Some Features of Surfactant Organization in DNA Solutions at Various NaCl Concentrations

The photosensitive azobenzene-containing surfactant C4-Azo-OC6TMAB is a promising agent for reversible DNA packaging in a solution. The simulation of the trans-isomer surfactant organization into associates in a solution with and without salt as well as its binding to DNA at different NaCl concentrations was carried out by molecular dynamics. Experimental data obtained by spectral and hydrodynamic methods were used to verify the results of simulation. It was shown that head-to-tail aggregates with close to antiparallel orientation of surfactant molecules were formed at certain NaCl and surfactant concentrations (below critical micelle concentration). Such aggregates have two positively charged ends, and therefore, they can be attracted to negatively charged DNA phosphates far located along the chain, as well as those that belong to different molecules. This contributes to the formation of intermolecular DNA–DNA contacts, and this way, the experimentally observed precipitation of DNA can be explained.


■ INTRODUCTION
Currently, compounds containing azobenzene, 1−6 including the charged ones, are of great interest because of their ability to photoisomerization. When exposed to ultraviolet radiation, the azobenzene group transfers from a stable trans form to a metastable cis form.
The cationic surfactant C 4 -Azo-OC 6 TMAB ( Figure 1) is an amphiphilic compound containing an azobenzene fragment in its hydrophobic tail, which is connected to a charged head group by a short alkyl chain.
It had been shown earlier 7−11 that azobenzene-containing surfactants bind to DNA phosphates and can form micelle-like structures in solutions with low NaCl concentration and with the surfactant concentration lower than the critical micelle concentration (CMC). This follows from a similar change in the absorption spectra of surfactants in a DNA solution and in micelles. The shrinkage of the DNA coil in the 0.005 M NaCl solution can be induced by C 4 -Azo-OC 6 TMA + (hereinafter abbreviated as Azo) binding at a charge ratio z < 1 (z is the ratio of the surfactant and DNA phosphate molar concentrations). At z > 2, one can observe the formation of discrete nanosized particles. It should be noted that DNA precipitation in the form of loose flakes at certain z values (1 < z < 2) was observed in such solutions. The boundaries of these zones (a drop in the volume of a DNA molecular coil, DNA precipitation, and the formation of discrete nanoparticles) are described by phase diagrams. 7 The UV irradiation of the surfactant solution induces a trans− cis conformational transition of the azobenzene group, which is easily monitored by a change in the absorption spectra. 8,12 Same UV-induced isomerization of surfactants after formation of their complexes with DNA causes an increase in DNA volume because of lower possibility of cis-isomer to produce DNA packaging at the same concentration as for trans-isomer. 7,8 This property of the surfactant can be used in light-induced reversible compaction−decompaction of DNA to create bionanostructures for various purposes. 5,13−15 The Azo has a positive charge at neutral pH. The ionic strength of surfactant solution determines the CMC 11 and the ability of the surfactant to form associates with different orientation of molecules (head-to-tail or head-to-head). 9 It is known that the ionic strength of solution has a great influence on the DNA−surfactant interaction. At a high salt concentration (>0.5 M NaCl), the surfactant does not induce DNA shrinkage and DNA condensation. 7,16 It was surprising that the colloidal instability in DNA− surfactant solutions with the NaCl concentration in the area of 0.01−0.5 M was observed in a wide range of z value at [Azo] < CMC. 9 It was suggested that the stability of DNA−surfactant solutions depends on the state of the surfactant molecules at different salt concentrations. An assumption was made that the predominantly head-to-tail type of surfactant associates was formed in solutions at intermediate salt concentrations (0.01− 0.5 M) and Azo concentrations C(Azo) < CMC. It was assumed that such associates have two positively charged ends, and therefore, they can be effectively attracted to the negatively charged DNA phosphates located far along the chain, as well as to those belonging to another DNA molecule. Such binding induces DNA aggregation and precipitation. 9 In other words, head-to-tail orientation of surfactant molecules in the associates contributes to the formation of intermolecular DNA−DNA contacts, resulting in phase separation in DNA−surfactant solutions. Nevertheless, the experimentally observed difference in the behavior of DNA−surfactant systems at different NaCl concentrations requires more detailed studies.
Experimental methods provide the information on average values of conformational parameters of DNA and surfactant absorption. Therefore, it is difficult to investigate the behavior of individual molecules. Computer simulation provides an additional information on a molecular level that cannot be extracted from the experiment. 7,17 The aim of this work is to study by simulation the behavior of the Azo cation in trans-conformation being present in aqueous solutions and to detect the possibility of surfactants to form head-to-tail aggregates. We are going to determine the structure of the formed aggregates and to analyze the effect of the ionic strength on aggregation and on the surfactant interaction with DNA. In addition, it is interesting to find the reason of the experimentally observed colloidal instability in surfactant−DNA solution within a certain salt concentration range. Experimental data were also obtained to verify the results of simulation.

■ MODEL AND SIMULATION METHOD
An Azo cation (Figure 1) consists of a trimethylammonium "head" with a charge +1(e) and a hydrophobic "tail" containing the azobenzene group. The charge of the head is distributed on its hydrogen atoms. In this work, only the trans-isomer of the Azo cation is investigated. In our simulations, the bromine anion was substituted by the chlorine anion.
Four solvents with different ionic strengths (see Table 1) were used for simulations. They were: (1) aqua, (2) saline (NaCl aqua solution with the physiological salt concentration, 0.15 M), (3) medium concentrated (0.23 M) NaCl aqua solution, and (4) highly concentrated NaCl aqua solution (0.8 M). For the simulations, the explicit models of Na + and Cl − ions were added to model water. Electrostatic forces between ions, partially charged atoms of water molecules, atoms of Azo cations, and DNA were calculated implicitly with the Ewald method. 18 In the Table 1, ionic strengths of the systems are presented as strengths of the solvent. The addition to the ionic strength by Azo chloride and DNA was not counted.
Three systems with Azo chloride (marked 1−3) and four systems with Azo chloride and DNA (marked with I−IV) were investigated. Systems 1−3 contained 18 Azo cations, and 18 Cl − anions in solvents of different ionic strengths (Table 1) were investigated on the first stage.
Then, the interaction of Azo cations with a fragment of double-stranded DNA with the length of 18 base pairs d(GCCCAGCATTTCACCCAG) and with a total charge −34(e) was introduced. The four systems that differed by the number of Azo cations and by the NaCl concentration (see Table 1) were simulated. In two systems (I and III), the charge of the DNA was partially compensated by the charge of Azo cations (z = 0.5) and in the rest of the systems (II and IV), the compensation was complete (z = 1).
Simulation was carried out by molecular dynamics (MD) in AKMD program 19 using the AMBER 20 force field and SPC/E model for water; parameters from work 21 were used for Na + and Cl − . The charges at the atoms of the head group and of the neighboring −CH 2 − group were calculated in GAMESS 22,23 (see Table 2). Geometry optimization of the isolated Azo cation was carried out in the B3LYP/6-311G**++ semi-empirical DFT method with wide basic set of Gaussians. In optimized geometry, restricted Hartree−Fock ab initio calculations with 6-31G basis set was used for the partial charge calculation within the Mulliken method.
The obtained values of the charges essentially differ from the values used in work, 24 where the N atom of the head has a positive charge. According to Pauling, 25−28 the electronegativity of N is equal to 3.04 compared to that of C, 2.55, and of H, 2.20. Therefore, the N charge must be less than C charge, and C charge must be less than H charge. Partial charges on atoms calculated in GAMESS almost satisfied the relationship between the electronegativities of the elements. H charge turns out to be greater than the C and N charges, but C charge is more negative than N charge.
First, each prepared system was equilibrated in the NpT ensemble. The further simulation was carried out in the NVT ensemble with the time step 2fs. The temperature was maintained at 21°C by the Nose−Hoover thermostat. 29 The average pressure during the simulation and the total time of the productive run are presented in Table 1. The electrostatic interactions were treated with the Ewald method. 18 The lengths of the bonds were maintained with the SHAKE algorithm. 30 Using the obtained MD trajectories, the following characteristics were calculated: distribution of Azo cations over the size of the aggregate, the order parameter of the Azo cation in the aggregate, and the cylindrical distribution function (CDF) for atoms of the Azo cation.
■ RESULTS AND DISCUSSION Surfactants in Solution with and without Salt. Despite the fact that the C 4 -Azo-OC 6 TMAB does not form a liquid crystal (LC) phase under the considered conditions, the compounds containing an azobenzene group are able to form ordered structures in a solution and used to feed the liquid crystal phase in the presence of polymers or without them, as it follows from a number of studies. 31,32 Azobenzene LCs have been created in which azobenzene moieties play roles both as mesogens and as photosensitive chromophores. Azobenzene LC films showed a nematic phase for trans isomers and no LC phase for cis isomers. Trans−cis photoisomerization of azobenzene with a laser pulse resulted in a nematic-to-isotropic phase transition with a rapid optical response of 200 ms. 32 Note that we do not consider this ability of trans−cis isomerization for the surfactant used in this work.
Both in unsalted (1) and in salted (2, 3) solutions (Table 1), the Azo cations stick together into aggregates which do not look like micelles and have another shape. Surfactant cations in each aggregate were oriented nearly parallel or antiparallel to each other as in liquid crystals. The aggregates are stable, and they do not dissolute in the simulation process. Generally, if surfactant cations stuck together, they remained so until the end of simulation time.
To study these aggregates in detail, the order parameter was introduced as follows. For each Azo cation, the principal axes of the inertia tensor were calculated. The long molecular axis was chosen as the axis of minimal inertia following the method for rod-like nematic liquid crystals. 19 Averaged direction of the long axes over all Azo cations in an aggregate was set as the director. The order parameter (S = ⟨(3cos 2 θ − 1)/2⟩, where θ is the angle between the director and the long axis of the molecule) 33 has values in the range of 0.5 to 0.8 (see Table S1 in the Supporting Information), depending on the size of the aggregate.
The order parameter falls with the growth of the aggregate size. It indicates that surfactants in large aggregates are not as ordered as in small ones. In small aggregates, the Azo cations are arranged without a shift along the long axis that resembles a fragment of the smectic phase layer. In large aggregates, cations move easier along the long axis and can deviate from the average direction at the periphery of the aggregate, so that it decreases the order parameter. With the increase of the number of Azo cations in aggregates, a tendency is observed of their turning into spherical micelles. It leads to increasing the entropy term in the energy of the aggregate.
The Aggregates are formed because of the hydrophobic interactions. 7 Certainly, the association of hydrophobic "tails" of the surfactants interacting with DNA does contribute to DNA condensation (along with the screening of DNA phosphates by positively charged heads of surfactants), as far as it affects the polymer−solvent interaction. In addition, the attraction of hydrophobic "tails" of surfactants associated with deleted DNA segments contributes to the packaging of macromolecules.
DNA−Surfactant Interactions in Solution with Different Salt Concentrations. Surfactant binding to DNA induces spectral changes similar to those observed for surfactants in micelles (see Figure 2, spectrum 2). It indicates that surfactants    Figure S1 in the Supporting Information). form a micelle-like structure on a DNA molecule. DNA interaction with the surfactant in solutions at high salt concentrations (>0.5 M NaCl) was not observed. 7,8,16 Simulation shows that the interaction of Azo cations with DNA is not observed in system III, that contains DNA, NaCl, and 17 Azo cations (see Table 1). Azo cations stick to the ends of DNA that is not interesting for us ( Figure S1 in the Supporting Information). In all other systems (I, II, and IV), Azo cations form a large aggregate on DNA (see Figure 4). Azo cations in aggregates are oriented parallel or antiparallel to each other (see Table 1 in the Supporting Information) similar to that observed in the absence of DNA. This result is in good agreement with the spectral data. Positively charged heads of Azo cations are attracted to the negatively charged phosphate groups on DNA.
The interaction of Azo cations with DNA decreases at high salt concentrations because of the screening of the DNA and Azo charges by salt ions. Indeed, in salted solution (system IV), the aggregate consists of fewer ions than in the unsalted ones (system II). It should be emphasized that the effect of salt on the organization of the surfactant in associates differs from that in the presence of DNA.
It is interesting to note that in system I, the charged heads of Azo almost penetrate into the major groove of DNA near the AT base pair. Nevertheless, the circular dichroism DNA spectra in the presence of surfactants show that DNA bases do not interact with surfactants, and DNA−surfactant binding does not affect the secondary structure of the macromolecule (see Figure S2 in the Supporting Information).
In system I, all positively charged ions (Azo and Na + ) must form an ionic atmosphere around DNA. In this case, Azo cations interact with DNA via electrostatic interactions.  Table 1). Oxygen atom (not shown) of the central Azo cation is on top. On the density scale, the unit corresponds to the average concentration of Azo cations multiplied by 64, that is, 252 mM.
In system IV which contains DNA, NaCl, and 34 Azo cations, an Azo cation penetrates into the minor groove of DNA and resides there up to the end of the simulation time, its charged head being out of the groove. Such a result was observed during the simulations earlier. 7 At the same time, the experiments show the absence of Azo in the minor groove of DNA in Azo−DNA complexes. 7,9 For detailed consideration of the aggregate structure, the CDFs 34,35 were obtained. For system I, the CDFs for the atoms of Azo cations are shown in Figure 5. It can be seen that benzene rings of the neighboring Azo cations in the aggregate mostly have T-shaped orientation with a slight shift. The distance between the lines on which the centers of the benzene rings of neighboring Azo cations in the aggregate are located is approximately 5 Å. This value is consistent with the distance between the centers of neighboring benzene molecules in the dimer of T-shape configuration. 36 CDF for the oxygen atom ( Figure 5d) shows that neighboring Azo cations in the aggregate have predominantly antiparallel orientation. Also, it was shown that orientation of benzene rings in aggregates is independent of salt concentrations and of the presence of DNA in solution.
The absorption spectrum of the surfactant in complexes with DNA in 0.005 M NaCl coincides with that for micelles. It is natural to assume that the state of the surfactant molecules in complexes with DNA at 0.005 M NaCl is the same as in micelles with predominantly head-to-head orientation of neighboring molecules and parallel association of hydrophobic "tails." A high instability in DNA−surfactant solutions at a salt concentration around 0.15−0.5 M NaCl and at a DNA concentration higher than 0.002% was observed also because of the viscosimetric data.
Indeed, the dependence of the reduced viscosity of DNA solutions on the z value (Figure 6a The data show that the only change in the DNA persistent length cannot explain the observed decrease in (α 1 − α 2 ). The predominant orientation of the "tails" of surfactants in the complexes with DNA does not coincide with the normal to the axis of the DNA helix but is observed at a certain angle to this axis. The model of the DNA interaction with the surfactant obtained by simulation (system Isalt-free and half compensated DNA phosphates by Azo cations) is in good agreement with this conclusion.
In contrast, at a significantly high salt concentration (1 M NaCl), the presence of the surfactant does not affect the intrinsic viscosity of DNA and its optical anisotropy (graph 4 in Figure 6a and graph 2 in Figure 6b) that demonstrates the absence of interaction.
The binding of surfactants to DNA can promote the attachment and orderly placement of DNA segments (side by side). Unfortunately, our study of birefringence in DNA solutions with surfactants did not reveal the formation of such structures (in contrast, one could see a decrease in DNA optical anisotropy). Of course, the usage of short double-stranded molecules and large concentrations of DNA should lead to the appearance of ordered structures and self-assembling systems, as was observed in studies. 37−39 A surfactant would certainly contribute to the formation of such structures. The usage of photosensitive surfactants together with short DNA can lead to the formation of amazing systems. 15 DNA Aggregation at Intermediate Salt Concentrations. It turns out that we could not determine DNA intrinsic viscosity in solutions with the surfactant in the range of ionic strength about 0.15−0.5 M NaCl because of the high instability in the DNA−surfactant solution. Indeed, a large data scatter in determining the reduced viscosity and the nonlinear concentration dependence of the reduced viscosity of DNA solutions on the DNA concentration prevents obtaining the correct result. The dependence of the reduced viscosity on the z value in 0.15 M NaCl (see Figure 6a) and its type indicates the emergence of intermolecular macromolecular associates of DNA induced by surfactant linkages with different DNA molecules. It requires the presence of surfactant aggregates with head-to-tail orientation.
As a result, surfactant binding to DNA in 0.15 M NaCl induces thermodynamic instability in solutions. Indeed, sometimes the partial precipitation of DNA in the form of loose flakes was observed.
It is useful to note here that the precipitation of DNA from the solution in the presence of multivalent polyamine cations (spermine or spermidine) was observed in a work. 40 In our case, the head-to-tail aggregates of Azo can be considered as effective "bridges" between DNA segments like multivalent cations.
As it was shown earlier, 9 two states of surfactants can be distinguished at different salt concentrations for surfactant concentrations below CMC. The spectra were divided into two clearly distinguishable intensity levels for low (<0.01 M) and for intermediate (>0.05 and <0.15 M) salt concentrations without a band shift. The first state, mainly monomeric, exists at very low NaCl concentrations. In such an environment, an insufficient shielding of cationic heads prevents formation of associates of hydrophobic tails. At higher NaCl concentration, the convergence of surfactant molecules becomes possible, and the second state of the surfactants as nonmicellar associates of surfactants was observed. It can be assumed that the arrangement of molecules in such aggregates is different from that realized in micelles. In addition, in this region of the salt concentration, the instability of the DNA solutions with a tendency to precipitate in this region of the salt concentration may indicate the formation of a special kind of aggregates, which promotes the formation of intermolecular DNA−DNA contacts. The structure of the aggregates is considered in detail by simulation in the section "Surfactants in Solution with and without Salt." What is the mechanism of colloidal instability in the DNA− surfactant solution that was observed in experiments? Simulation has shown the existence of head-to-tail aggregates, where Azo cations are oriented antiparallel to each other. Such an aggregate has two positively charged ends, like a multivalent cation, and can act as a sticker for two negatively charged DNA molecules.
This can explain the observed precipitation 9 of DNA molecules in solution with an intermediate NaCl concentration Because of the small Debye radius (<3 nm for NaCl >0.01 M), electrostatic repulsion between charged groups is suppressed. Formed associates cause instability in DNA solutions that is fixed by the behavior of viscosity (Figure 6a).
Types of Aggregates. Usually two typical modes of association are considered for aggregation of hydrophobic molecules in water: micelle formation and isodesmic aggregation. 41 A common model for amphiphilic molecules assumes that only two species, namely, monomers and micelles, are found in solutions depending on the concentration. These two states are separated by the CMC. An alternative model (isodesmic aggregation or EK-model) considers a more gradual transition to aggregates with their stepwise formation. Each step of such an association is characterized by the same association constant. At the same time, other models of the self-association in such systems are also considered. For example, the existence of several thermodynamic models of micelle formation are observed: pseudo-phase separation model, closed association model, isodesmic model, and cooperative aggregation model. 42 The pseudo-phase separation model 43 considers monomers and micelles to be distinct pseudo-phases in equilibrium with each other; the closed-association model, also referred to as the "reversible two-state model"; 44 micellization as a stoichiometric association reaction; the isodesmic model or the cooperative aggregation model 45,46 assumes stepwise aggregate formation; and the cooperative aggregation model 47 assumes dimer formation to be more difficult than all subsequent association steps.
In our work, we separate the systems, according to the NaCl concentration: • does not prevent the formation of associates with head-to-tail orientation and does not allow associates with head-to-head orientation. This is due to the correlation in the Debye screening radius (that depends on the NaCl concentration) and the distance between charged surfactant groups in such associates.
Self-assembly of amphiphilic molecules into associates in water solution requires noncovalent interactions (electrostatic interactions, hydrogen bonds, and van der Waals forces). Because of the "hydrophobic effect," the aggregation of alkyl chains of surfactants in an aqueous environment oriented molecules and turned them together into ordered associates. This became possible only at a certain NaCl concentration in a solution, which provided a screening of charged groups of surfactant molecules that would keep them together in associates with head-to-tail orientation and at the same time allow these associates to be attracted to DNA (at a salt concentration above 0.5 M, NaCl surfactant does not interact with DNA phosphates). Determination of aggregation types depending on the salt concentration requires further study.

■ CONCLUSIONS
The effect of the ionic strength on the aggregation of the azobenzene-containing surfactant and on its interaction with DNA was considered.
Simulation shows that in solution without DNA, the increase of the ionic strength promotes the aggregation of surfactant cations and leads to increasing the size of the formed aggregates. This

■ EXPERIMENTAL METHODS
Because the most important effect of the surfactant on the structure of DNA is the compaction of a macromolecule in solution, it is clear that it is most convenient to use the high molecular DNA in the experiment. Comparison of the experimental data with the results of computer simulation confirms that surfactants bind to DNA via nonspecific electrostatic interactions, and the sequence of nucleotides does not affect this binding. Indeed, surfactants interact with phosphate groups with the location around the double helix. The calf thymus DNA (Sigma-Aldrich) was used in the experiments. The molecular mass of the calf thymus DNA M = 10 7 was determined using DNA intrinsic viscosity ([η] in dL/ g) in 0.15 NaCl according to DNA was dissolved in ultrapure water (Milli-Q) and after 5 days of storage at 4°C, a salt solution was added to achieve [NaCl] = 0.005 M, followed by filtration or centrifugation of the DNA solution. The concentration of DNA in the stock solution was determined using hydrolyzed DNA (boiling in 6% HClO 4 at 100°C for 20 min). The concentration was determined from the difference in UV absorption at two wavelengths (270 and 290 nm). 48 This approach allows us to control the stability of the secondary structure of DNA (double helix) using the value of the molar extinction coefficient E 260 , as determined from the absorption of DNA solutions at 260 nm, taking into account the concentration determined for denatured DNA A surfactant C 4 -Azo-OC 6 TMAB was synthesized at the University of Potsdam (Laboratory of Prof. S. Santer). DNA− surfactant complexes were prepared by mixing equal volumes of DNA and surfactant solutions in 0.005 M NaCl or at other salt concentrations.
The absorption spectra in the UV and in visible regions were recorded using an SF-56 spectrophotometer (LOMO, Russia). In addition, a viscometric method was used with a low-gradient Zimm-Crozer-type viscometer. The measurements were carried out at a temperature of 21°C.
The flow birefringence method 49 was applied for determining DNA segmental optical anisotropy (the difference in the polarizabilities of Kuhn's segment along and normal to the axis of the DNA helix). Circular dichroism spectra were registered by Dichrograph Mark 4 (Jobin Ivon). An atomic force microscope NanoScope 4a (Veeco) was used for the imaging of Azo−DNA complexes on a fresh mica surface via direct scanning mode. DNA fixation was carried out with spin coating.
Order parameter S zz for Azo cation aggregates and results of simulation for system III and experimental data (CD spectra, viscosity data, and AFM images) (PDF) Formation of an aggregate on DNA in unsalted solution (system I) (MP4)