure is developed for studying the B-A transition in DNA using gel electrophoresis. The starting point has been the idea that the junction between the A and B sections, which appear within the transition interval would increase the mobility of the DNA molecules. Indeed, the mobility of DNA in a gel is shown to increase in the middle of the B-A transition due to the formation of the largest possible number of boundaries between the B and A forms. The middle of the B-A transition in supercoiled DNA appears to be shifted against the middle of the transition in open circular (as well as linear) DNA by about 1.3% towards lower ethanol concentrations under the influence of the superhelical stress.     

Mechanochemical study of NaDNA and NaDNA-netropsin fibers in ethanol-water and trifluoroethanol-water solutions.

Highly oriented calf-thymus NaDNA fibers, prepared by a wet-spinning method, were complexed with netropsin in ethanol-water and trifluoroethanol (TFE)-water solutions. The relative fiber length, L/L0, was measured at room temperature as a function of ethanol or TFE concentration to obtain information on the B-A conformational transition. The B-A transition point and transition cooperativity of the fibers were calculated. The binding of netropsin to NaDNA fibers was found to stabilize B form and to displace the B-A transition to higher ethanol concentration, as indicated by its elongational effect on the fiber bundles. An increased salt concentration was found to reduce netropsin binding. In netropsin-free ethanol solution, the dissociation of bound netropsin from the DNA fibers was observable. Pure B-NaDNA fibers were found to be more stable in TFE solution than in ethanol solution. This was interpreted as being due to a different steric factor and a larger polarity of TFE compared with ethanol, resulting in its smaller capacity to reduce the water activity and dielectric constant of the medium in the immediate vicinity of DNA fibers. Therefore, the effect of netropsin binding on the B-A transition of NaDNA fibers became less obvious in TFE solution. In another series of experiments, L/L0 was measured as a function of temperature to obtain information on the helix-coil transition, or melting, as well as the B-A transition of NaDNA and NaDNA-netropsin fibers. The melting temperature and helix-coil transition width were calculated from the melting curves. A phenomenological approach was used to describe the melting behavior of the fibers in and around the B-A transition region. The effect of netropsin on the melting of DNA fibers was attributed mainly to the stabilization of B-DNA and to a higher melting cooperativity in the B-DNA region.     

CC/GG Contacts Facilitate the B to A Transition of DMA in Solution

Abstract

Self-complementary decadeoxynucleotides, CCGATATCGG, CCAGATCTGG, CCCTG- CAGGG, GGGGGCCCCC, were designed and synthesized to estimate the A-philic free energy of CC/GG contacts.

First, regions of temperature-stability of the double-stranded conformation were determined for each 10-mer. Then, circular dichroism spectra were recorded for the B-family forms at different temperatures, counter-ion concentrations and trifluoroethanol contents.

A cooperative change typical of the B-A transition is observed in the CD spectra at a trifluoroethanol content specific for each duplex. The positions of half-transition points were functions not only of the nucleotide sequence but of the duplex length as well: the B to A transitions were hindered in these 10-mers in comparison with a lengthy DNA. The B-phility value was estimated to be 3 kcal/mol of 10-mer.

The B-A transition point was shown to drop with an increase in the number of CC/GG contacts in a duplex. The designed 10-mers made it possible to estimate quantitatively the A- phility of CC/GG contact as compared with an average DNA: (F_A-F_B)CC=0.2 Kcal/mol, (F_A-F_B)DNA=0.7 Kcal/mol.     

Peculiarities of the B to A Transition of the λ Phage Regulatory Site OR3 and of Its Fragment

Abstract

Conformations of the synthetic deoxyoligonucleotide 17 base pairs long, which is an OR3 operator of λ phage, and of its 9-b.p. fragment were studied by the circular dichroism method (CD). The regions of stability of the double-stranded state were determined for these duplexes. A comparison of the CD spectra for these oligonucleotides with the CD for a lengthy DNA showed the conformation of these short DNA pieces to belong to the B-family.

A cooperative change in the CD spectra is observed in trifluoroethanol (TFE) solutions at a TFE concentration specific for each oligonucleotide, which is supposed to stem from a B to A transition. The length of the fragment was found to affect the ability for the B-A transition. The transition into the A form is hindered by 13% TFE for the short 9-nucleotide in comparison with the 17-nucleotide. We suggest that this is due to the B form stabilization by terminal base pairs (B-phility of the ends).     

A Linear Biopolymer in the Vicinity of the Triple Point The Homopolymer Case

Abstract

This is a theoretical study of a situation where each residue of a linear biopolymer may adopt one of three conformational states. Such a situation exists in the case of DNA, since it may be in helical A, B,…, Z forms as well as the melted state. In the vicinity of the triple point in the phase diagram three states, e.g. the A form, the B form and the denatured state, co-exist within a given molecule. We present an exact analytical solution of the simplest homopolymer model. Theory predicts that the presence of two helical states in one molecule should affect the helix-coil transition in two ways. The melting temperature experiences an upward shift and the melting range width is increased, by a factor of √2 as a maximum.     

Three-state diagram for DNA

Experimental phase diagrams (A form, B form, Coil) were built in the coordinates (a, alcohol fraction: T, temperature) for the natural DNAs and poly d(A-T). The main parameter of the B-A transition,-cooperativity length, nu o, was estimated by the slopes of the branches A-B, A-Coil, B-Coil in the vicinity of the triple point: nu o = 10-20 base pairs, which corresponds to the energy for the B/A junction of 1.2-1.8 kcal/mol. We discovered two new effects which are due to the coexistence of the three different conformations in one polymeric molecule: an increase in the melting temperature above that for the 'ideal' triple point (i.e. for the case of the ideal phase transitions); a widening of the melting curve within the B-A transition range.     

A study of DNA melting in concentrated water-alcohol solutions

The DNA melting profiles with high resolution have been studied for conditions corresponding to the B and A conformations of DNA in water-alcohol solutions. The melting profiles of the A-form and B-form DNA, their mean melting temperatures and melting range width were found to differ. DNA was shown to be heterogeneous in respect of the B-A transition, the GC-rich regions more readily converting into the A form than AT-rich ones. The presence of boundaries between the A and B sections within the transition zone did not smooth off the fine structure of melting profiles.     

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.     

A study of the B-A transition in DNA by gel electrophoresis

A procedure is developed for studying the B-A transition in DNA using gel electrophoresis. The starting point has been the idea that the junction between the A and B sections, which appear within the transition interval would increase the mobility of the DNA molecules. Indeed, the mobility of DNA in a gel is shown to increase in the middle of the B-A transition due to the formation of the largest possible number of boundaries between the B and A forms. The middle of the B-A transition in supercoiled DNA appears to be shifted against the middle of the transition in open circular (as well as linear) DNA by about 1.3% towards lower ethanol concentrations under the influence of the superhelical stress.     

Mechanochemical study of conformational transitions and melting of Li-, Na-, K-, and CsDNA fibers in ethanol–water solutions

Highly oriented fibers of Li-, Na-, K-, and CsDNA were prepared with a previously developed wet spinning method. The procedure gave a large number of equivalent fiber bundle samples (reference length, L₀, typically = 12–15 cm) for systematic measurements of the fiber length L in ethanol–water solutions, using a simple mechanochemical set up. The decrease in relative length L/L₀ with increasing ethanol concentration at room temperature gave evidence for the B-A transition centered at 76% (v/v) ethanol for NaDNA fibers and at 80 and 84% ethanol for K- and CsDNA fibers. A smaller decrease in L/L₀ of LiDNA fibers was attributed to the B-C transition centered at 80% ethanol. In a second type of experiment with DNA fibers in ethanol–water solutions, the heat-induced helix–coil transition, or melting, revealed itself in a marked contraction of the DNA fibers. The melting temperature T_m, decreased linearly with increasing ethanol concentration for fibers in the B-DNA ethanol concentration region. In the B-A transition region, Na- and KDNA fibers showed a local maximum in T_m. On further increase of the ethanol concentration, the A-DXA region followed with an even steeper linear decrease in T_m. The dependence on the identity of the counterion is discussed with reference to the model for groove binding of cations in B-DNA developed by Skuratovskii and co-workers and to the results from Raman studies of the interhelical bonds in A-DNA performed by Lindsay and co-workers. An attempt to apply the theory of Chogovadze and Frank-Kamenetskii on DNA melting in the B-A transition region to the curves failed. However, for Na- and KDNA the T_m dependence in and around the A-B transition region could be expressed as a weighted mean value of T_m of A- and B-DNA. On further increase of the ethanol concentration, above 84% ethanol for LiDNA and above about 90% ethanol for Na-, K-, and CsDNA, a drastic change occurred. T_m increased and a few percentages higher ethanol concentrations were found to stabilize the DNA fibers so that they did not melt at all, not even at the upper temperature limit of the experiments (～ 80°C). This is interpreted as being due to the strong aggregation induced by these high ethanol concentrations and to the formation of P-DNA. Many features of the results are compatible with the counterion–water affinity model. In another series of measurements, T_m of DNA fibers in 75% ethanol was measured at various salt concentrations. No salt effect was observed (with the exception of LiDNA at low salt concentrations). This result is supported by calculations within the Poisson–Boltzmann cylindrical cell model. © 1994 John Wiley & Sons, Inc.     

B-Z Cooperativity and Kinetics of Poly(dG-m5dC) are Controlled by an Unfavorable B-Z Interface Energy

Abstract

Thermodynamic and kinetic properties of the B-Z transition of poly(dG-m⁵dC) were investigated using polynucleotide samples ranging in length from 11000 to 300 base pairs. Van't Hoff enthalpy values increase with increasing polymer length for the B-Z transition in 0.35 mM MgCl₂, 50 mM NaCl, 5 mM TRIS, pH 8. Rates of the B to Z transition increase with increasing polymer length for a jump of 0 to 3 mM MgCl, in 50 mM Nad, 5 mM TRIS, pH 8. The activation energy of the B to Z transition equals 7.9 ± 0.3 kcal/mol and is length independent Thermodynamic and kinetic data were fit to a model that simulates distribution of B- and Z-form tracts at the midpoint of B-Z equilibrium as a function of polymer length. A cooperative length of 1000 ± 200 base pairs is estimated for the B-Z transition. A direct relationship between rates of the B to Z transition and the square of the van't Hoff enthalpy values of the B-Z transition reflects a dependence of kinetics and cooperativity upon the energy of the nucleation event Faster B to Z transition rates with increasing polymer length can be explained by a mechanism rate limited by nucleation within the polymer instead of the ends.     

Fusion of complexes of DNA and extended ligands]

The theory of melting of DNA complexes with extended ligands (ties) is considered. Influence of ties interacting electorally with certain DNA regions and influence of extender ties, interacting unelectorally on the helix coil transition parameters is compared. It has been shown that both types of ties cause, coincided qualitatively, but differed quantatively, shifts of melting temperature and change of the melting range width of DNA. Comparison of theory with experiment in the case of DNA complexes with ribonuclease is given.     

Melting of DNA in ethanol–water solutions

The thermal denaturation curves of DNA have been investigated over a wide range of temperatures T and ethanol concentrations C by CD and uv absorption methods. The phase diagram of conformational DNA states has been constructed in T,C plane. The range of the A, B, and coiled forms of DNA was determined. The behavior of the DNA denaturation curve in the neighborhood of the triple point shows that the conformational transition B → A is realized by short parts of the DNA double helix. This is the reason for the coincidence of the melting temperatures of the A and B forms of DNA throughout the range of their coexistence.     

Theory of DNA melting in the interval of B-A transition

Theory of DNA melting within the B--A transition range is presented. The phase diagram in coordinates alcohol--temperature is plotted. The temperature shift of DNA helix--coil transition in the B--A transition point is predicted to be delta T = 3 degrees. The temperature rise of DNA melting in the range of B--A transition is caused by the presence of junctions between regions in B and A forms in helical sections.     

The denaturation transition of DNA in mixed solvents

The helix-to-coil denaturation transition in DNA has been investigated in mixed solvents at high concentration using ultraviolet light absorption spectroscopy and small-angle neutron scattering. Two solvents have been used: water and ethylene glycol. The "melting" transition temperature was found to be 94 degrees C for 4% mass fraction DNA/d-water and 38 degrees C for 4% mass fraction DNA/d-ethylene glycol. The DNA melting transition temperature was found to vary linearly with the solvent fraction in the mixed solvents case. Deuterated solvents (d-water and d-ethylene glycol) were used to enhance the small-angle neutron scattering signal and 0.1M NaCl (or 0.0058 g/g mass fraction) salt concentration was added to screen charge interactions in all cases. DNA structural information was obtained by small-angle neutron scattering, including a correlation length characteristic of the inter-distance between the hydrogen-containing (desoxyribose sugar-amine base) groups. This correlation length was found to increase from 8.5 to 12.3 A across the melting transition. Ethylene glycol and water mixed solvents were found to mix randomly in the solvation region in the helix phase, but nonideal solvent mixing was found in the melted coil phase. In the coil phase, solvent mixtures are more effective solvating agents than either of the individual solvents. Once melted, DNA coils behave like swollen water-soluble synthetic polymer chains.     

Melting of Cross-Linked DNA IV. Methods for Computer Modeling of Total Influence on DNA Melting of Monofunctional Adducts,Intrastrand and Interstrand Cross-Links Formed by Molecules of an Antitumor Drug

Abstract

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.