DNA Thermal Denaturation Research Articles

Targeting Abasic Sites in DNA by Aminoalkyl‐Substituted Carboxamidoacridizinium Derivatives and Acridizinium–Adenine Conjugates

AbstractTwo isomeric acridizinium–adenine conjugates, along with three model compounds (i.e. two 9‐(N‐alkylcarboxamido)acridizinium salts and one 9‐{N‐[(dimethylamino)alkyl]carboxamido}acridizinium salt) were prepared from the corresponding carboxyacridizinium salts. The interaction of these compounds with calf thymus DNA was studied by spectrophotometric and viscosimetric titrations, LD spectroscopy, and thermal DNA denaturation experiments. Both of the acridizinium–adenine conjugates and the N‐alkylcarboxamides intercalate into calf thymus DNA with moderate affinity [K ≈ 104 M–1 (DNA concentration in bp)]. In contrast, the N‐[3‐(dimethylamino)propyl]‐substituted carboxamide exhibits a significantly higher binding constant under identical conditions (K ≈ 105 M–1). The association of the acridizinium derivatives with abasic‐site containing DNA was assessed by thermal denaturation experiments with synthetic double‐stranded oligonucleotides, which contained one (TX) or no abasic site (TA). The acridizinium–adenine conjugates stabilize the DNA with the abasic position slightly more than they do the regular duplex, as indicated by the difference of the induced melting‐temperature shifts between the TX and TA duplexes (ΔΔTm ≈ 4 °C) at a ligand/DNA ratio r of 0.5. Most notably, a significantly more efficient stabilization (ΔΔTm = 9.6 °C) of the abasic duplex is achieved by theaminoalkyl derivative, namely 9‐{N‐[3‐(dimethylamino)propyl]carboxamido}acridizinium salt, which does not carry an adenine substituent. These results indicate that the design of abasic‐site‐targeting ligands must not necessarily involve the attachment of a nucleic acid base to the DNA intercalator.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

DNA binding and oxidative cleavage activity of ternary ( l-proline)copper(II) complexes of heterocyclic bases

Ternary copper(II) complexes [Cu( l-pro)(B)(H 2O)](NO 3) ( 1, 2) where l-pro = l-proline, B is a N,N-donor heterocyclic base, viz. 2,2′-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), are synthesized, characterized, and their DNA binding and cleavage activity studied. The bpy complex ( 1) is structurally characterized by single-crystal X-ray crystallography. The complexes show the presence of a distorted square-pyramidal (4 + 1) CuN 3O 2 coordination geometry. Complex [Cu( l-pro)(bpy)(H 2O)](NO 3) ( 1) crystallizes in the triclinic space group P1 with unit cell parameters: a = 7.082(3) Å, b = 10.483(5) Å, c = 11.581(5) Å, α = 89.700(7)°, β = 83.488(8)°, γ = 84.109(8)° and V = 849.7(7) Å 3. The one-electron paramagnetic complexes display a d–d band near 600 nm in water and show a cyclic voltammetric response due to Cu(II)/Cu(I) couple near 0.1 V (versus SCE) in Tris–HCl buffer–0.1 M KCl. Binding interactions of the complexes with calf thymus (CT) DNA have been investigated by emission, absorption, viscosity and DNA thermal denaturation studies. The phen complex displays significant binding propensity to the CT DNA giving an order: 2 (phen) ≫ 1 (bpy). The bpy complex does not show any apparent binding to the DNA and hence poor cleavage efficiency. Complex 2 shows efficient oxidative cleavage of SC-DNA in the presence of 3-mercaptopropionic acid (MPA) involving hydroxyl radical species as evidenced from the control data showing inhibition of DNA cleavage in the presence of DMSO and catalase.

1,2,4-Benzothiadiazine linked pyrrolo[2,1- c][1,4]benzodiazepine conjugates: Synthesis, DNA-binding affinity and cytotoxicity

Benzothiadiazine–pyrrolobenzodiazepine conjugates linked through different alkane spacers have been prepared. These new classes of hybrid molecules exhibit cytotoxicity against many cancer cell lines. Their DNA thermal denaturation studies have been carried out and one of the compounds ( 4b) elevates the DNA helix melting temperature of the CT-DNA by 6.7 °C after incubation for 36 h.

Influence of the Polypyridyl (pp) Ligand Size on the DNA Binding Properties, Cytotoxicity and Cellular Uptake of Organoruthenium(II) Complexes of the Type [(η6‐C6Me6)Ru(L)(pp)]n+ [L = Cl, n = 1; L = (NH2)2CS, n = 2

AbstractThe DNA binding of polypyridyl (pp) (η6‐hexamethylbenzene)ruthenium(II) complexes of the type [(η6‐C6Me6)RuCl(pp)](CF3SO3) (pp = phen, tap, dpq, dppz, dppn) 1–5 and [(η6‐C6Me6)Ru{(NH2)2CS}(pp)](CF3SO3)2 (pp = dpq, dppz, dppn) 6–8 has been studied by UV/Vis spectroscopy, circular dichroism and viscosity measurements. Complexes 3–5, 7 and 8 are potent cytotoxic agents towards the human cancer cell lines MCF‐7 and HT‐29. Stable intercalative binding into CT DNA is indicated for the dpq and dppz complexes by large increases ΔTm of 12–25 °C in the DNA thermal denaturation temperature for r = [complex]/[DNA] = 0.1. Large viscosity increases for DNA in the presence of 3 and 4 are also in accordance with this binding mode as are the pronounced hypochromic UV/Vis shifts for the π–π* transitions of the dppz ligands of 4 and 7 in the range 360–400 nm. A small ΔTm value of 2 °C and effectively unchanged viscosity suggest that Ru–N (nucleobase) coordinative binding is thermodynamically preferred for the larger polypyridyl ligand of 5 under equilibrium conditions, as is also the case for the small ligands phen and tap in complexes 1 and 2. CD spectra indicate that the B DNA conformation is essentially retained for interaction with the chloro complexes 1–5 but that very significant distortions occur for the thiourea complexes 6–8. The in vitro cytotoxicities of the chloro complexes 3–5 are dependent on the size of the polypyridyl ligand with IC50 values increasing in the order dppn < dppz < dpq: for instance IC50 values of 11.1, 2.12, and 0.13 μM were determined for 3–5 towards MCF‐7. These values correlate well with the cellular uptake efficiency which increases from 1.1 over 146.6 to 906.7 ng(Ru)/mg (protein) within the series 3–5. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

An Experimental and Computational Analysis on the Differential Role of the Positional Isomers of Symmetric Bis-2-(pyridyl)-1H-benzimidazoles as DNA Binding Agents

Three symmetrical positional isomers of bis-2-(n-pyridyl)-1H-benzimidazoles (n=2, 3, 4) were synthesized and DNA binding studies were performed with these isomeric derivatives. Like bisbenzimidazole compound Hoechst 33258, these molecules also demonstrate AT-specific DNA binding. The binding affinities of 3-pyridine (m-pyben) and 4-pyridine (p-pyben) derivatized bisbenzimidazoles to double-stranded DNA were significantly higher compared to 2-pyridine derivatized benzimidazole o-pyben. This has been established by combined experimental results of isothermal fluorescence titration, circular dichroism, and thermal denaturation of DNA. To rationalize the origin of their differential binding characteristics with double-stranded DNA, computational structural analyses of the uncomplexed ligands were performed using ab initio/Density Functional Theory. The molecular conformations of the symmetric head-to-head bisbenzimidazoles have been computed. The existence of intramolecular hydrogen bonding was established in o-pyben, which confers a conformational rigidity to the molecule about the bond connecting the pyridine and benzimidazole units. This might cause reduction in its binding affinity to double-stranded DNA compared to its para and meta counterparts. Additionally, the predicted stable conformations for p-, m-, and o-pyben at the B3LYP/6-31G* and RHF/6-31G* levels were further supported by experimental pKa determination. The results provide important information on the molecular recognition process of such symmetric head to head bisbenzimidazoles toward duplex DNA.

Synthesis of a novel C2-aryl pyrrolo[2,1- c][1,4]benzodiazepine-5,11-dione library: Effect of C2-aryl substitution on cytotoxicity and non-covalent DNA binding

A 23-member C2-aryl pyrrolo[2,1- c][1,4]benzodiazepine-5,11-dione (PBD dilactam) library has been synthesized using Suzuki coupling, and the effect of base upon racemisation at the C11a-position during the cross-coupling reaction studied. Three library members ( 21, 30 and 33) were sufficiently cytotoxic in the NCI’s preliminary screen to warrant further evaluation, and one ( 30, R = p-Br) was found to be cytotoxic at the sub-micromolar level in the A498 renal cancer cell line. DNA thermal denaturation studies suggested that this activity may be associated with non-covalent DNA interaction, and also demonstrated that introductin of C2–C3 unsaturation and addition of C2-aryl functionalities to the PBD dilactam skeleton significantly enhanced helix stabilisation compared to the unsubstituted PBD dilactam ( 6).

Synthesis, Characterization, and DNA‐Binding Properties of the Chiral Ruthenium(II) Complexes Δ‐ and Λ‐[Ru(bpy)2(dmppd)]2+ (dmppd = 10,12‐Dimethylpteridino[6,7‐f] [1,10]phenanthroline‐11,13(10H,12H)‐dione; bpy = 2,2′‐Bipyridine)

AbstractTwo novel chiral ruthenium(II) complexes, Δ‐[Ru(bpy)2(dmppd)]2+ and Λ‐[Ru(bpy)2(dmppd)]2+ (dmppd = 10,12‐dimethylpteridino[6,7‐f] [1,10]phenanthroline‐11,13(10H,12H)‐dione, bpy = 2,2′‐bipyridine), were synthesized and characterized by elemental analysis, 1H‐NMR and ES‐MS. The DNA‐binding behaviors of both complexes were studied by UV/VIS absorption titration, competitive binding experiments, viscosity measurements, thermal DNA denaturation, and circular‐dichroism spectra. The results indicate that both chiral complexes bind to calf‐thymus DNA in an intercalative mode, and the Δ enantiomer shows larger DNA affinity than the Λ enantiomer does. Theoretical‐calculation studies for the DNA‐binding behaviors of these complexes were carried out by the density‐functional‐theory method. The mechanism involved in the regulating and controlling of the DNA‐binding abilities of the complexes was further explored by the comparative studies of [Ru(bpy)2(dmppd)]2+ and of its parent complex [Ru(bpy)2(ppd)]2+ (ppd = pteridino[6,7‐f] [1,10]phenanthroline‐11,13 (10H,12H)‐dione).

Comparative Studies on the DNA-Binding Properties of Linear and Angular Dibenzoquinolizinium Ions

The interaction of the linear dibenzo[b,g]quinolizinium (5a) and the angular dibenzo[a,f]quinolizinium (6) with DNA was studied in detail in order to evaluate the influence of the shape of polycyclic quinolizinium ions on their DNA-binding properties. First, the synthesis and the thermally induced dimerization of 5a were reinvestigated because the preparation and isolation of the bromide salt of 5a according to literature procedures turned out to be problematic. The dibenzo[b,g]quinolizinium bromide [5a(Br)] tends to dimerize in solution with a highly selective and unprecedented formation of the corresponding anti-head-to-head dimer. Nevertheless, it was observed that careful exclusion of bromide ions from the reaction mixture suppresses the formation of the dimer. Moreover, the dimer may be transformed to the monomer by a remarkably rapid photoinduced electron-transfer reaction with 1-methoxynaphthalene. The association of 5a and 6 with nucleic acids was investigated by spectrophotometric and spectrofluorimetric DNA titrations, CD and LD spectroscopy, DNA thermal denaturation studies, and competition-dialysis techniques. Both dibenzoquinolizinium ions 5a and 6 exhibit an intercalative mode of binding to double-stranded DNA with moderate binding constants (K = 1-7 x 10(5) M(-1)) and a slight preference for association with GC-rich DNA regions. The structures of the intercalation complexes were calculated by molecular modeling methods. Competition-dialysis studies reveal that the isomers 5a and 6 bind selectively to triple-helical DNA (poly[dA]-poly[dT]2) as compared to selected synthetic and native double-stranded nucleic acids. Notably, the selectivity of the linear dibenzo[b,g]quinolizinium 5a toward triplex DNA is higher than the one of the angular derivative 6. In contrast, the DNA thermal denaturation studies reveal a higher stabilization of triple-helical DNA in the presence of 6 (DeltaTm3-->2 = 28 degrees C at r = 0.5) as compared to the stabilization by 5a (DeltaTm3-->2 = 14 degrees C at r = 0.5). This comparison emphasizes the importance of the extended pi system for the interaction of annelated quinolizinium ions with DNA. Moreover, the comparison between 5a and 6 demonstrates the significant influence of the shape of the pi system on the duplex- and triplex-stabilizing properties of the dibenzoquinolizinium ions.

Scaling laws at the phase transition of systems with divergent order parameter and/or internal length: The example of DNA denaturation

We used the transfer-integral method to compute, with an uncertainty smaller than 5%, the six fundamental characteristic exponents of two dynamical models for DNA thermal denaturation and investigate the validity of the scaling laws. Doubts concerning this point arise because the investigated systems (i) have a divergent internal length, (ii) are described by a divergent order parameter, and (iii) are of dimension 1. We found that the assumption that the free energy can be described by a single homogeneous function is robust, despite the divergence of the order parameter, so that Rushbrooke's and Widom's identities are valid relations. Josephson's identity is instead not satisfied. This is probably due to the divergence of the internal length, which invalidates the assumption that the correlation length is solely responsible for singular contributions to thermodynamic quantities. Fisher's identity is even more wrong. We showed that this is due to the d=1 dimensionality and obtained an alternative law, which is well satisfied at DNA thermal denaturation.

The relevance of nonlinear stacking interactions in simple models of double-stranded DNA

Single molecule DNA experiments provide interesting data that allow a better understanding of the mechanical interactions between the strands and the nucleotides of this molecule. In some sense, these experiments complement the classical ones about DNA thermal denaturation. It is well known that the original Peyrard-Bishop (PB) model by means of a harmonic stacking potential and a nonlinear substrate potential has been able to predict the existence of a critical temperature of full denaturation of the molecule. In the present paper, driven by the findings of single molecule experiments, we substitute the original harmonic intra-strand stacking potential with a Duffing type potential. By elementary and analytical arguments, we show that with this choice it is possible to obtain a sharp transition in the classical domain wall solution of the PB model and the compactification of the classical solitary wave solutions of other models for the dynamics of DNA. We discuss why these solutions may improve our knowledge of the DNA dynamics in several directions.

Direct Observation of Large Temperature Fluctuations during DNA Thermal Denaturation

In this Letter, we report direct measurement of large low frequency temperature fluctuations in double stranded DNA when it undergoes a denaturation transition. The fluctuation, which occurs only in the temperature range where the denaturation occurs, is several orders more than the expected equilibrium fluctuation. It is absent in single stranded DNA of the same sequence. The fluctuation at a given temperature also depends on the wait time and vanishes in a scale of a few hours. It is suggested that the large fluctuation occurs due to coexisting denaturated and closed base pairs that are in dynamic equilibrium due to the transition through a potential barrier in the scale of 25-30kBT0 (T0=300 K).

Effect of diethylsulfoxide on the thermal denaturation of DNA

DNA thermal denaturation has been investigated in aqueous solutions of diethylsulfoxide (DESO) by means of UV-vis and densimetry methods. It is suggested that, on the one hand, the structural change of entire solutions and, on the other hand, a direct interaction of DESO with DNA are responsible for the observed peculiar behavior. The results obtained were compared with those of dimethylsulfoxide (DMSO), also known from literature.

Thermal denaturation of double-stranded DNA: Effect of base stacking

We study the thermal denaturation of double-stranded DNA, i.e., separation of its two strands upon heating. A simple homo-polymer model is used to account for the effect of base stacking on the thermal stability of DNA. We find that stacking influences the stability in a nontrivial way: It not only enhances the stability but also makes the denaturation transition sharp. While stacking between bound monomers stabilizes DNA as does base pairing, stacking in unbound parts (or loops) rather destabilizes DNA--the overall stability is, however, enhanced by stacking.

Synthesis and DNA-binding ability of pyrrolo[2,1- c][1,4]benzodiazepine-azepane conjugates

A series of pyrrolobenzodiazepine–azepane conjugates linked through different alkane spacers have been prepared and their DNA thermal denaturation studies have been carried out. One of the compound ( 4b), elevates the DNA helix melting temperature of the CT-DNA by 2.0 °C after incubation for 36 h at 37 °C.

Dynamical model based on finite stacking enthalpies for homogeneous and inhomogeneous DNA thermal denaturation

We present a nonlinear dynamical model for DNA thermal denaturation, which is based on the finite stacking enthalpies used in thermodynamical nearest-neighbor calculations. Within this model, the finiteness of stacking enthalpies is shown to be responsible for the sharpness of calculated melting curves. Transfer-integral and molecular dynamics calculations are performed to demonstrate that the proposed model leads to a good agreement with known experimental results for both homogeneous and inhomogeneous DNA.

DNA binding potential and cytotoxicity of newly designed pyrrolobenzodiazepine dimers linked through a piperazine side-armed-alkane spacer

New pyrrolobenzodiazepine (PBD) dimers have been developed that are composed of two DC-81 subunits tethered to their C8 positions through piperazine moiety side-armed with alkaneoxy linkers (composed of 2–5 carbons). DNA thermal denaturation studies show that after 18 h of incubation with calf thymus DNA at a 1:5 ligand/DNA ratio, one of them, 6a, increases the Δ T m value by 24.0 °C. Thus, incorporation of a piperazine moiety instead of an inert alkanedioxy linker alone significantly enhances the DNA binding ability, and the analogous dimer 4 that lacks a piperazine moiety in the linker spacer elevates melting by only 15.1 °C under identical experimental conditions. This illustrates the effect of introducing a piperazine ring in the middle of such an alkanedioxy linker which produces several hydrophobic interactions and could also achieve a superior isohelical fit within the DNA minor groove. Interestingly, these dimers 6a– d are significantly more cytotoxic than 4 in a number of human cancer cell lines, in particular, compound 6c is highly potent for almost all the nine human cancer cell lines.

Relationship between phase transitions and topological changes in one-dimensional models

We address the question of the quantitative relationship between thermodynamic phase transitions and topological changes in the potential energy manifold analyzing two classes of one dimensional models, the Burkhardt solid-on-solid model and the Peyrard-Bishop model for DNA thermal denaturation, both in the confining and nonconfining version. These models, apparently, do not fit [M. Kastner, Phys. Rev. Lett. 93, 150601 (2004)] in the general idea that the phase transition is signaled by a topological discontinuity. We show that in both models the phase transition energy v(c) is actually noncoincident with, and always higher than, the energy v(theta) at which a topological change appears. However, applying a procedure already successfully employed in other cases as the mean field phi4 model, i.e., introducing a map M:v-->v(s) from levels of the energy hypersurface V to the level of the stationary points "visited" at temperature T, we find that M (v(c))=v(theta). This result enhances the relevance of the underlying stationary points in determining the thermodynamics of a system, and extends the validity of the topological approach to the study of phase transition to the elusive one-dimensional systems considered here.

Topological origin of the phase transition in a model of DNA denaturation.

The hypothesis that phase transitions originate from some topological change of the critical level hypersurface of the potential energy receives direct evident support by our study of the Bishop-Peyrard model of DNA thermal denaturation.

A Calorimetric and Spectroscopic Study of DNA at Low Hydration

Differential scanning calorimetry (DSC) and infrared spectroscopy (IR) were used to study the thermal behavior of DNA as a function of water content up to 12 wpn (water molecules per nucleotide). The DSC and IR results show cooperativity of thermal DNA denaturation at 12 wpn. In the 3.3 to 7.2 wpn range, an early structural change involving slight disruption of base stacking arises, followed at higher temperature by a denaturation process where the major changes in backbone conformation occur. DNA thermal denaturation in the 3.3 to 12 wpn range is irreversible. However, structural disruptions caused by dehydration are fully reversible. The loss of the DNA ordered conformation upon dehydration leads to a decrease in the endothermic maximum that characterizes the thermal denaturation of DNA. At 3.3 wpn, the DNA loses most of its ordered conformation and undergoes a glasslike transition similar to the transition that denatured DNA experiences during reheating. The calorimetric manifestation of the glass tran...

Fractal aggregation of DNA after thermal denaturation

DNA thermal denaturation was observed by atomic force microscopy (AFM) on the surface of newly cleaved mica. It was found that at temperatures higher than the melting point, denaturation of DNA molecules took place and globular particles with size distribution were formed, and these particles could aggregate together to form fractal structures, which followed the diffusion limited aggregation (DLA) model. At 100 °C, degradation of DNA took place and small particles of about 20 nm in size were formed, and they also aggregated in fractal structures with a lower dimension. Evaporating speed of water affects the fractal dimension.