Liquid Crystalline Phase Behavior of High ... - ACS Publications

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Biomacromolecules 2008, 9, 1860–1869

Liquid Crystalline Phase Behavior of High Molecular Weight DNA: A Comparative Study of the Influence of Metal Ions of Different Size, Charge and Binding Mode Neethu Sundaresan,† Cherumuttathu H. Suresh,‡ Thresia Thomas, T. J. Thomas,§,| and C. K. S. Pillai*,†,⊥ Chemical Sciences and Technology Division and Computational Modeling and Simulation Section, National Institute for Interdisciplinary Science and Technology (formerly Regional Research Laboratory), Council of Scientific and Industrial Research, Thiruvananthapuram 695019, India, and Departments of Environmental and Occupational Medicine and Medicine, The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, New Jersey 08903 Received January 31, 2008; Revised Manuscript Received April 16, 2008

The ability of Li+, Na+, K+, Rb+, Cs+, Mg2+, Ca2+, Sr2+, Ba2+, Cu2+, Cd2+, Al3+, V4+, Hg2+, Pd2+, Au3+, and Pt4+ to provoke liquid crystalline (LC) phases in high molecular weight DNA was investigated. The alkali and alkaline earth metal ions provoked typical cholesteric/columnar structures, whereas transition metal ions precipitated DNA into solid/translucent gel-like aggregates. Heavy metal ions reduced viscosity of DNA solution, disrupting rigid, rod-like DNA structure necessary for LC textures. Three-layer quantum mechanical-molecular mechanical (QM/MM) studies of Li+, Na+, K+, Mg2+, and Ca2+ binding DNA fragment suggested several possible binding modes of these ions to the phosphate groups. The dianion mode of metal binding, involving the phosphate groups of both strands of DNA, allowed for higher DNA binding affinity of the alkaline earth metal ions. These results have implications in understanding the biological role of metal ions and developing DNA-based sensors and nanoelectronic devices.

Introduction Several metal ions are essential for living organisms to maintain their metabolic activity, while many others are toxic.1 The affinity of a metal ion for a specific site on DNA is a function of its charge, free energy of hydration, coordination geometry, and coordinate bond forming capacity.2–5 DNA is a polyelectrolyte and requires cations to neutralize the negative charge on the phosphate anions.6 Metal ions interact with DNA in a hydrated state and act as hydrogen bond donors, in addition to their role as stabilizers of the double helix by reducing intermolecular repulsion.7–9 Cation-DNA interaction has several consequences: stabilization of the helical structures (duplex, triplex, and quadruplex) and conformational transitions in sequence-specific DNA structures.10–15 Metal ions can also provoke a distortion of the double helix by base binding, leading to loss of rigidity, rod-like shape, and induction of condensed/ aggregated structures.16–18 Cation-induced DNA condensation to liquid crystalline (LC) textures has become an active area of research due to its potential applications in therapeutics (gene delivery),19–21 in the design of biosensing units,22 and DNA chips.23,24 Safinya and colleagues19,20 conducted detailed sincrotron X-ray diffraction * To whom correspondence should be addressed. Telephone: 91-471 252 0361/234 0801. Fax: 91-471 234 1814. E-mail: [email protected]. † Chemical Sciences and Technology Division, National Institute for Interdisciplinary Science and Technology. ‡ Computational Modeling and Simulation Section, National Institute for Interdisciplinary Science and Technology. § Department of Environmental and Occupational Medicine, The Cancer Institute of New Jersey. | Department of Medicine, The Cancer Institute of New Jersey. ⊥ Present address: Sree Chitra Tirunal Institute for Medical Sciences and Technology, Biomedical Technology Wing, Poojappura, Thiruvananthapuram 695012, India.

and optical absorption spectroscopic studies of cationic liposome- and divalent cation-condensed DNA and found twodimensional hexagonal lattice and other liquid crystalline structures. In the case of divalent cations, the repulsive forces between DNA chains reversed from repulsive to attractive forces. Recent measurements of repulsive and attractive forces demonstrated that the attractive forces between DNA double helices were approximately 2.3-fold higher than the repulsive force in multivalent cation condensed DNA.25 The behavior of semirigid polymers to form ordered LC phases above a critical concentration (first described by Onsager and later elaborated by Flory and others) has been well established for synthetic polymers,26–28 but the LC organization of polyelectrolytes such as DNA has not been investigated in detail, except in the case of low molecular weight, fragmented DNA.29–32 In the case of synthetic, semirigid, high molecular weight polymers, which are polydisperse, LC domain ordering occurs spontaneously at high concentrations to minimize the macromolecular excluded volume.6 As a strong polyelectrolyte, DNA is surrounded by a counterion layer, which determines its effective particle radius and, hence, its effective axial ratio and excluded volume. Therefore, condensed DNA assumes different degrees of order and packaging, depending upon the concentration of DNA in solution and the nature of the counterion. These compact LC states are important to allow proteins to access the DNA template for a multitude of biological tasks, including replication and transcription.33 Rill and co-workers have shown that the critical concentration, CD, above which the LC phase appears, depends on the chain length of DNA.34 However, most of the investigations of the LC behavior of DNA have been conducted with low molecular weight fragmented or sonicated DNA of ∼150 bp length.31 Using such DNA, Livolant and colleagues

10.1021/bm800101x CCC: $40.75  2008 American Chemical Society Published on Web 05/30/2008

Metal Ions and Liquid Crystalline DNA

demonstrated that the natural polyamines, spermidine and spermine, and a metal complex, cobalt hexamine (Co(NH3)6Cl3) were capable of provoking multiple LC phases.35–38 These and other studies on the condensation of DNA showed that DNA has a tendency to assume the hexagonal LC texture. Saminathan et al.39 showed that natural and synthetic polyamines could provoke and stabilize LC textures of high molecular weight DNA in a concentration- and structuredependent manner. Structural specificity effects of polyamines have been reported in the induction and stabilization of Z-DNA, triplex DNA, and DNA nanoparticle formation.13–15,40–43 A similar investigation on the interaction of high molecular weight DNA with metal ions of different binding modes, size, and charge is expected to shed light on the supramolecular organization of DNA in vivo and also on the functioning of DNA in the condensed states. Our study documents the requirements and conditions leading to the initiation of LC phases of DNA by metal ions, their time-dependent evolution, and stability. Our results provide a better understanding of the role of metal ions in cellular functions and define the stability of LC phases of DNA under the influence of different metal ions.

Materials and Methods Metal Salt Solutions. LiCl, NaCl, KCl, RbCl, CsCl, MgCl2, CaCl2, BaCl2, SrCl2, CuCl2, CdCl2, Al(NO3)3, VOSO4, HgCl2, PdCl2, HAuCl4, and H2PtCl6 were used in this study. The salts were of the highest purity and were purchased from SD. Fine Chemicals, Mumbai, India (alkali and alkaline earth metal salts), or Sigma Aldrich, Milwaukee, WI (transition metal salts). Preparation of DNA. High molecular weight calf thymus DNA was purchased from Worthington Biochemical Corporation (Freehold, NJ) and used without further purification. Millipore water was used as the medium in our experiments. DNA was dissolved in 0.1 M NaCl (pH 7) and dialyzed it against the same solution four times. The observed A260/A280 ratio of the DNA solution was 1.88, indicating that the DNA was free of protein contamination.39 The weight average molecular weight of the DNA was 6 × 106, as determined by multiangle laser light scattering and Zimm plot. The second virial coefficient was 6 ( 1 × 10-4 mol mL/g2. It had a root-mean-square radius of 238 ( 3 nm. The concentration of calf thymus DNA was determined by measuring the absorbance at 260 nm and using the molar extinction coefficient () of 6900 M-1 cm-1. The final concentration of DNA was 7.14 mg/mL. A homogeneous DNA solution of 7.14 mg/mL concentration was used as the stock solution in the present set of experiments. Preparation of higher concentrations of DNA faced difficulty in dissolution and attaining homogeneity. Preparation of Samples for Polarized Light Microscopy. DNA stock solutions (7.14 mg/mL) were diluted with 0.1 M NaCl for the required lower DNA concentrations. Also stock 1 M metal ion solutions, prepared in 0.1 M NaCl, were diluted with the same to obtain further lower metal ion concentrations. Appropriate DNA solutions were then mixed with selected metal ion solutions and allowed to attain equilibrium for 3 h at 26 °C to prepare the metal-DNA complex. Preliminary experiments indicated that cacodylate buffer and phosphate buffer have interactions with various metal ions. Acetate/citrate buffer was noted to have minimal interactions. The best results were obtained when DNA was dissolved in 0.1 M NaCl (pH 7). Similar results were obtained with acetate/citrate buffer and with 0.1 M NaCl at pH 7. The LC behavior of DNA in the presence of alkali and alkaline earth metal ions was studied under the following conditions: (i) varying DNA concentrations, while keeping the metal ion concentration (1 M) constant to determine the critical DNA concentration required to induce LC behavior in the presence of each metal ion and (ii) varying the metal ion concentration, while keeping the DNA concentration constant (7.14 mg/mL) to examine the effect of different metal ion concentrations on the LC organization of DNA. In the case of alkali and alkaline earth

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metals, there was no precipitation of DNA, when metal ion solutions were mixed and equilibrated with DNA. However, the same methodology could not be used in the case of multivalent and transition metal ions because Cu2+, Cd2+, V4+, and Al3+ precipitated DNA to a solid gel/translucent gel-like mass, which did not have the order and mobility required to exhibit anisotropy. In certain cases, heavy metal ions drastically reduced the viscosity of DNA solution up to a certain metal ion concentration. Hence, we determined the critical metal ion concentration (CM) of transition metal ions as the concentration at which the metal ion did not precipitate DNA. Hence, we determined the critical metal ion concentration (CM) of transition metal ions as the concentration at which the metal ion did not precipitate DNA and CM of heavy metal ions as the concentration at which the metal ion did not reduced the viscosity of DNA required for anisotropy. Polarized Light Microscopy. We performed microscopic experiments with a Nikon Optiphot Polarized Light Microscope, equipped with a Nikon camera. Microscopic glass slides and coverslips were soaked in chromic acid and further rinsed with deionized water and dried using Analar acetone prior to use. Desired concentrations of metal ions and DNA were mixed in an Eppendorf tube, vortexed for 15 min, and then allowed to equilibrate at room temperature (26 °C) for 3 h. A total of 20 µL of each solution of metal ion-DNA complex was sandwiched between a clean microscopic glass slide and a coverslip, and the coverslip was sealed with DPX mountant (a neutral solution of polystyrene and plasticizers in xylene used in microscopy work, M/s. Nice Chemicals Ltd., Mumbai) to prevent dehydration of the sample.44 The preparations were then incubated at 37 °C for extended time periods to observe phase changes until crystallization or complete darkening (isotropization) occurred. The preparations were monitored periodically for phase changes under the microscope, and photographs were taken when the phases became prominent and distinct. The phases and granular boundaries were clear and sharp when the sample was incubated at 37 °C. A triplicate of each sample was made to ensure reproducibility of the phase changes. The results were reproducible in at least three separate experiments. The following parameters were noted: (a) CD, critical DNA concentration required to exhibit anisotropy; (b) CM, critical metal ion concentration below which anisotropic behavior is exhibited; (c) Tiso, time required for the LC phases to darken and disappear (ie., isotropization); and (d) Ttr, time required for cholesteric to columnar phase transition. Molecular Modeling. Theoretical results for the metal ion phosphate interactions were obtained with the ONIOM technique of Morokuma et al.,45–47 which is a hybrid approach that combines the methods of quantum mechanics and molecular mechanics (QM/MM) to model large molecules. The three-layer ONIOM model selected for the study is presented in Scheme 1. In Scheme 1, the binding region of the metal ion to the phosphate anion was treated with the QM-based density functional theory method of B3LYP/6-31G(d) (high layer), the cytosine-guanine base pair interactions were modeled using the semiempirical PM3 method (medium layer), and the steric effects of the sugar units were incorporated through the MM method (low layer) by selecting the universal force field (UFF). A dianion (two anionic phosphate groups, as in Scheme 1) and an anion model (the phosphate group in the medium layer is neutral) were considered. Sundaresan et al.48,49 have recently published a detailed account of the methodology used in the molecular modeling of metal ion-DNA interaction. The calculated energetic values using the QM/MM model have an error of (10%.

Results Effects of Alkali Metal Ions on the Structure of DNA. Among alkali metal ions, Na+ and K+ have been shown to induce a complex and polymorphic phase behavior in DNA.24 The mode of interaction involves a multimolecular assembly of DNA molecules by electrostatic interaction between the negatively charged phosphate groups of DNA and positively

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Scheme 1. Representation of the Three-Layer ONIOM Model Selected for the Study

charged metal ions. To compare the efficacy of different alkali metal ions to induce the LC phase in DNA, we determined the critical DNA concentration (CD) required to reach LC textures by polarized light microscopy (Figure 1). The critical DNA concentration required to exhibit anisotropic behavior was the same for all the monovalent ions except that for Li+ ion.50 All alkali metal ions exhibited the typical cholesteric and columnar textures, but the cholesteric to columnar phase transition (Ttr) was facilitated by increased size of the metal ion (Ttr Li+ < Ttr Na+ < Ttr K+ < Ttr Rb+ < Ttr Cs+; Figure 2). The Li+ ion, with its high water of hydration51 and a tendency for binding to DNA bases, exhibited a highly stable (for more than two months) biphasic cholesteric-columnar arrangement. Effects of Alkaline Earth Metal Ions on the Structure of DNA. Figure 1 also shows the CD values measured in the presence of 1 M alkaline earth metal ions (Mg2+, Ca2+, Sr2+, and Ba2+). Among these ions, the CD of Ca2+ was much higher than that of Sr2+ and Ba2+. A comparison of the CD values showed that alkali metal ions provoked LC textures at much lower DNA concentrations than that necessary for the alkaline earth metal ions. As in the case of alkali metal ions, alkaline earth metal ions having the lowest ionic radius showed the highest CD value. Thus, the ionic radius is an important factor in the efficacy metal ions to induce and stabilize the LC phase of DNA. It is interesting to note that Mg2+ could not induce the LC phase at 1 M concentration. The LC phases initially appeared to flow spontaneously, typical of the cholesteric phase with their characteristic fingerprint pattern and tear drops in certain textures, which turned to the more ordered columnar phase over time, with fan-shaped textures and striated patterns. Because 1 M Mg2+ could only provoke isotropic behavior in DNA, the

Figure 1. Critical DNA concentration (CD) required for exhibiting anisotropy in the presence of alkali and alkaline earth metal ions (1 M). At this concentration, Mg2+ did not provoke the formation of LC phase.

concentration of Mg2+ was lowered from the 1 M level to that ranging from 0.03 to 0.75 M (at a constant DNA concentration of 7.14 mg/mL). Under these conditions, DNA showed a weakly birefringent fluidic texture, similar to the cholesteric ordering. Therefore, this concentration range was used to determine the comparative efficacy of alkaline earth metal ions in inducing LC phases. As shown in Figure 3, Ba2+ and Sr2+ were more efficacious than Ca2+ and Mg2+ in inducing the columnar hexagonal phase of LC DNA. The LC phase transitions occurred not only as a concentrationdependent phenomenon, but also as a time-dependent phenomenon. It is important to note here that the time-dependent changes in LC textures of DNA occurred under conditions at which solvent evaporation was prevented by sealing the glass slides with a neutral solution of polystyrene and plasticizers in toluene. Therefore, the observed changes were a consequence of the reorganization of DNA strands under the influence of metal ions. Figure 3 shows the time-dependent changes in the texture of LC phases under the influence of different metal ions. The time required for cholesteric to columnar phase transition (Ttr) varied from hours to days (Ttr Mg2+ ) Ca2+ > Ba2+ > Sr2+). (Ttr for Ba2+ is higher than Ttr for Sr2+ only for metal concentrations below 0.75 M.) Figure 3b,c shows that, at 1 M concentration, both Ba2+ and Sr2+ provoked higher ordered columnar hexagonal phase directly. The columnar phase could easily be identified from its typical fan shaped textures with striated patterns. On the other hand, Mg2+ and Ca2+ exhibits the typical lower ordered cholesteric phase at similar concentrations (0.75 M in the case of Mg2+), indicating that the size of the ion played an important role in the stabilization of the LC phases of DNA. It can be noted from Figure 3 that the transition from cholesteric to columnar hexagonal (Ttr) is found to be timedependent, based on the size of the ion (Ttr Mg2+ ) Ttr Ca2+ > Ttr Ba2+ > Ttr Sr2+). The Ttr values varied from hours to days and the nature of the phases obtained initially were birefringent cholesteric planar like textures, which adopted a more ordered columnar phase gradually with a biphasic region, sometimes with restricted fluidity coexisting. Effects of Transition and Multivalent Metal Ions on the LC Behavior of DNA. In contrast to the behavior of alkali and alkaline earth metal ions, 1 M concentrations of Cu2+, Cd2+, Al3+, and V4+ precipitated DNA into solid/translucent, gel-like aggregates, which lacked the mobility required for the LC behavior. As ionic strength is one of the parameters affecting critical DNA concentration, it appeared that the CD required to exhibit LC ordering could not be achieved for the high molecular weight DNA. So, we reduced the metal ion concentration to a level at which DNA did not form gels. The samples were incubated at 37 °C and the phase transitions were monitored at different time points with a polarizing microscope, with crossed



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Figure 2. Time-dependent phase transitions of LC DNA (7.14 mg/mL) in the presence of alkali metal ions [(a) Li+, (b) Na+, (c) K+, (d) Rb+, and (e) Cs+]. Concentration of DNA dissolved in 0.1 M NaCl (pH 7) is ∼4 mg/mL. All the phases were obtained under controlled conditions (Y-axis scales are not fixed arbitrary units).

Figure 3. Time-dependent phase transitions of liquid crystalline DNA in the presence of alkaline earth metal ions: (a) Mg2+, (b) Ca2+, (c) Sr2+, and (d) Ba2+.

polars. The critical metal ion concentration (CM) at which the sample had enough viscosity to exhibit LC behavior was as follows: Cu2+, 0.625 mM; Cd2+, 5 mM; Al3+, 0.625 mM; and V4+, 0.625 mM. Interestingly, we found identical CMs for Cu2+, Al3+, and V4+, in spite of the different electronic configurations of these metal ions. Figure 5 shows the time-dependent changes in the LC textures of Cu2+, Cd2+, Al3+, and V4+. In the presence of Cu2+, DNA

initially exhibited a cholesteric phase below the CM (0.625 mM), with two pointed brush defects (Figure 6a) on which fingerprint pattern, with antiparallel arrangement, developed (Figure 6b). This phase later assumed a herringbone pattern of the columnar hexagonal phase (Figure 5). At very high metal ion concentrations (1-5 mM), DNA aggregation and precipitation occurred in the presence of Cu2+, V4+, and Al3+. However, Cd2+ showed a weakly birefringent cholesteric phase initially (below its CM

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Figure 4. Liquid crystalline phases of DNA obtained in the presence of alkaline earth metal ions: panel (a) shows the broken fan shaped textures, typical of columnar phase from the fluidic cholesteric phase, obtained in the presence of 0.06 M Mg2+; panel (b) shows texture typical of columnar phase obtained with 0.06 M SrCl2, obtained after 7 h of incubation at 37 °C; and panel (c) shows a texture typical of columnar hexagonal phase obtained with 0.06 M BaCl2 (10×). Concentration of DNA is ∼7 (mg/mL).

Figure 5. Time-dependent phase transitions of liquid crystalline DNA in the presence of multivalent metal ions: (a) Cu2+, (b) Cd2+, (c) Al3+, and (d) V4+.

of 5 mM), which darkened within 8 h (Figure 5) without transforming into a higher ordered columnar hexagonal phase. Al3+ gave a highly birefringent and fluidic cholesteric phase (Figure 6c) initially below the CM (0.625 mM), but it became unstable and darkened after 24 h (Figure 6d), yielding an isotropic phase without transformation to the higher-ordered columnar hexagonal phase. Similarly, V4+ precipitated DNA at concentrations >0.625 mM; however, at lower concentrations, it provoked LC phases in a two-step manner. A birefringent fluidic cholesteric phase was formed initially, and threaded structures, typical of nematic phase, developed (Figure 6e), probably due to the anchoring effects of the glass slide. After prolonged incubation, the LC phase transformed into a silkwormlike texture of columnar phase (Figure 6f) and remained stable for 5 days (Tiso) at 0.625 mM V4+, and up to 2 weeks at lower metal ion concentrations (0.3 mM). Effect of Heavy Metal Ions on the LC Behavior of DNA. We next examined the effects of four base binding heavy metal ions, Hg2+, Pd2+, Au3+, and Pt4+ on the LC behavior of DNA. These ions produced a drastic decrease in the viscosity of DNA. At a concentration of 7.14 mg/mL of DNA in 0.1 M

NaCl and 100 mM metal ions, the solution started flowing spontaneously and appeared to be totally dark under the polarizing microscope. In the case of all four metal ions, the formation of a mesophase was observed at concentrations below the CM of 12 mM (see Table 1). The CM for the induction of the LC phase appeared independent of the charge they carry; however, as the metal ion concentration was further lowered to 10 mM, the sample showed birefringent domains with homogeneous illumination of cholesteric ordering under the polarized light microscope (Figure 7). With Hg2+, Pd2+, Au3+, and Pt4+ (for 10 mM metal ion concentration), the LC textures were mainly cholesteric planar, with homogeneous illumination, irrespective of the nature of the metal ion. The LC texture also changed to isotropic phase after 24 h (36 h in the case of Pt4+) of sample preparation, indicating a high level of instability (Tiso Hg2+ ≈ Tiso Pd2+ < Tiso Au3+ < Tiso Pt4+) of the LC phase due to possible distortion of the double helix. There was no transition to the higher ordered columnar hexagonal phase; however, a columnar hexagonal phase (Figure 7) was observed at 1 mM concentration, which might be due to electrostatic interaction, and double helix stabilization at low metal ion concentration. The heavy metal ions thus showed the highest level of instability in the LC phase behavior in comparison to alkali, alkaline earth, and other multivalent ions studied. Three-Layer QM/MM ONIOM Calculations. The threelayer QM/MM ONIOM calculations have revealed both the outer sphere and the inner sphere coordination behaviors of Li+, Na+, K+, Mg2+, and Ca2+ ions to the phosphate group of a DNA fragment in its anion and dianion states.41,42 It was found that in the anion model, the outer sphere binding of Li+ ion (Figure 8) is highly preferred (binding energy ) 112.5 kcal/ mol) compared to its other modes of interaction. In addition, binding affinity of Li+ to the phosphate group of DNA was significantly higher than those of Na+ and K+ ions in the anion and dianion models. On the other hand, the binding behavior of alkaline earth metal ions to anion and dianion forms of DNA model systems indicated higher affinity of the phosphate group of the DNA to these metal ions compared to that of alkali metal ions.42 A monodentate binding mode was the most stable structure observed for both the Mg2+ and the Ca2+ ions in the anion model, while the binding interactions to the dianion model of the DNA fragment gave rise to significantly larger structural deformation at the basepair region, which led to the formation of “ring” structures. In Figure 9, the outer sphere bonding mode of the hexahydrated Mg2+ ion to the dianion model of the DNA fragment is given, which also illustrates the form of the “ring” structure. Another notable point is that in both the anion and the dianion models, Mg2+ bound structures were considerably more stable than that of the Ca2+ bound structures. For instance, in the dianion models, the binding energy values of Mg2+ and Ca2+ structures were 304.2 and 286.1 kcal/mol, respectively.



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Figure 6. Time-dependent liquid crystalline phase transitions of DNA obtained in the presence of multivalent ions: (a) cholesteric fluidic phase formed after 3 h and (b) fingerprint pattern with antiparallel arrangement appeared on the cholesteric phase after 7 h of incubation with 0.625 mM Cu2+ at 37 °C; (c) cholesteric phase and (d) isotropisation of the cholesteric phase in the presence of 0.625 mM Al3+; (e) cholesteric and nematic threaded texture of DNA, which flowed spontaneously under the microscope, obtained with 0.625 mM V4+; and (f) the same phase transformed into silkworm like texture probably of columnar phase in the presence of Al3+ (10×). Concentration of DNA was ∼7 (mg/mL). Similar results were obtained in three separate experiments. Table 1. Critical Metal Ion Concentrations Required by Multivalent Metal Ions To Induce Liquid Crystalline Behavior metal ions

critical metal ion concentration (mM)

2+

Cu Cd2+ Al3+ V4+ Hg2+ Pd2+ Au3+ Pt4+

0.62 5 0.625 0.625 12 12 12 12

In addition, unlike Ca2+ ion, the charge of the DNA fragment appeared to be crucial in deciding the binding strength as well as the binding mechanism of Mg2+ ion.

Discussion Previous studies of liquid crystalline phase transitions of DNA were carried out with low molecular weight DNA fragments, preparedeitherbysonicationormicrococcalnucleasedigestion29,34–38 of high molecular weight DNA. High molecular weight DNA is useful because the results can shed light on the supramolecular

organization of DNA in the cell. It has been estimated that the local concentration of DNA in a cell is not lower than 50 mg/ mL and could reach a concentration of up to 400 mg/mL.52 In the present study, the concentration of DNA necessary to exhibit the LC phase is much lower (1-7.14 mg/mL) than that used in previous reports with low molecular weight DNA (>150 mg/ mL). However, it should be noted that LC phases were observed at 1 M concentration of alkali or alkaline earth metal ions, at concentrations far higher than the physiologically achievable concentrations. Nevertheless, the LC phases and textures observed in our study are comparable to the DNA mesophases obtained with low molecular weight DNA. This result is in agreement with the observation of Merchant and Rill27 who showed a dramatic decrease in the critical concentration of DNA for liquid crystal formation, as the molecular weight of DNA increased. Measurements of CD in the presence of alkali metal or alkaline earth metal ions demonstrated that the ionic radius of the metal ion is an important factor governing the efficacy of induction of LC phases. Within the alkaline earth metal family, Ca2+, with an ionic radius (ri) of 0.099 nm required higher DNA concentrations compared to Sr2+ (ri ) 0.113 nm) and Ba2+ (ri

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Figure 9. Three-layer QM/MM level optimized geometry of the dianion model of DNA fragment showing the outer sphere interaction of the hexahydrated Mg2+ ion. Description of the three layers follows the same order as that given for Figure 8.

Figure 7. Time-dependent phase transitions of liquid crystalline DNA in the presence of heavy metal ions: (a) Hg2+, (b) Pd2+, (c) Au3+, and (d) Pt4+. Arbitrary units are used on the Y-axis.

Figure 8. Three-layer QM/MM level optimized geometry of the anion model of DNA fragment showing the outer sphere interaction of the tetrahydrated Li+ ion. Ball and stick model represents the QM layer wherein the B3LYP/6-31G(d) level of density functional theory is applied. The stick representation is used for the QM layer treated at the semiempirical PM3 level. Big spheres correspond to the atoms in the MM layer wherein the UFF force field is used in the calculations.

) 0.135 nm), which have larger ionic radii. Similarly, within the alkali metal family, Li+ (ri ) 0.06 nm) required the highest DNA concentration compared to Na+, K+, and Cs+ with ionic radii in the range of 0.095 to 0.167 nm. Metal ions with larger ionic radius might be able to interact with DNA strands at greater distance and thereby require less DNA concentration for the induction of the LC phase. We observed multiple LC phases with different textures, sometimes diffused and unstable or otherwise mainly distinct and clear, which in turn depended on the type, nature, and concentration of metal ions by mixing metal ions with DNA. Two main phases, cholesteric and columnar hexagonal, were found in our study, either separately or in coexistence, with slight variations in texture, depending on local conditions. It is interesting to note that the previously reported precholesteric blue phases,53,54 also classified as blue phases, which indicated a transition from the isotropic to the cholesteric phase, were

not observed with any of the metal ions used in this investigation. This might be attributed to the ability of metal ions to directly order the DNA molecules to the simple twist configuration of the more stable cholesteric phase. Among the alkaline earth metals, Sr2+ and Ba2+ showed the largest range of metal ion concentration (0.03-1 M) to stabilize columnar hexagonal phase. On the other hand, Ca2+ showed the maximum range for stabilizing cholesteric phase although it also induces the more stable hexagonal phase. Among the transition metal ions tested, V4+ yielded a unique profile with nematic Schlieren phase at 3 h and stable columnar hexagonal phase for almost 2 weeks. On the other hand, cholesteric and columnar hexagonal phases induced by heavy metals, Au2+, Hg2+, Pd 2+, and Pt4+ were relatively less stable, lasting only 24-36 h. Thus, metal ions may be selectively used to stabilize specific LC phases. Interestingly, it has been reported that the DNA complex with metal ions, Zn2+, Ni2+, or Co2+ forms a DNA conformation called M-DNA, shown to be a better conductor of electron transport, raising the possibility of its use in nanoelectronics.55–57 Several recent X-ray studies on cation and cationic lipid binding to DNA have revealed the formation of the liquid crystalline phases. Structural polymorphism of liquid crystalline textures has been reported from these studies, suggesting the existence of a higher density hexagonally packed region and a lower density cholesteric region, with fluid-like positional ordering, in the presence of metal ions and dehydrating solvents.58–61 It is well-known that the induction of liquid crystalline phase and the state of hydration of DNA are inter-related. Counterion binding to DNA is usually associated with the removal of water molecules from neighboring DNA strands, which brings ordered alignment of DNA molecules required for LC behavior.62–64 The enthalpy of hydration of alkali and alkaline earth metal ions is as follows:51 Li+, -124; Na+, -97; K+, -77, Rb+, -70; Cs+, -63; Mg2+, -459; Ca2+, -377; Sr2+, -344; and Ba2+, -312 kcal/mol. The enthalpy of hydration of Mg2+ and Ca2+ are much higher than that of Sr2+ and Ba2+ ions and that might explain the difference in CD between the ions of high ionic radii (Ba2+ and Sr2+) and those of low ionic radii (Mg2+ and Ca2+). High-resolution X-ray studies indicate that Mg2+ binds to the DNA molecule through a water bridge, stabilizing the hydrogen bonded water network.65 An analysis of Figure 1 indicates that Mg2+ might exhibit LC phases at a higher CD, which could not


be achieved in our experimental conditions because of the difficulty in preparing a homogeneous solution of the high molecular weight DNA at concentrations higher than 10 mg/ mL. It should also be noted that Li+ required a high critical concentration of DNA than that required by other alkali metal ions. This behavior is similar to that of Mg2+ and correlates to the diagonal relationship in the periodic table. A very interesting finding from this study is that Ba2+ and 2+ Sr give higher ordered columnar hexagonal phase directly (see Figure 2b,c). The columnar phase could easily be identified from its typical fan-shaped textures with striated patterns. On the other hand, Mg2+ and Ca2+ exhibit the typical lower ordered cholesteric phase at similar concentrations (0.75 M in the case of Mg2+), indicating that the size of the ion also plays an important role in the stabilization of the LC phases of DNA. It can be noted from Figure 2 that the transition from the cholesteric to the columnar hexagonal (Ttr) phase is timedependent, based on the size of the ion Mg2+ ) Ca2+ < Ba2+ < Sr2+ (Ttr Mg2+ ) Ttr Ca2+ > Ttr Ba2+ > Ttr Sr2+, for low metal ion concentrations (for 0.25 M concentration, Ttr Ba2+ ) Ttr Sr2+). The Ttr values vary from hours to days, and the nature of the phases obtained initially are birefringent cholesteric planar-like textures, which can adopt a more ordered columnar phase gradually with a biphasic region, sometimes with restricted fluidity coexisting. All the phases obtained in the presence of alkaline earth metal ions started darkening/homeotropisation after 8 days (Tiso) of sample preparation. The formation of columnar textures in the presence of Mg2+ and Ca2+ also suggests their importance in inducing the most efficient packaging of DNA molecules in vivo.66 It appears that the size dependency could be of use in trapping a particular texture of DNA for applications in designing DNA-based devices such as biosensors and chips.50 In contrast to the behavior of alkali and alkaline earth metal ions, Cu2+, Cd2+, Al3+, and V4+ ions precipitated DNA into a solid gel at 1 M metal ion concentration and produced translucent gel-like aggregates, lacking mobility at intermediate concentrations (0.625 mM to 1 M). The DNA aggregation observed with Cu2+, Cd2+, Al3+, and V4+ ions could be due to rapid multistep, interstrand/intrastrand cross-linking or chelating of metal ions to phosphate groups and bases.67–69 While experiments using transition and heavy metal ions failed to demonstrate the critical DNA concentration (CD), they required much lower concentrations of the metal ions to induce LC phases. Thus, millimolar concentrations of these ions provoked the LC phase, whereas molar concentrations of alkali and alkaline earth metals were required to accomplish this. However, LC textures produced by heavy and transition metal ions were unstable compared to the LC textures formed in the presence of alkali and alkaline earth metal ions. One of the reasons for the difference in CM observed may be the difference in their binding mode to DNA. Unlike alkali and alkaline earth metal ions, transition metal ions are known to have strong baseaffinity.70 They can chelate or coordinate directly to the nucleophilic atoms of the bases.71–77 (In the IR region, the bases of nucleic acids absorbs at the 1400-1800 cm-1 region and the main absorption bands are as follows: 1580 cm-1, the vibrations of C-N7 of guanine; 1650 cm-1, the vibration of C2-O of cytosine; and 1680 cm-1, the vibrations of C6-O of guanine and C4-O of thymine.77 IR spectra of Cu2+, V4+, and Cd2+ ions showed significant changes in the base absorption region 1400-1800 cm-1 and slight changes in the phosphate absorption regions, which are indicative of both phosphate and base interactions.) This mode of interaction may perturb the


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hydrogen bonding between base pairs, resulting in the destabilization of DNA or the distortion of rigid rod-like behavior required for LC ordering. Although Cu2+ is known to destabilize the double helical structure of DNA, it appears that, below a certain concentration, it mainly binds to phosphate groups and reduces the charge density and intermolecular repulsion, thereby bringing the ordered alignment required to exhibit anisotropic behavior.78 Unlike Al3+ and Cd2+, Cu2+ exhibited a columnar phase with a herringbone pattern (Figure 5), indicative of higher ordering. These textures were the most stable compared to LC phases induced by the four ions studied. Cadmium is well-known for its toxicity in biological systems.79 Langlais et al.70 have interpreted Raman spectra of calf thymus DNA in the presence of Cd2+ to indicate binding to phosphates at AT regions, as well as to the N7 atom of guanine. Catte et al.80 reported the modification of the LC phase in the presence of Cd2+ with low molecular weight fragmented DNA. The effect of Cd2+ on the LC behavior of high molecular weight DNA was more drastic than that observed with Cu2+ and V4+. Only the cholesteric phase with weak birefringence was obtained below the CM (5 mM), and it got darkened within ∼8 h (Figure 4), without transforming into a higher ordered columnar hexagonal phase. Cd2+ is reported to inflict damage on DNA through the induction of single strand breaks, which will distort the rigidity of the DNA molecule. This could be the reason for the weak birefringence and reduced stability of the LC phases. Figure 5 demonstrates that the order of stability of the LC phase is in the following order: Tiso Cu2+ > V4+ > Al3+ > Cd2+. The range of interactions of the metal ions used in the present study varied from nonspecific electrostatic phosphate binding (Li+, Na+, K+, Rb+, Cs+, Mg2+, Ca2+, Sr2+, Ba2+), partly electrostatic phosphate and partly covalent base interactions (Cu2+, Cd2+, Al3+, V4+), to mainly covalent base binding (Hg2+, Pd2+, Au3+, and Pt4+). Our data, thus, revealed very interesting phase behavior in terms of metal ion concentration, size, charge, and binding mode, when metal ions are allowed to interact with DNA. The mobility of the phases in the case of alkali and alkaline earth metal ions indicated that the mode of binding of these ions should be on the phosphate moieties along the DNA strands instead of interstrand cross-linking, which can lead to an arrest of molecular mobility. Recent data also support binding of Li+, Na+, K+, Rb+, and Cs+ to the phosphate groups of DNA, stabilizing the double helix.50,81 Theoretical studies have revealed a significantly higher binding affinity of Li+ to the phosphate group of DNA when compared to those of Na+ and K+ ions.48 These results support the observation of a unique LC behavior of DNA in the presence of Li+ (Figure 8). Similarly, in the case of alkaline earth metal ions, the high binding power of Mg2+ ion to the dianion model of DNA can be correlated with the higher CD required for LC behavior of DNA in the presence of Mg2+ ion.49 In the cases of multivalent, transition, and heavy metal ions, the inter- or intrastrand cross-linking perturbs the supramolecular order disturbing the stability of the phases drastically. Thus, Al3+,52 mainly a phosphate binder, that is expected to stabilize DNA showed a highly unstable cholesteric phase (Figure 5), indicating the destabilization of DNA. The destabilization of DNA in the presence of Al3+ can be explained on the basis of a possible cross-linking (because of the multiple valence of Al3+ ion) to another phosphate moiety of the same strand of DNA. This type of interaction can disrupt the rigidity of DNA, required for its LC ordering and transformation to a columnar phase. Our IR spectra (unpublished results) of Al3+-DNA interaction

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did not provide evidence for base binding of Al3+. It is a matter of conjecture to relate the ability of Al3+ to induce destabilization of the supramolecular assembly of DNA to the reported neurotoxic effect of this metal and its link to Alzheimer’s disease.82 Unlike transition metal ions, the behavior of heavy metal ions with DNA is interesting because of the ability of these metals to cause a drastic reduction in the viscosity of DNA solution. The heavy metal ions are known to form bridges between the stacked bases and between the strands,74,76 which will eventually perturb the double helix leading to the collapse of the helical structure resulting in the drastic decrease in viscosity. The viscous nature and ordered alignment required for exhibiting liquid crystalline behavior presumably might have lost at high metal ion concentration. Hence, it was extremely difficult to optimize the metal ion concentration to get an LC phase. So, the metal ion concentration was brought down where the DNA possesses the viscosity required for LC ordering. The data presented herein also indicate that fluidity as well as the order of the phases are governed by the charge density and the size of the ion. For example, transition to columnar hexagonal texture from the more fluidic cholesteric lower ordered phase of Ca2+ and Sr2+ are definitely determined by the size of the ions (Figure 3). This is interesting because the hexagonal arrangement is the most efficient form of packing in the formation of the toroids.39 Our finding of the hexagonal arrangement of DNA that can be induced by controlling the size and charge of the cations indicates the possibility of controlling the phase structures in applications wherever it is required.

Conclusions In summary, multiple LC phases of DNA could be generated by metal ions at DNA concentrations that are far less than that required for low molecular weight (150 bp length) DNA. Two main phases, cholesteric and columnar hexagonal, are found either separately or in coexistence, with variations in the stability of the phases, depending on local conditions and time. The overall phase behavior is dependent on several parameters, including the concentration, size, charge, and binding mode of the metal ion and time of treatment. One of the important findings of this study is that the CD required to exhibit anisotropy is higher for alkaline earth metals than that for alkali metals, suggesting the importance of size and charge of the metal ion in inducing and stabilizing LC textures DNA. Multivalent, transition, and heavy metal ions differed considerably from alkali and alkaline earth metal ions in their induction and stabilization/ destabiliosation of LC phases, which could be due to their different binding modes. The binding studies of Li+, Na+, K+, Mg2+, and Ca2+ with a DNA fragment, using three-layer quantum mechanical-molecular mechanical (QM/MM) approach have shown that the dianion mode of metal binding, involving the phosphate groups of both strands of DNA, allows for higher DNA binding affinity of the alkaline earth metal ions. It is important to note that highly stable DNA mesophases may find application in the field of nanoelectronics and biosensors. This may stimulate similar studies, with potential application of DNA liquid crystalline structures to gene therapy, and development of gene targeted drugs, DNA based device, and biomarkers. Future studies may find correlations of DNA LC textures to environmental factors involved in the mechanisms of causation of human diseases, caused by an excess or deficiency of the metal ions in the body.

Sundaresan et al.

Acknowledgment. Council of Scientific and Industrial Research, Government of India (to N.S.) and National Institutes of Health (CA080163 to T.J.T. and T.T.). Note Added after ASAP Publication. There was an error in the caption of Figure 9 and refs 23, 25, and 60 in the version published ASAP May 30, 2008; the corrected version was published ASAP June 11, 2008.

References and Notes (1) Bhattacharya, P. K. Metal Ions in Biochemistry; Alpha Science International, Ltd.: Oxon, U.K., 2005. (2) Subirana, J. A.; Soler-Lopez, M. Annu. ReV. Biophys. Biomol. Struct. 2003, 32, 27–45. (3) Denisov, V. P.; Halle, B. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 629– 633. (4) Williams, L. D.; Maher, L. J., III Annu. ReV. Biophys. Biomol. Struct. 2000, 29, 497–521. (5) Hud, N. V.; Polak, M. Curr. Opin. Struct. Biol. 2001, 11, 293–301. (6) Manning, G. S. Q. ReV. Biophys. 1978, 11, 179–246. (7) Schneider, B.; Patel, K.; Berman, H. M. Biophys. J. 1998, 75, 2422– 2434. (8) Munoz, J.; Sponer, J.; Hobza, P.; Orozco, M.; Luque, F. J. J. Phys. Chem. B 2001, 105, 6051–6060. (9) Pastor, N. Biophys. J. 2005, 88, 3262–3275. (10) McFail-Isom, L.; Sines, C. C.; Williams, L. D. Curr. Opin. Struct. Biol. 1999, 9, 298–304. (11) McFail-Isom, L.; Shui, X.; Williams, L. D. Biochemistry 1998, 37, 17105–17111. (12) Heddi, B.; Foloppe, N.; Hantz, E.; Hartmann, B. J. Mol. Biol. 2007, 368, 1403–1411. (13) Thomas, T. J.; Messner, R. P. J. Mol. Biol. 1988, 201, 463–467. (14) Thomas, T.; Thomas, T. J. Biochemistry 1993, 32, 14068–14074. (15) Vijayanathan, V.; Lyall, J.; Thomas, T.; Shirahata, A.; Thomas, T. J. Biomacromolecules 2006, 6, 1097–1103. (16) Eichhorn, G. I.; Shin, Y. A. J. Am. Chem. Soc. 1968, 90, 7323–7328. (17) Kuklenyik, Z.; Marzilli, L. G. Inorg. Chem. 1996, 35, 5654–5662. (18) Duguid, J. G.; Bloomfeild, V. A.; Benevides, J. M.; Thomas, G. J., Jr Biophys. J. 1995, 69, 2623–2641. (19) Koltover, I.; Salditt, T.; Radler, J. O.; Safinya, C. R. Science 1998, 281, 78–81. (20) Koltover, I.; Wagner, K.; Safinya, C, R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97 (26), 14046–14051. (21) Kabanov, A. V.; Kabanov, V. A. Bioconjugate Chem. 1995, 6, 7–20. (22) Yevdokimov, Y. M.; Salyanov, V. I. Biosens. Bioelectron. 1996, 11, 889–901. (23) Cognard, J. Mol Cryst. Liq. Cryst. Suppl. 1982, 78, 1. (24) Sigel, R. K. O. Angew. Chem., Int. Ed. 2007, 46, 654–656. (25) Todd, B. A.; Parsegian, V. A.; Shirahata, A.; Thomas, T. J.; Rau, D. C. Biophys. J. 2008, 94, 4775–4782. (26) Onsager, L. Ann. N. Y. Acad. Sci. 1949, 51, 627–659. (27) Flory, P. J. Proc. R. Soc. London, Ser. A 1956, 243–273. (28) Flory, P. J. Molecular theories of liquid crystals In Polymer Liquid Crystals ; Ciferri, A., Krigbaum, W. R., Meyer, R. B., Eds.; Academic Press: New York, 1982. (29) Rill, R. L. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 342–346. (30) Minton, A. P. Biopolymers 1981, 20, 2093–2120. (31) Livolant, F.; Leforestier, Prog. Polym. Sci. 1996, 21, 1115–1164. (32) Michi, N.; Giuliano, Z.; Brandon, D. C.; Christopher, D. J.; Julie, O. C.; Ronald, P.; Tommaso, B.; Noel, A. C. Science 2007, 318, 1276–1279. (33) Williams, D.C., Jr.; Cai, M.; Clore, G. M. J. Biol. Chem. 2004, 279, 1449–1457. (34) Merchant, K.; Rill, R. L. Biophys. J. 1997, 73, 3154–3163. (35) Livolant, F.; Levelut, A. M.; Doucet, J.; Benoit, J. P. Nature 1989, 339, 724–726. (36) Rill, R. L.; Livolant, F.; Aldrich, H. C.; Davidson, M. W. Chromosoma 1989, 98, 280–286. (37) Livolant, F. J. Mol. Biol. 1991, 218, 165–181. (38) Pelta, J.; Livolant, F.; Sikorav, J. L. J. Biol. Chem. 1996, 271, 5656– 5662. (39) Saminathan, M.; Thomas, T.; Shirahata, A.; Pillai, C. K. S.; Thomas, T. J. Nucleic Acids Res. 2002, 30, 3722–3731. (40) Saminathan, M.; Antony, T.; Shirahata, A.; Sigal, L. H.; Thomas, T.; Thoams, T. J. Biochemistry 1999, 38, 3821–3830. (41) Vijayanathan, V.; Thomas, T.; Shirahata, A.; Thomas, T. J. Biochemistry 2001, 40, 13644–13651.

Metal Ions and Liquid Crystalline DNA (42) Mahtab, R.; Sealey, S. M.; Hunyadi, S. E.; Kinard, B.; Ray, T.; Murphy, C. J. Inorg. Biochem. 2007, 101, 559–564. (43) Vijayanathan, V.; Thomas, T.; Thomas, T. J. Biochemistry 2002, 41, 14085–14094. (44) Sikorav, J. L.; Pelta, J.; Livolant, F. Biophys. J. 1994, 67, 1387–1392. (45) Svensson, M.; Humbel, S.; Froese, R. D. J.; Matsubara, T.; Sieber, S.; Morokuma, K. J. Phys. Chem. 1996, 100, 19357–19363. (46) Vreven, T.; Morokuma, K.; Farkas, O.; Schlegel, H. B.; Frisch, M. J. J. Comput. Chem. 2003, 24, 760–769. (47) Morokuma, K. Philos. Trans. R. Soc. London, Ser. A 2002, 360, 1149– 1164. (48) Sundaresan, N.; Suresh, C. H. J. Chem. Theory Comput. 2007, 3, 1172– 1182. (49) Sundaresan, N.; Pillai, C. K. S.; Suresh, C. H. J. Phys. Chem. A 2006, 110, 8826–8831. (50) Sundaresan, N.; Thomas, T.; Thomas, T. J.; Pillai, C. K. S. Macromol. Biosci. 2006, 6, 27–32. (51) Cotton, F. A.; Wilkinson, G. AdVanced Inorganic Chemistry, 3rd ed.; Wiley: Eastern Mumbai, 1992. (52) Pelta, J., Jr.; Durnad, D.; Doucet, J.; Livolant, F. Biophys. J. 1996, 71, 48–63. (53) Strzelecka, T. E.; Davidson, M. W.; Rill, R. L. Nature 1998, 331, 457–460. (54) Strzelecka, T. E.; Rill, R. L. Biopolymers 1990, 30, 57–71. (55) Wettig, S. D.; Li, C. Z.; Long, Y. T.; Kraatz, H. B.; Lee, J. S. Anal. Sci. 2003, 19, 13–26. (56) Clever, G. H.; Kaul, C.; Carell, T. Angew. Chem., Int. Ed. 2007, 46 (33), 6226–6236. (57) Wettig, S. D.; Wood, D. O.; Aich, P.; Lee, J. S. J. Inorg. Biochem. 2005, 99 (11), 2093–2101. (58) Skuridin, S. G.; Gulyaeva, J. G.; Yevdokimov, Yu.M. Mol. Biol. 2006, 40 (6), 961–969. (59) Tresset, G.; Cheong, W. C. D.; Tan, Y. L. S.; Boulaire, J. R.; Yeng, M. L. Biophys. J. 2007, 93 (2), 637–644. (60) Bruni, P.; Pisani, M.; Amici, A.; Marchini, C.; Montani, M.; Francescangeli, O. Appl. Phys. Lett. 2006, 88, 073901–3. (61) Podgornik, R.; Strey, H. H.; Gawrisch, K.; Rau, D. C.; Rupprecht, A.; Parsegian, V. A. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4261– 4266.


1869

(62) Komyshev, A. A.; Lee, D. J.; Leikin, S.; Wynveen, A. ReV. Mod. Phys. 2007, 79, 943–996. (63) Garner, M. M.; Rau, D. C. EMBO J. 1995, 14, 1257–1263. (64) Strey, H. H.; Podogornik, R.; Rau, D. C.; Parsegian, V. A. Curr. Opin. Struct. Biol. 1998, 8, 309–313. (65) Mrevlishvili, G. M.; Sottomayor, M. J.; Ribeiro da Silva, M. A. V. Thermochim. Acta 2002, 394, 83–88. (66) Achard, M. F.; Klenman, M.; Nashtishin, Y. A.; Nguyen, H. T. Eur. Phys. J. E 2005, 16, 37–47. (67) Bridgewater, L. C.; Manning, F. C.; Patierno, S. R. Carcinogenesis 1994, 15, 2421–2427. (68) Duguid, J. G.; Bloomfield, V. A. Biophys. J. 1995, 69, 2633–2639. (69) Gao, Y. G.; Sriram, M.; Wang, A. H. Nucleic Acids Res. 1993, 21, 4093–4101. (70) Langlais, M.; Tajmir-Riahi, H. A.; Savoie, R. Biopolymers 1990, 30, 743–752. (71) Duguid, J. G.; Bloomfield, V. A.; Benevides, J.; Thomas, G. J. Biophys. J. 1993, 65, 1916–1928. (72) Eichhorn, G. L.; Shin, Y. A. J. Am. Chem. Soc. 1968, 90, 7323–7328. (73) Pillai, C. K. S.; Nandi, U. S. Biopolymers 1977, 17, 709–729. (74) Pillai, C. K. S.; Nandi, U. S. Biopolymers 1973, 12, 1431–1435. (75) Pillai, C. K. S.; Nandi, U. S.; Levinson, W. Bioinorg. Chem. 1977, 7, 151–157. (76) Pillai, C. K. S.; Nandi, U. S. Biochim. Biophys. Acta 1977, 474, 11– 16. (77) Andrushchenko, V. V.; Kornilova, S. V.; Kapinos, L. E.; Hackl, E. V.; Galkin, V. L.; Grigoriev, D. N.; Blagoi, Y. P. J. Mol. Struct. 1997, 408-409, 225–228. (78) Tajmir-Riahi, H. A.; Langlais, M.; Savoie, R. Nucleic Acids Res. 1988, 16, 751–762. (79) Giaginis, C.; Gatzidou, E.; Theocharis, S. Toxicol. Appl. Pharmacol. 2006, 213, 282–290. (80) Catte, A.; Cesare-Marincola, F.; Van der Maarel, J. R.; Saba, G.; Lai, A. Biomacromolecules 2004, 5, 1552–1556. (81) Badisa, V. L.; Latinwo, L. M.; Odewumi, C. O.; Ikediobi, C. O.; Badisa, R. B.; Ayuk-Takem, L. T.; Nwoga, J.; West, J. EnViron. Toxicol. 2007, 22, 144–151. (82) Anitha, S.; Rao, K. S. J. Structure and Bonding; Springer: Berlin/ Heidelberg, 2002.

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