DNA-Surfactant Interactions. Compaction, Condensation, Decompaction and Phase Separation

Journal of the Chinese Chemical Society, 2004, 51, 447-469 447 Invited Paper DNA-Surfactant Interactions. Compaction, Condensation, Decompaction and Phase Separation Rita Dias a,b, Mónica Rosa a,b, Alberto Canelas Pais a, Maria Miguel a and Björn Lindman* a,b a Departament of Chemistry, University of Coimbra, 3004-535 Coimbra, Portugal b Department of Physical Chemistry 1, Centre for Chemistry and Chemical Engineering, P. O. Box 124, S-22100 Lund, Sweden Recent investigations of the interaction between DNA and alkyltrimethylammonium bromides of various chain lengths are reviewed. Several techniques have been used such as phase map determinations, fluorescence microscopy, and electron microscopy. Dissociation of the DNA-surfactant complexes, by the addition of anionic surfactant, has received special attention. Precipitation maps for DNA-cationic surfactant systems were evaluated by turbidimetry for different salt concentrations, temperatures and surfactant chain lengths. Single-stranded DNA molecules precipitate at lower surfactant concentrations than double-helix ones. It was also observed that these systems precipitate for very low concentrations of both DNA and surfactant, and that the extension of the two-phase region increases for longer chain surfactants; these observations correlate well with fluorescence microscopy results, monitoring the system at a single molecule level. Dissociation of the DNA-cationic surfactant complexes and a concomitant release of DNA was achieved by addition of anionic surfactants. The unfolding of DNA molecules, previously compacted with cationic surfactant, was shown to be strongly dependent on the anionic surfactant chain length; lower amounts of a longer chain surfactant were needed to release DNA into solution. On the other hand, no dependence on the hydrophobicity of the compacting agent was observed. The structures of the aggregates formed by the two surfactants, after the interaction with DNA, were imaged by cryogenic transmission electron microscopy. It is possible to predict the structure of the aggregates formed by the surfactants, like vesicles, from the phase behaviour of the mixed surfactant systems. The compaction of a medium size polyanion with shorter polycations was furthermore studied by means of Monte Carlo simulations. The polyanion chain suffers a sudden collapse as a function of the condensing agent concentration and of the number of charges on the molecules. Further increase of the concentration gives an increase of the degree of compaction. The compaction was found to be associated with the polycations promoting bridging between different sites of the polyanion. When the total charge of the polycations was lower than that of the polyanion, a significant translational motion of the compacting agent along the polyanion was observed, producing only a small-degree of intrachain segregation. However, complete charge neutralization was not a prerequisite to achieve compacted forms. Keywords: DNA; Surfactant; Vesicles; Compaction; Decompaction; Precipitation; Fluorescence microscopy; Monte Carlo simulations. INTRODUCTION The compaction of DNA, together with the reduction of its charges, is believed to facilitate the uptake of nucleic acids through the cellular membrane. 1-5 Since the strong binding of cationic surfactants to DNA allows these two effects to be fulfilled, it is not surprising that the complexation with cationic lipids is one strategy for delivery of DNA to cells. However, synthetic cationic surfactants per se cannot be used for this purpose, since the complexes of DNA and cationic micelles do not result in effective transfection. It is a common viewpoint to explain this low transfection by the cytotoxicity of surfactants and a low stability of these complexes upon a change in the environment. 6 In spite of this, quaternary ammonium surfactants can be used, in small amounts, for charging of neutral liposomes, thereby improving their transfection efficiency; they have the advantage of lower cost when compared with other synthetic lipids. 6,7 The degree of compaction is not often discussed but it is believed to be important for the delivery of DNA to cells. Interestingly, opposite to what could be expected, not always the most compact complexes are the most efficient. 8

448 J. Chin. Chem. Soc., Vol. 51, No. 3, 2004 Dias et al. After delivery, DNA must become accessible to the enzymatic machinery of the cell. Since lipid complexation is known to inhibit at least certain DNA processing enzymes such as DNAse, 9-11 it is likely that the transfected DNA can become active only by release from the lipid complex. In vitro such release can be accomplished by addition of anionic species, like surfactants, which bind the cationic lipid and release the DNA, 12,13 and there are indications that such a mechanism may play a role also in vivo, 14 at least for oligonucleotides. Bhattacharya and Mandal 12 have shown by circular dichroism, electrophoresis and the DNAse protection assay, that after the release the DNA is in its native B-form. Because of the growing interest in this field and numerous applications of the DNA-cationic surfactant systems, several studies have been presented in the literature. The strong association displayed by DNA and cationic surfactant systems is well-known, and it is related to some applications: in 1967 a procedure was first described 15 that used quaternary ammonium surfactants to precipitate DNA for its extraction and purification. A few years later, cetyltrimethylammonium bromide (CTAB) was also used for the precipitation and counting of small quantities of DNA. 16 The CTAB-DNA precipitation method is still in use, with more or less complicated modifications. 17-20 Binding of cationic surfactant to DNA occurs at concentrations well below the CMC of the surfactant and the binding isotherms have a sigmoidal shape which demonstrates the cooperative binding. 21-24 The binding isotherms were shown to be strongly dependent on the surfactant chain length, 21 suggesting that hydrophobic interactions are important for the interaction and that it was analogous to the formation of micelles. Also, the binding constants were shown to change with the salt concentration, 21,22 indicating strong electrostatic interactions between the negatively charged DNA and the oppositely charged surfactants. The complex formation between short DNA fragments and dodecyltrimethylammonium bromide (DTAB) was studied by using a combination of several techniques: dynamic light scattering, capillary electrophoresis and surfactant selective electrodes 23 and the surfactant was shown to bind in two stages. In the first stage, the surfactant exchanges with the counter-ions condensed on the surface of DNA, not changing the effective charge on the surface of DNA. In the second stage, further binding occurs, without exchange of counter-ions, which leads to a dramatic change in the effective charge of DNA. Phase separation occurs for higher DNA concentrations. A number of studies using fluorescence probes and techniques like fluorescence quenching or fluorescence spectroscopy have also been presented. 25-27 Ethidium bromide, for example, is known to be displaced from DNA molecules as the surfactant binding starts and, even though the process is not very sensitive, it has been used in several studies. For example, an investigation was conducted on the dissociation of DNA-cationic surfactant complexes by the addition of salt, 27 and it was shown that the amount of salt required to dissolve the complexes was not dependent on the surfactant chain length but markedly decreased with the increase in the number of methyl groups in the head of the surfactant. It was suggested that the distance between the DNA and surfactant charges is an important factor that determines the stability of the complexes. The structure of the complexes has been studied by small angle X-ray scattering (SAXS). While for the DNA- DTAB systems, the structure is not well known, 28 the structure with CTAB is believed to be hexagonal. 29,30 The first picture, given by Ghirlando et al., 29 shows hexagonal packing of DNA and rod-like CTAB micelles, in a normal hexagonal packing. However, also the formation of inverted phases with the DNA coated with surfactant and the hydrophobic tails facing the solution has been suggested. 31 The formation of these hydrophobic particles is counterintuitive since this structure would not be stable in solution as was indicated in molecular simulations. 32 A recent study by Leal et al., based on geometrical considerations, as well as hydration and NMR measurements, 33,34 has proved that the DNACTA complex, when fully hydrated, has a normal hexagonal structure. In this work 33 it was also suggested that the DNA is in the A-form inside the complexes, since the electrostatic repulsions between the phosphate groups are screened, a similar behaviour as to that observed with the addition of salt. The A-form conformation of DNA is also preferred due to packing considerations. Fluorescence microscopy studies on large DNA molecules, several kilo base pairs (kbp), have shown that cationic surfactants induce a discrete collapse from DNA coil to a compact globular form. For intermediate concentrations of surfactant a region is observed where both DNA coils and globules coexist. 24 This coexistence region is a common phenomenon for DNA molecules on the addition of condensing agents such as organic solvents, 35 flexible polymers, 36 and multivalent ions. 37-39 The coil-globule transition of long DNA molecules is then discrete, a (quasi-) first-order transition for individual chains, but continuous for their ensemble average. 35,37 Compaction of DNA is believed to be driven by attractive interactions between different parts of the mole-

DNA-Surfactant Interactions J. Chin. Chem. Soc., Vol. 51, No. 3, 2004 449 cule, by ion correlation effects arising from the presence of multivalent ions, for example, 40,41 leading to the formation of a nucleation centre in the DNA chain that grows along the molecule chain. 42 Due to the hydrophobic interactions between the cationic surfactant molecules, these will self-assemble and act as multivalent ions, inducing DNA compaction. The importance of DNA compaction and packaging in living cells was mentioned above. It is believed that DNA has three levels of compaction in cells. First, the nucleosomes, in which DNA is wrapped around spherical positively charged proteins, the histones. Part of the DNA is not bound and will link several nucleosomes forming a structure like beads on a string. The winding of DNA around the histones contributes to its packaging by reducing its extension; nevertheless the packing ratio of the nucleosome is only of around 7. The second level of compaction appears when these DNA-protein complexes, nucleosomes, are arranged in a helical array, the chromatin fibre, which can achieve a condensation degree of about 40. The folding of such solenoid fibres into long loops attached to a nonhistone scaffold forms tertiary structures such as the chromonema fibres which provide additional condensation. 43-45 Also the packaging of DNA in bacteriophage capsids is quite remarkable. Several studies have been made to shed light on the arrangement of DNA in phage capsids or simply in a spherical cavity. 46,47 A series of other positively charged agents within the cell are believed to also play a role. Polyamines, like spermidine and spermine, constitute a group of cell components and even though their functions are mostly unknown, it is believed that they are important in the regulation of cell proliferation and differentiation. Since they are always found in association with nucleic acids it is also widely accepted that they act as helpers in DNA packaging. Interestingly, the most compact DNA is found in sperm heads where the condensing agents are protamines, argininerich linear proteins. The interactions between polyamines and DNA started to be studied some time ago 48,49 and the interest for these systems has grown steadily. Polyamines interact predominantly by electrostatic interactions with DNA molecules 50 inducing their compaction, 51 aggregation 52 and precipitation. 53,54 Although these systems have been much studied for decades, there are a number of interesting points which only recently have been explained. Spermine, for example, is used in high concentrations as a crystallizing agent; nevertheless, spermine molecules are usually not detected in X-ray studies. It has been proposed recently 55 that the flexible polyamine molecules interact in an irregular manner with the DNA molecule, with no definite binding sites. One of the most interesting techniques to study the interaction between DNA and condensing agents is fluorescence microscopy since it enables us to visualize and follow the conformational changes of single DNA molecules in solution. A number of studies on the interaction between DNA and polyamines (and related agents such as chitosans, see e.g. Ref. (56)) have been presented in the literature. 57-59 There are a number of interesting aspects of these systems. One is the fact that the compaction of DNA is quite critical. That is, either the macromolecule is in an extended conformation exhibiting a slow worm-like motion (coil), or it is in the compact state, characterized by a higher fluorescence intensity and a long-axis length of less than 1.0 m (globules); no intermediate states are usually found. For intermediate polyamine concentrations, both populations coexist. Even though the amounts required to compact DNA are rather small (10 M of spermine, for example) it should be noticed that it corresponds to a large excess of positive charges when compared to the concentration of DNA nucleotides, 0.1 M. 58 It was also observed that the concentrations of the flexible polycations, spermidine, with three positive charges, and spermine, with four charges, required to induce compaction of DNA, differ by about one order of magnitude. 58,59 From the studies presented in the literature concerning the interaction between DNA and positive agents, 24,38,39,60 only when longer polycations were used, the mixing ratio for compaction between positive and negative charges was 1 61 or below. 57 Varying degrees of compaction have been predicted through molecular dynamics simulations for oppositely charged chains of the same length, 62 depending on the strength of the interaction. Also, the characterization of complexes in solutions containing positive and negative chains has been carried out by Monte Carlo simulation, 63,64 showing that the nature of the complexes varies with the linear charge density of the chains. Stability of the DNA molecules in solution. Temperature and salt dependence DNA molecules in their native state adopt the B conformation. However, slight changes in the conditions may lead to a loss of the secondary structure and the melting of the double-helix can occur. DNA melting is the mechanism of separating the two strands in the double-stranded DNA molecules (dsdna) into two single strands (ssdna). This transition is thermally induced and the temperature at the middle point of

450 J. Chin. Chem. Soc., Vol. 51, No. 3, 2004 Dias et al. the transition is called melting temperature, T m. The dsdna to ssdna transition, or the melting temperature, is dependent on the salt concentration and on the base composition of DNA. It is natural to think that if the melting temperature is dependent on the salt concentration in solution then it should also be sensitive to the DNA concentration, since an increase leads to an increase on the concentration of counter-ions in solution. In fact, Korolev and co-workers have performed a detailed investigation on salt-free Na-DNA solutions and obtained the following relation, 65 T m = 18.3 log C p + 90.2 (1) where C p is the DNA concentration (in M) over the studied concentration range (C p from6 10-5 to 4.5 10-2 M). Studies were conducted to assess the conformation of DNA in solution at various DNA and salt concentrations, as well as temperatures, by circular dichroism, and melting curve determinations by UV/Vis absorbance and differential scanning calorimetry. From circular dichroism it could be concluded that for solutions with low DNA concentration, 0.1 mm, the addition of 1 mm of salt is sufficient to stabilize the DNA secondary structure in its native B-form conformation. When using as little as 0.01 mm of salt for this temperature, 25 C, and DNA concentration, the double-helix molecules undergo denaturation to single-stranded DNA molecules. The denaturation of nucleic acids leads to an increase in the UV absorption; in this way the melting transition can be followed by UV/Vis spectroscopy. The resulting spectra are typically called melting curves and indicate the melting temperature of the system at the mid point of the transition. It was found, as observed with circular dichroism, that as little as 1.0 mm of salt is sufficient to stabilize the double-helix DNA molecules. Also we observed that for the samples with no salt or with a lower amount of it (0.01 mm) there is no melting transition. Differential Scanning Calorimetry (DSC) is another technique that can be used for the determination of melting temperatures. It has the advantage over the UV/Vis spectrometry, that solutions with relatively high concentrations of DNA can be measured. We started by studying dilute DNA solutions, 0.5 mm, and varying NaBr concentrations. The results were quite similar to the ones obtained by the melting curves determined by UV/Vis absorption. No transitions were obtained for the DNA solutions with no, or little (0.01 mm) salt. Again, for the samples with 1.0 and 10.0 mm of salt the curves are identical, and the melting occurred at 68.7 C for both. We performed the same studies for more concentrated solutions of DNA, about 6 mm. In this case we found that even for the salt-free DNA solution there is a peak corresponding to the DNA melting, indicating that the presence of the DNA counterions is sufficient to stabilize the double-helix conformation at room temperature. In general we can say then that for dilute solutions of DNA with no addition of salt, the DNA molecules are in the single-stranded conformation, whereas the addition of a small amount of NaBr, 1 mm, is sufficient to stabilize the double-helix DNA. For concentrated solutions, of around 6 mm of DNA, no addition of salt is necessary to stabilize the secondary structure. Condensation and precipitation of DNA with cationic surfactants General aspects Because of the growing interest in this field and numerous applications, several studies have been presented in the literature. It was demonstrated through the use of cationic surfactant selective electrodes that cationic surfactants bind to the negatively charged DNA macromolecule in a cooperative manner. 21-24 Many techniques like X-ray scattering, fluorescence quenching, gel electrophoresis and fluorescence microscopy have been used to study the complex formation of DNA with quaternary ammonium surfactants, as well as structures of complexes formed, 23-26,29,66-69 giving rise to a model of the formation of surfactant aggregates on the DNA surface. Despite the interest in these systems, there has not been a systematic study on the precipitation behaviour of DNA with the addition of oppositely charged surfactants, even though this information is very important for many applications: for separation it is obviously necessary, while for other applications the formation of a precipitate might be disastrous. Above we discussed the stability of the DNA double-helix with changes in temperature and salt concentration. Here we focus on the interactions between DNA and the cationic surfactant, DTAB and discuss the differences in the precipitation behaviour of dsdna and ssdna with the addition of the lipid. Unless stated otherwise, all experiments were conducted with 10 or 100 mm of NaBr so that DNA is in the double helix conformation. Also the role of the hydrophobic interactions in these systems is discussed on the basis of a study of the interactions between DNA and alkyltrimethylammonium bromide surfactants of various chain lengths. A

DNA-Surfactant Interactions J. Chin. Chem. Soc., Vol. 51, No. 3, 2004 451 discussion about the composition and the structure of the precipitate is also provided. Differences between single- and double-stranded DNA We consider here the precipitation behaviour of DNA with the addition of DTAB. This is a surfactant with a relatively short alkyl chain which allows for a rather fast equilibration of the samples; also this surfactant forms a micellar solution up to relatively high surfactant concentrations. 70 A schematic representation of the ternary phase diagram is shown in Fig. 1a. We could conduct our study up to 40 wt% of DTAB, but samples with DNA concentrations higher than 4 wt% were not prepared because of the extremely high viscosity of the solution. As expected, the aqueous mixture of DNA and cationic surfactant phase separates associatively into a dilute phase and one phase concentrated in both polyelectrolyte and surfactant, a precipitate. The formation of precipitate has been Fig. 1. (a) Schematic representation of the isothermal pseudo-ternary phase map for the system DNA- DTAB-water. There is a phase separation in almost the entire considered region. (b) Expanded view of the water corner of the system. Open symbols correspond to clear one-phase solutions and filled symbols to two-phase samples.t=25 C. Redrawn from Ref. (60). reported for several systems involving alkyltrimethylammonium bromides and polyanions, like poly(methacrylic acid), 71 sodium hyaluronate, 72,73 or sodium polyacrylate. 74,75 The electrostatic interactions between the components are obviously strong and lead to a strong association. Surfactant aggregates induced by the polymer act as its counter-ions, thereby reducing the charge of the complex and the entropic driving force for mixing as well as the interpolymer repulsions. 76 However, contrary to other polyelectrolyte-surfactant systems, 73,74,77,78 the precipitate does not redissolve with an excess of surfactant, at least in the examined, very broad, interval of concentrations. The difficulty of the redissolution of complexes composed of very highly charged polymers has already been mentioned in some studies. 77,79,80 Further information drawn from the phase map is that the precipitate is formed at very low amounts of DNA and low surfactant concentrations, far below the surfactant critical micelle concentration, CMC. This is a logical observation, since the polyelectrolyte-oppositely charged surfactant systems are known for a critical aggregation concentration, CAC, lower than the CMC of free surfactants, often by orders of magnitude. The fact that the cationic surfactant binding occurs preferentially to anionic polyelectrolytes of high charge density 81-83 underlines this behaviour. We performed a rigorous study of the two-phase border for these systems. For this, turbidity studies were conducted and the results are presented in Fig. 1b as an expansion of the dilute region of the pseudo-ternary phase map. This solubility diagram is presented in a simplified two-dimensional representation. Since the amount of water in these systems is extremely high, this type of representation provides a better visualization. One interesting point is that the precipitate region shows an asymmetry with respect to the surfactant and DNA axes (note the difference in scale of the axes). This was observed for other systems (Refs. (72, 84-85), for example) and means that an addition of small amounts of DNA to a surfactant solution will cause precipitation, while a larger amount of surfactant will be required to cause phase separation of a more concentrated polyelectrolyte solution. We were interested in the differences in behaviour between single- and double-stranded DNA molecules when interacting with cationic surfactants. We performed then two different studies within this system, salt dependence and temperature dependence. Effect of salt As mentioned above, the melting of DNA is dependent

452 J. Chin. Chem. Soc., Vol. 51, No. 3, 2004 Dias et al. on the salt concentration, as well as on the composition of the bases. For solutions with a low concentration of DNA and no addition of simple salt, the molecules present a singlestranded conformation, whereas as low addition as 1 mm of NaBr is sufficient to keep the DNA molecules in their native double-helix state. We performed precipitation studies of DNA solutions by the addition of DTAB in the absence and presence (1.0 and 100.0 mm) of NaBr. The results are presented in Fig. 2. We can see that for the ssdna solutions a lower amount of surfactant is necessary to induce phase separation than for the dsdna solutions. It can be argued that the behavior is a general feature for polyelectrolyte-oppositely charged surfactant systems. Thus it is commonly accepted that the CAC of polyelectrolyte-oppositely charged surfactant systems increases on addition of salt. 86 This is due to a weakened interaction between the polymer and surfactant induced by the stabilization of (free) micelles and a screening of the electrostatic interactions. This delays the precipitation of the system and decreases the two-phase region. It should be stressed, however, that in our case we observe a crossing of the two precipitation lines, which is novel and not expected from previous work; furthermore, there are no noticeable differences in the phase separation of the system in the presence of 1.0 or 100.0 mm, suggesting that the system is not very sensitive to the salt concentration changes for these intermediate concentrations. It is our suggestion that the differences in behaviour arise from the fact that we have different DNA conformations. To make this point clear we studied the temperature dependence of the same system. Effect of temperature For the temperature studies we used a fixed concentration of NaBr, 10-5 M, and performed the studies at 4, 25 and 50 C. Under these conditions we have at 4 C double-helix DNA solutions and for the two higher temperatures singlestranded DNA molecules. The precipitation diagram is presented in Fig. 3. We can see the same features as in the precipitation map of Fig. 2. For the two higher temperatures, the precipitation regions overlap, less surfactant is required to induce the phase separation of the DNA-DTA complexes. For the samples mixed and kept at 4 C, higher concentrations of DTAB are necessary for precipitation. These two studies, salt and temperature dependences, agree well not only qualitatively but also quantitatively. When comparing the solubility diagrams of the system with salt and of the one at lower temperature (Fig. 4), for example, we see that the precipitation lines overlap, except for the most dilute part. We can conclude that the precipitation of the DNAcationic surfactant systems does depend on whether the DNA is in the single- or double-stranded conformation. The fact 6.0 5.0 [DTAB] (mm) 4.0 3.0 2.0 1.0 Fig. 2. Precipitation map for the system DNA-DTABwater at different salt concentrations: 1 and 100 mm NaBr (solid line) and 0 mm NaBr (dashed line). Like in the previous Fig., open symbols correspond to clear one-phase solutions and filled symbols to two-phase samples.t=25 C. From Ref. (117). 0.0 0.0 1.0 2.0 3.0 4.0 5.0 6.0 [DNA] (mm) Fig. 3. Phase map for the systems DNA-DTAB-water at different temperatures: 4 C (solid line); 25 and 50 C (dashed line). As before, open symbols correspond to clear one-phase solutions and filled symbols to two-phase samples. From Ref. (117).

DNA-Surfactant Interactions J. Chin. Chem. Soc., Vol. 51, No. 3, 2004 453 that the two systems show a different behaviour and that the ssdna molecules do precipitate for lower concentrations of surfactant is probably due to the fact that the ssdna molecules have the hydrophobic bases more exposed to the solution than the dsdna, which can lead to an additional interaction between these parts of the DNA molecules and the hydrophobic chains of the surfactant molecules, along with the already mentioned strong electrostatic interactions. Considering again the melting temperature of nucleic acids, it is possible to say that any small, or large, molecule which binds differentially to single strands and double strands will alter the melting temperature. 87 An increase in concentration of a molecule which binds preferably to the double strands will raise the T m, and the opposite effect on T m will be seen when the molecules bind preferentially to the single strands. The melting behaviour of DNA solutions in the presence of alkyltrimethylammonium bromide salts was studied by Orosz and Wetmur, 88 who observed that the T m decreased when the chain length and the concentration of salt was increased. This corroborates the idea that alkyltrimethylammonium surfactants bind preferentially to single-stranded DNA molecules. The fact that the two conformational states show a different behaviour brings interesting perspectives from a separation and purification point of view, as it seems that, with an appropriate choice of surfactant concentration it is possible to separate single- from double-stranded DNA molecules in solution. Effect of surfactant alkyl chain length The interaction between DNA and cationic surfactants of various chain lengths (CTAB, tetradecyltrimethylammonium bromide, TTAB, and DTAB) was investigated. The results for the different chain length surfactants are presented in Fig. 5a in a simplified two-dimensional representation. We can see that the extent of phase separation increases strongly with the surfactant alkyl chain length. For example, CTAB, the surfactant with the longer chain length, binds more strongly to DNA, leading to the formation of precipitate for even smaller amounts of the polymer than for DTAB. This demonstrates the importance of the hydrophobic interactions between the surfactant molecules, and it is an illustration that surfactant binds to DNA in the aggregated form. The same asymmetry of the two-phase region was ob- Fig. 4. Precipitation behaviour of DNA in its native form with the addition of DTAB. The solid line corresponds to samples prepared and stored at 4 C and the dashed line to the system with 100.0 mm of NaBr. From Ref. (117). Fig. 5. Phase maps for the systems DNA-CTAB-water (circles, lower curve), DNA-TTAB-water (triangles, middle curve) and DNA-DTAB-water (diamonds, upper curve), in the presence of NaBr (0.1 M), i.e. with dsdna molecules (a), and absence of salt i.e. with ssdna (b). Like before, open symbols correspond to clear onephase solutions and filled symbols to two-phase samples.t=25 C. From Ref. (60).

454 J. Chin. Chem. Soc., Vol. 51, No. 3, 2004 Dias et al. served for the surfactants of longer chain length, even though DTAB is the surfactant for which this behaviour is the most pronounced. The log-log scale used in the representation of these phase maps (Fig. 5) provides a better illustration of the phase separation. An analogous study was performed with ssdna and we can observe (Fig. 5b), like before, a significant decrease of the two-phase region for lower amounts of DNA. As discussed above, the fact that the system in the absence of salt precipitates for lower surfactant concentrations has probably to do with the fact that ssdna has some exposed hydrophobic parts, which increase its affinity to the surfactant molecules. Again, comparing the precipitation map of CTAB or TTAB with and without salt we observe the same crossing of the precipitation lines, as shown in Fig. 2 for DTAB. Associative phase behaviour is well documented for mixed aqueous systems of a polyelectrolyte and an oppositely charged surfactant 71,73,77,79-83,86,89,90 and so is also the effect of surfactant on the polyion conformation. 72-74,79,80,90 However, the findings for the flexible polyions of lower charge density differ qualitatively from what we find here for DNA. Thus in previously studied cases, like hyaluronan and polyacrylate, high concentrations of surfactant have to be added before phase separation occurs. Furthermore, the change in polyion extension is gradual, with no indication of two dominating states. For the flexible low charge density cases it can be inferred that while surfactant binding is cooperative, it results in surfactant self-assembly aggregates and is characterized by a well-defined critical association concentration, there is an essentially uniform distribution of surfactant aggregates among the different polyions. Phase separation takes place when the net charge of polyion-surfactant aggregates attains a low value and the distribution explains why rather high amounts of surfactant are needed for phase separation in these cases. For DNA, the very low values of surfactant concentration at which phase separation starts demonstrate a different binding situation and an all or none binding: as binding to one DNA molecule starts, further binding is facilitated and one DNA double helix molecule is saturated before binding starts at another, i.e. there is a double cooperativity. Characterization of the DNA-surfactant precipitate It is a common belief that when DNA and cationic surfactant are mixed at equal concentrations, a stoichiometric complex is formed 33 with the counter-ions of both components being released. 91 A study was conducted within the two-phase region to establish the dependence of the amount of the precipitate on the variation of the [DTAB]/[DNA] mixing ratio, R. The DNA concentration was fixed at 3 wt% and the surfactant concentration was increased stepwise until a maximum of R = 7. The samples were analyzed with respect to the precipitate and water in the supernatant. The results are plotted in Fig. 6. By observing the dependence of the amount of precipitate on [DTAB]/[DNA] we clearly see that the precipitate starts to form at very low concentrations of surfactant, as shown above, and that it increases steadily until it reaches a plateau. The plot of the water content in the supernatant contains more information. Also, the amount of water in the supernatant increases with the increase of the surfactant concentration, due to the formation of the precipitate, which decreases the concentration of DNA in solution. At a mixing ratio of approximately 1.0 the percentage of water in the supernatant reaches a maximum of Fig. 6. (a) Weight percent precipitate in the sample versus the mixing ratio between surfactant and DNA, [DTAB]/[DNA], at a constant 3 wt% DNA. (b) Dependence of the amount of water in the supernatant on the molar ratio [DTAB]/ [DNA] at 3 wt% DNA. The dashed line represents the decrease of the amount of water due to the increase of the concentration of surfactant. From Ref. (60).

DNA-Surfactant Interactions J. Chin. Chem. Soc., Vol. 51, No. 3, 2004 455 100. After that it decreases again, with the increase of surfactant in solution. We can deduce from this that the maximum amount of precipitate corresponds to a point close to the charge neutralization, equivalent to one surfactant molecule for each DNA negative charge. In Fig. 6b the dashed line represents the original amount of water in the samples. It is easily observed that the decrease of the amount of water in the supernatant is parallel to this line. This gives the indication that the surfactant added to the samples probably remains in the supernatant, possibly as free micelles, and that the amount of precipitate is constant in this region. The observation that the precipitate reaches a maximum amount near the charge neutralization and thereafter remains constant, at least within the studied region, is in good agreement with the non-redissolution of DNA-surfactant complexes with an excess of surfactant. Since there is no binding of the surfactant to the complex after its neutralization, an inversion of the complex charge is not observed, as is the case with many similar systems. 73-75,77 Structure of the DNA-surfactant precipitate To evaluate the structure of the DNA-surfactant aggregates we performed small-angle X-ray scattering studies. The results are presented in Fig. 7. We can see that for both CTAB and TTAB we have three diffraction peaks at positions 1, 3, and 2, which corresponds to hexagonal structures, with lattice spacing of 54.4 and 53.1 Å, respectively. The fact that the DNA-CTA complexes precipitate in an hexagonal structure is well known, 29,30 but there has been some discussion whether the CTAB is arranged in a normal or inverted hexagonal structure. As discussed above, the picture of the inverted hexagonal structure with the DNA coated with surfactant with the hydrophobic tails sticking to the water is a little counterintuitive. Simulations have shown that this is not the most stable conformation when compared with the picture of surfactant micelles on the DNA surface. 32 Recent work by Leal et al., based on geometrical considerations, as well as hydration and NMR measurements, 33,34 has directly confirmed that the structure is indeed a normal hexagonal one (Fig. 8). The preference for this structure is supported by the fact that CTAB in water forms rod-like micelles with an increase of the ionic strength; addition of DNA molecules will have a similar effect. Also the fact that DNA is a stiff polymer (in these experiments herring DNA was used with an average size of 200 nm, corresponding to only 4 times its persistence length) makes this rearrangement favourable. For TTAB, the same arguments apply while for DTAB the structure is manifestly different. Unfortunately, the diffractogram for this system is not so well resolved. The fact that we have a bump after the first diffraction peak indicates the existence of some type of long-range structure; however, the resolution is such that no structure can be reliably attributed. Whereas both CTAB and TTAB form without doubt an hexagonal structure with DNA, DTAB does not seem to form these extended aggregates with the same ease. We should keep in mind that the only difference between DTAB and TTAB, for example, is the size of the chain. When going from Fig. 7. Small angle X-ray diffractograms of precipitated DNA-cationic surfactant complexes. All the samples were prepared with equal concentrations of DNA and surfactant, 6.0 mm. The bottom curve corresponds to DNA-CTA precipitate, the middle one to DNA-TTA and the upper one to the complex DNA-DTA. T = 25 C. From Ref. (95).

456 J. Chin. Chem. Soc., Vol. 51, No. 3, 2004 Dias et al. CTAB, to TTAB, to DTAB we are making the hydrophobic chain length smaller, which makes the critical packing parameter (CPP) also smaller. This has a great influence on the structure of the formed surfactant aggregates; in this case the curvature is increasing, which facilitates the formation of spherical micelles. This is clearly seen in the binary phase diagram of DTAB with water since the micellar region is stable above 40 wt%, 70 while for CTAB a hexagonal phase starts around 20 wt%. 92 It is common to pass through a discrete micellar cubic phase for relatively high concentrations of surfactant, when going from a micellar solution to a hexagonal phase due to the packing of the spherical surfactant micelles. 93 It has been reported 94 that these cubic structures can be induced by the addition of polyelectrolytes. Having polymeric ions as counter-ions increases the net attraction between the micelles. However, these studies were performed with flexible polyelectrolytes and polyion bridging is the most important attractive force in these systems. The fact that DNA is not a flexible polyelectrolyte can explain the fact that a possible cubic structure will not be as ordered as for other similar systems. Compaction of DNA with cationic surfactant. Dilute regime Above was presented a study on the interactions of DNA with cationic surfactants at relatively high DNA concentrations, i.e., in a macroscopic study. Here we present a study of the same systems in the dilute regime. The low concentration of DNA used allows for the study of interactions on a single-molecule level which provides complementary insight. The method used was fluorescence microscopy. Fluorescence microscopy (FM) is a technique that has recently been used in the study of DNA conformational behaviour in the presence of various cosolutes, and its main advantage is to allow for the visualization of single molecules in solution. DNA molecules in aqueous solution present an extended conformation, migrating in the solution and exhibiting a relatively slow worm-like motion, i.e., they are in the unfolded coil conformation. When, for example, TTAB is added to the DNA solution above a certain concentration, in this case 2.0 10-5 M, we observe a coexistence region of some compact molecules in solution along with DNA coils. These compact molecules, that present a high fluorescence intensity, and a long-axis length less than 1.0 M, are denoted as DNA globules. With further increase of the surfactant concentration, [TTAB] = 8.0 10-5 M, we reach a region where only DNA globules are detected. In Fig. 9 we represent this conformational change Table 1. Characterization of the Interaction between DNA and Cationic Surfactants. C 0 Represents the Concentration at which Globules Were First Detected in the Solution, and C 1 - the Disappearance of a Last DNA Globule. ÄC is the Coexistence Interval Width. T = 25 ºC. Redrawn from Ref. (60) C 0 (ì M) C 1 (ì M) ÄC (ì M) C 1 /C 0 CTAB 8.0 24.0 16.0 3.0 TTAB 20.0 80.0 60.0 4.0 DTAB 80.0 300.0 220.0 3.8 by plotting the long axis length of the DNA molecules versus the surfactant concentration. These results along with those for DNA conformational behaviour in the presence of CTAB and DTAB are presented in Table 1. By looking at the conformational behaviour of DNA molecules in the presence of cationic surfactants of different chain lengths (Fig. 10), we firstly conclude that a larger amount of the shorter chain-length surfactant is needed to induce compaction of DNA macromolecules. We observed that the coexistence region begins for concentrations of 8.0 and 80.0 M for CTAB and DTAB, respectively. These results are in line with the results of phase diagram studies, where we observed that a higher concentration of DTAB is needed for the formation of the precipitate. The coexistence region, C, is narrower for DNA- CTAB systems and becomes wider for the shorter-chained surfactants. It was emphasized above that the binding of the surfactant molecules to DNA is highly cooperative as de- Fig. 9. Long-axis length, L, of T4DNA molecules, 0.5, versus the concentration of TTAB. Error bars indicate the statistical error in the distribution and are given by the standard deviation. T = 25 C. From Ref. (95).

DNA-Surfactant Interactions J. Chin. Chem. Soc., Vol. 51, No. 3, 2004 457 scribed by binding isotherms. 21 In the sigmoid-shaped plots, the amount of surfactant bound to the polymers, the binding degree,, is plotted against the free surfactant concentration. By using binding isotherms, Kwak et al., 21 monitored the effects of surfactant chain length on the DNA-surfactant interactions. For the longer surfactant chains, the binding isotherm has an abrupt behaviour, while for a short surfactant alkyl chain the plot has a less steep profile. It is not surprising that the slope of the cooperative part of the binding isotherm has been related to this coexistence interval. 90 If we look to the ratio C 1 /C 0 instead we realize that the values are very close for the three surfactants used, which tells that the differences between the systems are a consequence of the differences in hydrophobicity of the amphiphile molecules. It is important to emphasize the role of attractive interactions in compaction and phase separation. Compaction and phase behaviour are driven by attractive interactions between different parts of a DNA double helix and between double helices, respectively. As argued below we ascribe these interactions to attractive electrostatic interactions due to the correlation effects arising in the presence of multivalent counter-ions. 40 With surfactant binding occurring in the form of discrete micelles, this can be understood if the local perturbation of the conformation of the DNA chain facilitates micelle association in adjacent parts. However, surfactant binding to DNA leads to the formation of highly extended aggregates, like hexagonal phases. With preferred very large, unlimited, aggregates the highly cooperative DNA folding can be easily understood. However, we know that the cooperative Fig. 10. DNA conformational behaviour in the presence of cationic surfactants CTAB, TTAB, and DTAB. The DNA charge concentration was maintained at 0.5 M. Open circles correspond to the coil conformational state of DNA and filled ones to the presence of globular DNA molecules. Shaded circles represent the coexistence between elongated coils and compacted globules.t=25 C. From Ref. (95). folding of DNA is not limited to self-aggregating cationic surfactants; it also occurs for cationic polymers 56,57 and multivalent counterions. 37-39 It appears, therefore, that it is best to consider the role of the cationic surfactant self-assemblies in terms of attractive correlation interactions between different parts of a DNA molecule thus inducing a compaction; the role of ion-ion correlation effects in DNA compaction has been discussed elsewhere. 35 Concluding the study of the interactions between DNA and cationic surfactants by fluorescence microscopy we note that DNA exhibits a discrete phase transition in the presence of cationic surfactants from coils to globules. Experiments were made with cationic surfactants of different chain lengths and we concluded that CTAB, the longer-chained surfactant, is more efficient compacting DNA than the shorter-chained surfactant. We also studied the DNA-CTAB system by dynamic light scattering and observed the same coexistence between extended DNA coils (pure DNA) and compacted DNA globules. 95 Furthermore, we saw that the number of globules in solution was linearly dependent on the concentration of the surfactant which gave us a strong indication of double-cooperative binding. This is in line with the result that phase separation starts for lower concentrations of DNA and surfactant when compared with other polyelectrolyte-oppositely charged surfactant systems. In these last cases an even distribution of the surfactant amongst the polymer chains is usually expected whereas in the DNA-surfactant systems there is, in our opinion, a coexistence of surfactant saturated DNA molecules (precipitate) and naked DNA molecules in the supernatant. Decompaction of DNA-cationic surfactant complexes by the addition of anionic surfactants General As was mentioned above, the compaction of DNA is significant in processes like gene therapy and drug delivery. However, the dissociation of these complexes in the appropriate place is also crucial. The dissociation of DNA-surfactant complexes in highly diluted solutions was previously studied by the addition of various co-solutes, like monovalent salts, 30 synthetic polyacid, 90 and neutral liposomes. 96 Anionic species like multivalent ions and simple surfactants, 12 as well as negatively charged liposomes, 97 were shown to dissociate DNA-liposome complexes and release DNA into solution. Anionic surfactants were also shown to enhance the dissociation of cationic DNA-intercalated drugs. 98 Here we present a study of the decompaction of the

458 J. Chin. Chem. Soc., Vol. 51, No. 3, 2004 Dias et al. DNA-cationic surfactant complexes by the addition of anionic surfactants, by means of fluorescence microscopy. We investigate the influence of the anionic surfactant chain length as well as the role of the cationic amphiphile hydrophobicity on the dissolution of the DNA-cationic surfactant complexes. Mixtures of cationic and anionic surfactants are known as catanionic mixtures 99 and have, due to their rich phase behaviour and interfacial properties, been the subject of many studies (for reviews see Refs. (99, 100)). By varying the mixture composition, i.e., total surfactant concentration and mixing ratios, one can obtain aggregates with different geometries, ranging from spherical, to cylindrical and planar. The most notable structures these systems form are vesicles, and catanionic vesicles are believed to be thermodynamically stable; in fact they are formed spontaneously and reversibly and remain stable for a long period of time. 101 It was found that in the presence of positively charged catanionic vesicles, DNA undergoes a conformational change from elongated coil to compacted globular states and that the macromolecules adsorb onto the surface of the vesicle in a collapsed globular form. On the other hand, we observed no globule-coil transition in the single DNA molecules by the addition of negatively charged vesicles. 102 These results are plausible, taking into account the long range electrostatic repulsions between species with the same charge. However, since both cationic and anionic surfactants are present in a catanionic mixture, DNA could well associate with the cationic amphiphile and form compacted complexes. Thus, we decided to probe the stability of the DNA-cationic surfactant complexes and, after compacting DNA with cationic amphiphiles, we added a negatively charged surfactant. We found that the DNA globules then undergo decompaction. Dependence on the anionic surfactant chain length To study the effect of the hydrophobicity of the negatively charged surfactant on the decompaction of DNAcationic surfactant complexes we started by compacting DNA with DTAB. For the chosen concentration of surfactant, 3.16 10-4 M, we could observe, by fluorescence microscopy, that all DNA molecules were in the compacted state, according to the results presented above. These collapsed molecules presented a high fluorescence intensity and a long-axis length, L, of less than 1.0 µm. We then started by adding the anionic surfactant, sodium octylsulphate (SOS) for example. No visible effect was noted until a certain concentration, 3.80 10-4 M, where we observed a coexistence between globules and coils. Above an SOS concentration of 6.31 10-3 M only coils were observed in solution (Fig. 11). For sodium dodecylsulphate (SDS), a smaller amount of surfactant was required to induce decompaction. As observed in Fig. 11 a concentration of 8.71 10-5 M was enough to start the DNA unfolding and for 2.88 10-4 M, below charge neutralization, all DNA molecules were in the coil conformation. This is easy to understand from the chain length dependence of surfactant self-assembly. On adding the anionic surfactant to the solution of DNA and cationic surfactant, above a certain concentration it will associate and form mixed self-assemblies with the oppositely charged amphiphile and release DNA back into the solution as a coil. The onset of this association can be defined in terms of a critical micellar concentration for the mixture of the two surfactants (CMC mixt ). Since SDS is more hydrophobic than SOS, the CMC mixt for that surfactant will be smaller than the shorter chained one. Dependence on the cationic surfactant chain length We also performed experiments to determine the dependence of DNA decompaction on the chain length of the compacting amphiphile, CTAB, TTAB, and DTAB. Following the same procedure as above we started by compacting DNA with 3.16 10-4 M of CTAB, for example, and added the anionic amphiphile stepwise. Contrary to the experiments varying the anionic surfactant we found no variation on changing the cationic surfactant chain length (Fig. 12), that is, the amount of negatively charged surfactant necessary to unfold DNA is independent of the hydrophobicity of the compacting amphiphile. Fig. 11. Dependence of the conformational behaviour of single T4DNA molecules, 0.5 M in aqueous buffer solution and a constant DTAB concentration of 3.16 10-4 M, on the stepwise addition of SDS and SOS. Filled circles correspond to the globular DNA conformation, and shaded circles to the coexistence between elongated coils and compacted DNA molecules, whereas open circles correspond to the extended conformation of DNA. T=25 C. From Ref. (13).

DNA-Surfactant Interactions J. Chin. Chem. Soc., Vol. 51, No. 3, 2004 459 This is explained as follows. Above a certain CMC mixt, dependent on the anionic surfactant chain length, two types of structures can be formed in solution, DNA-cationic surfactant (DNA-S + ) globules and cationic-anionic surfactant aggregates (S + -S - ). In the case of CTAB and SOS, for example, when we observe an unfolding of DNA molecules, there is a dissociation of DNA-CTAB globules, with CTAB molecules leaving DNA-CTAB complexes and transferring to CTAB-SOS aggregates. Since this transfer of molecules is occurring between two different surfactant aggregates, it will not depend on the surfactant chain length, or alternatively expressed there is a cancellation in the alkyl chain length effects in forming the two types of aggregates. Surfactant aggregate structures We can note from the above results that the interaction between the oppositely charged surfactants is stronger than the one between DNA and cationic amphiphiles. A question that remains is then, what type of aggregates will the surfactants form. To answer this we performed some cryogenic transmission electron microscopy (cryo-tem) experiments, since this technique allows a good visualization of nm-size objects. We found structures of crystalline appearance in most of the samples (Fig. 13). This is not unexpected since both surfactants had the same chain length and approximately the same concentrations. It is known that most catanionic systems precipitate at equimolar concentrations even at very high water contents. 100 Along with the precipitate we observed, under other conditions, the formation of vesicles (Fig. 14). The formation and stability of vesicles are dependent on the surfactant chain type and length. Thus, vesicle regions are usually larger when one of the amphiphiles has a double chain or when two single chained surfactants have asymmetric chains, which has been attributed to optimal packing conditions. 103 The presence of vesicles in the sample is then due to a large excess of the anionic surfactant and an asymmetry in the surfactant chain lengths, SOS vs. DTAB. 101,104 These findings led us to enquire whether it was possible to predict the structures formed by the surfactant mixture, subsequent to DNA compaction and decompaction, if we had the knowledge of the phase diagram of the mixture. In particular we investigated this issue by additional cryo-tem experiments. We chose a point in the CTAB/SOS/water phase diagram, 105,106 with 9.88 mm of CTAB and 43.00 mm of SOS, which corresponds to the region of negatively charged vesicles. This region was selected since vesicles are self-assembly structures easy to observe and recognize as well as one of the most interesting for application purposes. In the solution, as expected, we observed only the presence of somewhat polydisperse unilamellar vesicles with sizes ranging from 20 to 100 nm (Fig. 15a). Fig. 12. Dependence of the conformational behaviour of single DNA molecules, 0.5 M in aqueous buffer solution at constant concentrations of cationic surfactants CTAB, TTAB and DTAB, 3.16 10-4 M, on the total SDS concentration. Symbols are the same as in Fig. 11.T=25 C. From Ref. (13). Fig. 13. Cryo-TEM images of the surfactant structures formed subsequent to the DNA compaction with DTAB and addition of the anionic amphiphile SDS (0.5 M of T4DNA in buffer solution; 3.16 10-4 M of DTAB). In (a), (b), and (c), [SDS] = 8.13 10-4 M, and in (d), [SDS] = 1.48 10-4 M. In all samples, crystals can be observed, as denoted by arrows. Redrawn from Ref. (13).

460 J. Chin. Chem. Soc., Vol. 51, No. 3, 2004 Dias et al. A sample with the same surfactant concentration was prepared using the normal FM procedure: we observed in the microscope that in a DNA buffer solution with 9.88 mm of CTAB, all DNA molecules presented a compact conformation; with the addition of the anionic surfactant, SOS at 43.00 mm, only coils were present in solution. These concentrations of amphiphiles were enough to induce compaction and decompaction of the DNA molecules. Observing then the surfactant aggregates, by cryo-tem, we realized that there was no visible difference between this sample and the previous one, prepared by mixing surfactants alone (Fig. 15b vs. Fig. 15a). Again, only small unilamellar vesicles were present in solution. Note that these findings provide support for thermodynamic stability of the vesicles, still a matter somewhat controversial. The ability of controlling the structures formed by the surfactants is significant and promising for a number of applications. For example, when using cationic surfactants to compact DNA for purification purposes, the addition of an amount of anionic surfactant would both release DNA back into solution and form a precipitate with the oppositely charged surfactant. This would enable a simple and efficient separation of DNA from the surfactants in solution. From another point of view, for an eventual application of these types of systems to gene therapy, the presence of crystals would be catastrophic for the cells. All of this discussion was conducted by focusing on the amphiphile aggregates and the DNA conformational behaviour. There is no indication that the DNA molecule undergoes denaturation and/or degradation during the compaction and release process. It has been reported 12 that the DNA released from cationic lipid complexes by the addition of anionic additives was in its native B-form conformation. It was accordingly observed that anionic amphiphiles can be used to unfold and release DNA previously compacted by cationic surfactants. We found that by using anionic surfactants with longer chains the decompaction of DNA is more efficient, while it does not depend on the hydrophobicity of Fig. 14. Cryo-TEM images of the surfactant structures observed after DNA, 0.5 M in buffer solution, interaction with DTAB, 3.16 10-4 M, and SOS, 7.94 10-3 M. The coexistence of crystals (white arrows) and vesicles (black arrows) can be observed. Redrawn from Ref. (13). Fig. 15. Cryo-TEM images of an aqueous solution of SOS, 4.3 10-2 M, and CTAB, 9.88 10-3 M in the absence (a) and presence (b) of DNA, 5.0 10-7 M; (a) and (b) unilamellar vesicles. Black arrows denote vesicles, whereas the white ones indicate crystallized water. Redrawn from Ref. (13).