Interactions between DNA and Nonionic Ethylene Oxide Surfactants

Jul 14, 2010 - P.O. Box 124, SE-221 00 Lund, Sweden, and §Department of Chemistry, University of Coimbra,. 3004-535 Coimbra, Portugal. Received April...
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Interactions between DNA and Nonionic Ethylene Oxide Surfactants are Predominantly Repulsive Alexandra H. E. Machado,*,†,‡ Dan Lundberg,*,§,‡ Antonio J. Ribeiro,† Francisco J. Veiga,† Maria G. Miguel,§ Bj€orn Lindman,§,‡ and Ulf Olsson‡ †

Laboratory of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, 3000-548 Coimbra, Portugal, ‡Division of Physical Chemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden, and §Department of Chemistry, University of Coimbra, 3004-535 Coimbra, Portugal Received April 8, 2010. Revised Manuscript Received June 24, 2010

In the present work, the interactions between double-stranded (ds) or single-stranded (ss) DNA and nonionic ethylene oxide (EO) surfactants, with special attention to the possible contributions from hydrophobic interactions, have been investigated using a multitechnique approach. It was found that the presence of ss as well as dsDNA induces a slight decrease of the cloud point of pentaethylene glycol monododecyl ether (C12E5). Assessment of the partitioning of DNA between the surfactant-rich and surfactant-poor phases formed above the cloud point showed that the polymer was preferably located in the surfactant-poor phase. Surface tensiometry experiments revealed that neither of the DNA forms induced surfactant micellization. Finally, it was shown by DNA melting measurements that another EO surfactant (C12E8) did not affect the relative stabilities of ss and dsDNA. To summarize, all experiments suggest that the net interaction between DNA and nonionic surfactants of the EO type is weakly repulsive, which can be attributed mainly to steric effects. In general, the results were practically identical for the ds and ss forms of DNA, except those from the cloud point experiments, where the decrease of the cloud point was less pronounced with ssDNA. This finding indicates the presence of an attractive component in the interaction, which can reasonably be ascribed to hydrophobic effects.

Introduction The fine details in the highly ordered structure of the familiar DNA double helix are directed and stabilized by hydrogen bonds and stacking of the flat bases. On a somewhat coarser level, however, the formation of double-stranded DNA (dsDNA) from the complementary single strands (ssDNA) is largely controlled by the balance of hydrophobic interactions between the bases, which promote assembly, and electrostatic repulsions between the phosphate groups, which counteract it.1 The sometimes neglected amphiphilic nature of DNA is manifested in a number of parallels to the behavior of more conventional amphipihiles, such as different aspects of the assembly of water-soluble surfactants into micelles. For instance, the stability of dsDNA in aqueous solution increases with an increasing ionic strength; in fact, if DNA is present in very low concentrations in pure water, the double helix spontaneously disassembles into ssDNA.2 Other similarities include a pronounced cooperativity in the double helix formation, which results in a narrow temperature range of coexistence of the ss and ds forms in denaturation experiments,1 and the disruption of dsDNA when it is transferred from an aqueous solution to a nonaqueous polar solvent.3 The phosphate groups present on each of the nucleotides render DNA a high negative charge. This can, in turn, induce a strong attractive interaction with oppositely charged molecules, which often leads to associative phase separation.4 Complexes *To whom correspondence should be addressed. (A.H.E.M.) E-mail: [email protected]. (D.L.) E-mail: [email protected]. (1) Evans, D. F.; Wennerstr€om, H. The Colloidal Domain Where Physics, Chemistry, Biology, and Technology Meet, 2nd ed.; Wiley-VCH: New York, 1999. (2) Rosa, M.; Dias, R.; Miguel, M. G.; Lindman, B. Biomacromolecules 2005, 6, 2164–2171. (3) Cui, S.; Yu, J.; K€uhner, F.; Schulten, K.; Gaub, H. E. J. Am. Chem. Soc. 2007, 129, 14710–14716. (4) Dias, R.; Mel’nikov, S.; Lindman, B.; Miguel, M. G. Langmuir 2000, 16, 9577–9583.

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formed by DNA and cationic amphiphiles have been extensively studied. One of the main incentives for understanding such systems is the widespread use of cationic amphiphiles in gene delivery formulations,5,6 but they have also been used in a range of other applications, for instance, in extraction and purification of DNA,7,8 renaturation and ligation of complementary strands,9 and separation, by precipitation fractionation, of native and denatured DNA.2 Most work on DNA-cationic amphiphile coassembly has been performed with dsDNA. However, there are a number of investigations that allow comparison between the behaviors of the ss and ds forms of DNA, and clear differences can indeed be observed. Complexes formed by dsDNA and cetyltrimethylammonium bromide (CTAB) reveal a 2D structure, with elongated micelles and practically straight DNA chains arranged in a hexagonal order,10,11 whereas the corresponding complexes with ssDNA adopt a micellar cubic structure with the surfactant aggregates decorated by the polymer.11 The difference in structure of the respective complexes can largely be attributed to a higher conformational flexibility of ssDNA, which has a persistence length in the range 1-6 nm,12-14 as compared to the relatively stiff (5) Lasic, D. D.; Templeton, N. S. Adv. Drug Delivery Rev. 1996, 20, 221–266. (6) Wasungu, L.; Hoekstra, D. J. Controlled Release 2006, 116, 255–264. (7) Trewavas, A. Anal. Biochem. 1967, 21, 324–329. (8) McLoughlin, D. M.; O’Brien, J.; McManus, J. J.; Gorelov, A. V.; Dawson, K. A. Bioseparation 2000, 9, 307–313. (9) Pontius, B. W.; Berg, P. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 8237–8241. (10) Leal, C.; Wads€o, L.; Olofsson, G.; Miguel, M.; Wennerstr€om, H. J. Phys. Chem. B 2004, 108, 3044–3050. (11) Zhou, S.; Liang, D.; Burger, C.; Yeh, F.; Chu, B. Biomacromolecules 2004, 5, 1256–1261. (12) Smith, S. B.; Cui, Y.; Bustamante, C. Science 1996, 271, 795–799. (13) Tinland, B.; Pluen, A.; Sturm, J.; Weill, G. Macromolecules 1997, 30, 5763– 5765. (14) Desruisseaux, C.; Long, D.; Drouin, G.; Slater, G. W. Macromolecules 2000, 34, 44–52.

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dsDNA, with a persistence length of about 50 nm at low ionic strengths.15 There are also a number of findings that indicate a stronger attractive interaction between cationic surfactants and ssDNA than with dsDNA. For instance, denatured DNA has been shown to precipitate with lower concentrations of dodecyltrimethylammonium bromide (DTAB) than the corresponding native DNA.2 Furthermore, the addition of cationic surfactants to cross-linked DNA gels reveals a more pronounced deswelling and collapse in the case of the denatured form of DNA.16 In related studies on DNA gel particles prepared with CTAB, assessment of DNA release kinetics shows faster release from particles containing dsDNA as compared to the case with gels prepared from ssDNA.17,18 Single-stranded DNA has lower linear charge density than dsDNA. The linear charge density of dsDNA is ∼0.59 negative charges/A˚,19 whereas it is roughly half for ssDNA, presuming that the length remains approximately unchanged, which means that the electrostatic attraction to cationic surfactant aggregates is expected to be lower in the former case than in the latter. This suggests that a stronger attractive interaction in the case of ssDNA can be attributed to a combination of the higher flexibility20 and a larger contribution from hydrophobic interactions. Very few studies have been dedicated to the study of interactions between the double- or single-stranded forms of DNA and nonionic surfactants, where strong electrostatic interactions are absent. In general, the interactions between polyelectrolytes and nonionic surfactants are weak. However, if the polyelectrolyte carries hydrophobic domains or groups, rather strong interactions between the components can be induced, which can, in turn, have a dramatic influence on the behavior of the system.21,22 For instance, a water-soluble polymer grafted with a small fraction of hydrophobic side chains, often referred to as a hydrophobically modified polymer (HMP), can induce a strong increase in the viscosity of a polymer-surfactant solution, as compared to the case with the unmodified polymer. This is due to a transient physical cross-linking of the surfactant micelles by the amphiphilic polymer.23 The strong adsorption of these polymers to nonionic surfactants is also reflected in its incorporation in lamellar24 or microemulsion phases25 formed in systems containing nonionic surfactant and by an increase in the cloud point.24 Thus, an investigation of the effects of ss and dsDNA on the behavior of nonionic surfactants can provide information on the importance of hydrophobic contributions to the interactions of DNA with amphiphiles. An assessment of the extent of hydrophobic interactions between DNA and amphiphiles is of interest from both technical and fundamental points of view. It has already been mentioned above that formulations of DNA containing amphiphiles are of great importance for pharmaceutical applications. Furthermore, (15) Lu, Y.; Weers, B.; Stellwagen, N. C. Biopolymers 2002, 61, 261–275. (16) Costa, D.; Miguel, M. G.; Lindman, B. J. Phys. Chem. B 2007, 111, 10886– 10896. (17) Moran, M. C.; Miguel, M. G.; Lindman, B. Biomacromolecules 2007, 8, 3886–3892. (18) Moran, M. C.; Miguel, M. G.; Lindman, B. Langmuir 2007, 23, 6478–6481. (19) Dias, R.; Lindman, B. DNA Interactions with Polymers and Surfactants; John Wiley & Sons: Hoboken, 2008. (20) Wallin, T.; Linse, P. Langmuir 1996, 12, 305–314. (21) Brackman, J. C.; Engberts, J. B. F. N. Chem. Soc. Rev. 1993, 22, 85–92. (22) Saito, S.; Anghel, D. F. In Polymer-surfactant systems; Kwak, J. C. T., Ed.; Marcel Dekker: New York, 1998; pp 357-408. (23) Holmberg, K.; J€onsson, B.; Kronberg, B.; Lindman, B. Surfactants and Polymers in Aqueous Solution, 2nd ed.; John Wiley & Sons: Chichester, 2003. (24) Iliopoulos, I.; Olsson, U. J. Phys. Chem. 1994, 98, 1500–1505. (25) Kabalnov, A.; Olsson, U.; Thuresson, K.; Wennerstr€om, H. Langmuir 1994, 10, 4509–4513. (26) Ledeen, R. W.; Wu, G. J. Lipid Res. 2004, 45, 1–8.

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the cell nucleus contains a significant fraction of polar lipids.26 The possible functions, in addition to their role as structural components, of the largely net-uncharged nuclear lipids are largely unknown. It is found, however, that a fraction of the nuclear lipids are colocalized with the chromatin and potential indications on strong interactions between DNA and nonionic amphiphiles can be taken to suggest a significance of direct interplay between DNA and lipids. In a larger perspective, findings on systems containing amphiphiles can, to a certain extent, be extrapolated to the importance of hydrophobic interactions between DNA and other entities carrying hydrophobic domains. In this study, the interactions between double-stranded or single-stranded DNA and nonionic surfactants with ethylene oxide (EO) headgroups were investigated by utilizing established methods for identifying polymer-surfactant interactions.19,23 The effects of the presence of ss or dsDNA on the behavior of the surfactant in aqueous solutions were assessed by determining changes in the cloud point of the surfactant, the partitioning of DNA between the surfactant-rich and surfactant-poor phases above the cloud point in samples of different compositions, and the dependence of the surface tension on surfactant concentration in the presence of DNA. Furthermore, the influence of the presence of surfactant on the melting temperature of DNA was investigated.

Experimental Section Materials. Deoxyribonucleic acid (DNA) sodium salt from salmon testes and Trizma (Tris) base (reagent-grade) were purchased from Sigma (USA). Pentaethylene glycol monododecyl ether (C12E5) and octaethylene glycol monododecyl ether (C12E8) were obtained from Nikko Chemicals (Japan); all surfactants were used as received. Seakem LE agarose and Gelstar nucleic acid gel stain were supplied from Cambrex (USA) and Tris acetate-EDTA (TAE) buffer 10 concentrate from Sigma (USA). 6 orange loading dye solution and ZipRuler express DNA ladder were kind gifts by Fermentas (Sweden). All solutions were prepared using water purified with Millipore Milli-Q equipment. Preparation of DNA Solutions and Characterization of the DNA. DNA stock solutions were extensively dialyzed (typically for 2 days) against water using dialysis tubing with a molecular weight cutoff of 6000-8000 (Spectrum, USA). The pH of the DNA solutions was adjusted to 7-8, by the addition of NaOH,2 before dilution with water and/or mixing with a Tris buffer concentrate. The pH of the buffer was adjusted, by the addition of HCl, to be 8 in the final solution in 2 mM Tris buffer. As will be further discussed in the Results and Discussion section, the size of the DNA was determined by agarose gel electrophoresis to be approximately 10 000 base pairs (bp). DNA concentrations were determined by measuring the absorbance at 260 nm, considering that the molar extinction coefficients are 6600 M-1.cm-1 for double-stranded DNA and 8700 M-1.cm-1 for singlestranded DNA.27 The average molecular weight of a nucleotide phosphate residue was considered to be 330 g/mol. The ratio A260/ A280 was determined to be higher than 1.8, which suggests that the DNA is essentially free from protein contamination.27 The solutions of ssDNA were prepared using a previously described procedure, where solutions of dsDNA were first heated at 90-95 °C for 15 min and then rapidly cooled on ice for 30 min.28 The fast cooling limits renaturation of the DNA. When preparing the ssDNA, the solutions of dsDNA were diluted to the desired final concentrations prior to denaturation. An efficient conversion to ssDNA was confirmed by an increase of (27) Sambrook, J.; Russell, D. W. Molecular cloning: a laboratory manual, 3rd ed.; Cold Spring Harbor Laboratory Press: New York, 2001; Vol. 1. (28) Yang, A. Y.; Rawle, R. J.; Selassie, C. R. D.; Johal, M. S. Biomacromolecules 2008, 9, 3416–3421.

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approximately 30% in the absorbance at 260 nm, as compared to the value for the original dsDNA solution.27 Since thermally denatured DNA will gradually renature (although slowly), all ssDNA solutions were used shortly after preparation. Gel Electrophoresis. Agarose gels (1%) were prepared in 1TAE buffer and stained with 2.5 μL of the dye Gelstar, which can detect both conformational forms of DNA with high sensitivity. This dye confers green and orange fluorescence to ds and ssDNA, respectively. DNA-containing samples were diluted with 2 mM Tris buffer to a concentration in the range 20-25 μg/mL and mixed with 2.5 μL of 6 loading dye. Fifteen microliters of sample were then added to each well. A DNA ladder (100 to 20 000 bp) was used for size reference. Electrophoresis was performed at 90 V, using 1TAE as running buffer. When completed, the gel was placed in a detector (Dark Reader, Clare Chemicals, USA) that allows visualization of the DNA bands by irradiation with ultraviolet light. Cloud Point Determination. The cloud points of C12E5 in the absence or presence of ds or ssDNA were determined by measuring the variation in turbidity at 530 nm, as the temperature was increased from 20 to 60 °C at a rate of 0.5 °C/min. The experiments were performed on a Cary 300 Bio UV-visible spectrophotometer (Varian, USA) equipped with a Peltier device for temperature control, and the sample was placed in a 1 cm cuvette. In a typical result from a cloud point experiment, one can generally observe an initial gradual increase in turbidity, which can be attributed to critical fluctuations when approaching phase separation, followed by a very steep increase in the slope as phase separation occurs. The cloud point is identified as the temperature at the point of intersection of the two linear portions of the plot (exemplified in Figure S1, Supporting Information).

Partitioning of DNA between the Surfactant-Poor and Surfactant-Rich Phases above the Cloud Point. Samples containing ds or ssDNA and 2.5 or 10 wt % C12E5 were placed in water baths at 34.5 and 38.3 °C for the low and high surfactant concentrations, respectively. Separation of the two phases in the respective samples was slow, which can be explained by small density differences and quite low surface tensions between the phases. Thus, the samples were left to equilibrate in the water baths for up to one month to obtain visually clear phases. After separation, the upper and lower phases of each sample were collected and diluted with 2 mM Tris buffer whereafter the amount of phosphorus in the respective samples was determined by inductively coupled plasma mass spectrometry (ICP-MS) (performed in the Ecology Department at Lund University, Sweden). The amount of DNA was calculated from the amount of phosphorus using eq 1 DNA mass ¼

av mol wt nucleotide  mass of P mol wt P

ð1Þ

The calculations were performed using an average molecular weight per nucleotide of 330.29 Surface Tension Measurements. Surfactant solutions with or without ds or ssDNA were vigorously stirred to ensure complete mixing and then transferred to Pyrex cells for equilibration. Surface tension measurements were performed using a du No€ uy ring tensiometer (Kr€ uss, Germany). All measurements were performed at 25 °C and in triplicate.

Influence of Surfactants on DNA Melting Temperature. DNA melting transitions in the absence or presence of C12E8 were investigated by monitoring the changes in the UV absorption at 260 nm as the temperature was gradually increased from 25 to 90 °C at a constant rate of 0.5 °C/min. The experiments were performed on a Cary 300 Bio UV-visible spectrophotometer (Varian, USA) equipped with a Peltier device for temperature control. The melting point was identified as the temperature (29) M€ulhardt, C. Molecular Biology and Genomics; Elsevier Academic Press: Burlington, 2007.

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corresponding to the midpoint of the slope in the melting curve (Supporting Information, Figure S2).

Results and Discussion Choice of Materials. In this study, the experiments were performed with nonionic surfactants carrying ethylene oxide (EO) polar headgroups. These surfactants, usually designated as CmEn, with m indicating the number of carbon atoms in the alkyl chain and n the number of EO units in the headgroup, show a characteristic temperature dependence of their physicochemical properties. With increasing temperature, the EO groups are less hydrated and the surfactant becomes more hydrophobic.23 This leads to lowered aqueous solubility at higher temperature. At the so-called cloud point temperature, there is a transition from a homogeneous micellar solution into two isotropic phases, one surfactant-rich and one surfactant-poor, which is manifested by an increased turbidity of the solution.23 The cloud point is often rather sensitive to the presence of cosolutes in the system, which can, by different mechanisms, cause increases as well as decreases in its value. For instance, the aforementioned HMPs, which associate to the surfactant aggregates, and thus stabilize the homogeneous micellar solution, can induce a considerable increase in the cloud point.24 A solute that is depleted from the micellar pseudophase, on the other hand, will promote phase separation and thus cause a decrease of the cloud point. Thus, an investigation of the effects of a certain cosolute on the cloud point of an EO surfactant provides a sensitive tool for identifying attractive or repulsive interactions between the components. C12E5 was chosen as the main surfactant in the investigations, since its behavior in aqueous systems is very well characterized and it shows a cloud point in a suitable temperature range, e.g., 31.9 °C at a concentration of 1 wt % in pure water.30 Literature values of the critical micelle concentration (CMC) of this surfactant are 64-65 μM, at 25 °C.23,31 In most experiments, the surfactant concentration was significantly higher than the CMC, implying that a major fraction of the surfactant resides in micelles. Since the cloud point of C12E5 is below the expected range of DNA denaturation temperatures and therefore can overlap with the melting curve of the DNA, another surfactant of the same class, C12E8, was used in the DNA melting experiments. The CMC of C12E8 is 71 μM, at 25 °C.23 Ideally, to simplify interpretation of the results, one would like the DNA used to be as well-defined and close to monodisperse as possible. However, considering the amount of DNA required in the herein discussed experiments this option was not reasonable. Therefore, a quality of DNA from salmon sperm, which has been previously used in a number of related studies, was chosen for this work. In order to remove salts, small fragments and low molecular weight impurities that may be present, the DNA was purified by dialysis. As was mentioned in the introduction, dsDNA is inherently unstable at very low concentrations. The limiting concentration for dsDNA stability without added salt has been reported to be 0.56 mM for DNA from herring sperm.2 Furthermore, dissolution of CO2 from the air might decrease the pH and thus affect the DNA protonation state.32 Thus, to ensure integrity of the DNA double helix and stability of the pH, most experiments were (30) Strey, R.; Schom€acker, R.; Roux, D.; Nallet, F.; Olsson, U. J. Chem. Soc. Faraday Trans. 1990, 86, 2253–2261. (31) Rosen, M. J.; Cohen, A. W.; Dahanayake, M.; Hua, X. Y. J. Phys. Chem. 1982, 86, 541–545. (32) Korolev, N. I.; Vlasov, A. P.; Kuznetsov, I. A. Biopolymers 1994, 34, 1275– 1290.

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Figure 2. Variation in the cloud point of 1 wt % C12E5 in 2 mM Tris buffer (diamonds) or in water (squares) with the concentration of double-stranded (solid symbols) and single-stranded DNA (open symbols). The DNA concentration is expressed in terms of phophate groups. The experimental error was estimated to be within (0.2 °C. Figure 1. Electrophoresis gel showing bands for dsDNA in the absence (lane 2) or presence (lane 4) of 1 wt % C12E5, ssDNA in the absence (lane 3) or presence (lane 5) of the same surfactant, and a reference ladder (lane 1).

performed in the presence of a Tris buffer of pH 8. On the other hand, the presence of salts in the system can affect the behavior, notably the cloud point, and it can reduce or eliminate effects relating to the osmotic pressure. Thus, to minimize its effects on the physicochemical behavior, the buffer concentration was kept as low as possible, specifically at 2 mM. In addition, certain experiments were performed both in buffer and in pure water in order to assess possible differences. Characterization of DNA and Verification of DNA Storage Stability. Before the interactions between DNA and nonionic surfactants were investigated, the DNA used was characterized with respect to its size, and it was verified that its conformational and/or storage stability was not affected by the presence of surfactant. These parameters were assessed by means of gel electrophoresis, which allows separation of molecules according to their size and assessment of changes in the DNA conformation. As is shown in Figure 1, the native DNA, which, as expected, causes the dye to fluoresce green, is large and shows a notable polydispersity, with a mean size of approximately 10 000 bp (lane 2). The denatured DNA, however, which gives orange fluorescence, shows a higher mobility and appears to be more polydisperse (lane 3). The higher mobility of ssDNA as compared to that of dsDNA can be ascribed to its higher flexibility. The extent of DNA degradation in the used temperature and time ranges is expected to be of minor influence.33 The apparent higher polydispersity can likely be attributed to the formation of a wide range of secondary structures within and between the ssDNA strands, by fractional intra- or intermolecular base pairing.34 This effect can reasonably be explained by similar arguments as the smearing associated with the transitory trapping of circular DNA, as compared to the band arising from linear DNA of the same size.35 As is also shown in Figure 1, the results on solutions of pure ds and ssDNA were compared with those obtained with (33) Freifelder, D.; Dewitt, R. Gene 1977, 1, 385–387. (34) Dimitrov, R. A.; Zuker, M. Biophys. J. 2004, 87, 215–226. (35) A˚kerman, B.; Cole, K. D. Electrophoresis 2002, 23, 2549–2561.

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corresponding solutions containing 1 wt % of C12E5. It can be seen that there are no significant differences in the bands obtained in the absence or presence of surfactant for dsDNA (lanes 2 and 4) and for ssDNA (lanes 3 and 5) and that the characteristic fluorescence of the bands is conserved in the presence of surfactant. These observations suggest that C12E5 does not notably affect the DNA conformation or stability at the conditions used. Moreover, DNA solutions with or without surfactant in different concentrations were shown to be stable for at least 2 months. The bands in the agarose gel remain practically unchanged, showing that no considerable degradation of the DNA occurs with time. Effect of Double-Stranded and Single-Stranded DNA on the Cloud Point of C12E5. As mentioned above, an investigation of possible changes in the cloud point of an oxyethylene surfactant on addition of a cosolute is a useful approach for identifying, and to some extent classifying, interactions between the components. The cloud point is usually detected by visual inspection of the surfactant solution as the temperature is increased, and identified as the temperature where the sample turns turbid. In our study, this parameter was determined by measuring turbidity in a UV-visible spectrophotometer, which allows a better control of the temperature gradient and is expected to give a more accurate value of the cloud point. Figure 2 shows the dependence of the cloud point of a solution of 1 wt % C12E5 (24.6 mM) in the presence of different concentrations of ds or ssDNA in either 2 mM Tris buffer or pure water. The values obtained for the cloud point of 1 wt % C12E5 in the absence of DNA are in accordance with literature values.30,36,37 The value obtained in buffer is slightly lower than the one in water (32.8 and 32.9 °C in 2 mM Tris buffer and water, respectively). It can be seen that both forms of DNA have only a small effect on the surfactant cloud point under the conditions used. When compared to what is generally found for highly charged polyelectrolytes, these findings are not unexpected, at least for dsDNA.23 However, for ssDNA, with its more distinct hydrophobic domains, the very limited effect on the cloud point is in significant contrast to what is found in corresponding (36) Feitosa, E.; Brown, W.; Hansson, P. Macromolecules 1996, 29, 2169–2178. (37) Fonseca, S. M.; Eusebio, M. E.; Castro, R.; Burrows, H. D.; Tapia, M. J.; Olsson, U. J. Colloid Interface Sci. 2007, 315, 805–809.

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mixtures with HMPs. For instance, hydrophobically modified poly(sodium acrylate) (HMPA) associates strongly with C12E5 aggregates and induces a considerable increase of the cloud point; at a concentration of 0.1% of a HMPA with 3 mol % of C18 chains randomly grafted to its backbone, an increase of the cloud point of approximately 5 °C was observed in a 2 wt % solution of C12E5 in D2O.24 The small effect of ssDNA on the cloud point of C12E5 in comparison to that of HMPs inevitably raises questions about the effective hydrophobicity of ssDNA. To explain the difference one needs to take a closer look at the structures of the respective polymers. In the case of a typical HMP, hydrophobic chains are grafted to the hydrophilic polymer backbone in a way that leaves them highly exposed to the surrounding and available to interact with the surfactant aggregates. Their hydrophobic moieties can, therefore, be efficiently anchored in the micelles. The singlestranded DNA, on the other hand, has a very different structure. Each nucleotide has a polar end at the phosphate group, whereas the base constitutes a more hydrophobic domain. When the nucleotides are connected into a polymer, however, this cannot necessarily be considered as a ribbon with one hydrophobic and one hydrophilic side. In fact, ssDNA is known to retain much of a helical structure due to base stacking interactions,38-40 which limits the effective exposure of its hydrophobic parts. This conformation could potentially be disrupted if the interaction with the surfactant was strong enough, but this does not seem to be the case in the herein studied system. In addition to the hidden hydrophobic groups, there is a steric effect associated with the bulky and highly hydrated headgroups of the surfactant, which can likely prevent the hydrophobic parts of the DNA from reaching the hydrophobic micellar core. It is probable that a combination of the factors discussed above impair the hydrophobic interactions between the ssDNA and the surfactant. Although the effect of ssDNA on the cloud point of C12E5 is, in comparison to the effects observed with typical HMP, practically negligible, some small but reproducible changes in the cloud point are observed and these differ somewhat between the different conditions studied. For all conditions used, there was first a slight decrease of the cloud point followed by an increase in its value with increasing DNA concentration. The weak minimum is likely a consequence of a subtle balance between several factors, and its origin was not further investigated. More importantly, there is in general a slight decrease of the cloud point in the presence of DNA, which can be attributed to a repulsive interaction between the surfactant and the polymer. This repulsion can reasonably be ascribed to steric interactions between the micelles and the DNA; the repulsion is weak, but strong enough to overshadow the loss in entropy expected on confinement of the DNA and its counterions in a smaller volume. Interestingly, one can note that, although the general trend for both ss and dsDNA is similar, the denatured DNA consistently gives slightly higher cloud point values than the native one in the whole investigated range of DNA concentration and in both media used. This observation can be taken to suggest that there might actually be a small contribution from hydrophobic attraction between ssDNA and the surfactant that partially counterbalances the steric repulsion. However, in the direct quantitative comparison between ss and dsDNA one should also recall that as

the DNA concentration is expressed in terms of bases, the concentration in terms of polymer chains is twice as high in ssDNA compared to dsDNA, which may affect, for example, the steric excluded volume interactions. In order to assess the contribution from electrostatics to the results obtained, the cloud point of C12E5 in the absence or presence of DNA was also determined in 0.5 M NaCl, where the electrostatic interactions between the DNA strands will be effectively screened. As expected, the cloud point of the surfactant decreased, to a value of 27.6 °C, in the salt solution, due to the salting-out effect of Cl-.41 In the presence of dsDNA (1.5 mM), the cloud point decreased even further to 22.6 °C. These findings can be explained by the entropic penalty from confining DNA in a separate phase being substantially reduced in the presence of added salt. Thus, the increase in ionic strength promotes phase separation. The slight decrease in the cloud point values in Tris buffer as compared to the values in water can likely be ascribed to the same mechanism. DNA Partitioning between Surfactant-Rich and Surfactant-Poor Phases Formed above the Cloud Point. As discussed in the previous section, the general decrease in the cloud point of the surfactant on the addition of ds or ssDNA suggests a weak net repulsion between the components, although there are indications of an attractive component in the case of ssDNA. To gain further understanding of the nature and relative importance of these components to the interaction between DNA and C12E5, we chose to assess the partitioning of DNA between the two phases formed above the cloud point. The partition coefficient is a sensitive parameter that is useful for investigating the balance between repulsive and attractive interactions between a polymer and a surfactant. Substances that associate with the surfactant aggregates, such as the previously mentioned HMP,42 are expected to show a preference for the surfactant-rich phase, whereas large molecules with no attractive interaction with the micelles or substances that are depleted from the micellar surface generally remain in the less crowded surfactant-poor phase. Partitioning between the two phases above the cloud point has been successfully utilized to separate biomolecules according to hydrophobicity and size.43,44 In order to obtain similar volumes of the surfactant-rich and the surfactant-poor phases, the samples were prepared with higher surfactant concentrations than those used in the cloud point experiments, specifically 2.5 and 10 wt % (which correspond to 61 and 246 mM). Samples with these surfactant concentrations were left to phase-separate at 34.5 and 38.3 °C, respectively.30 The use of two different C12E5 concentrations aimed to assess the possible dependence of partitioning on surfactant concentration. The presence of the surfactant was found to interfere with the 260 nm UV absorbance from DNA, likely due to scattering from the surfactant aggregates. To obtain accurate values of the DNA concentration, this was thus instead determined by elemental analysis of phosphorus, using ICP-MS. The partition coefficient of the DNA between the two phases (K) was calculated according to eq 2

(38) Arnott, S.; Chandrasekaran, R.; Leslie, A. G. W. J. Mol. Biol. 1976, 106, 735–748. (39) Camerman, N.; Fawcett, J. K.; Camerman, A. J. Mol. Biol. 1976, 107, 601– 621. (40) Vesnaver, G.; Breslauer, K. J. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 3569– 3573.

(41) 6230. (42) (43) (44) 192.

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K ¼

½DNAt ½DNAb

ð2Þ

Kabalnov, A.; Olsson, U.; Wennerstr€om, H. J. Phys. Chem. 1995, 99, 6220– Zhao, G.; Chen, S. B. Langmuir 2006, 22, 9129–9134. Hinze, W. L.; Pramauro, E. Crit. Rev. Anal. Chem. 1993, 24, 133–177. Liu, C. L.; Nikas, Y. J.; Blankschtein, D. Biotechnol. Bioeng. 1996, 52, 185–

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Article Table 1. Partitioning of DNA in the Surfactant-Rich and Surfactant-Poor Phases above the Cloud Pointa

sample

medium

temperature (°C)

total conc. C12E5 (wt %)

partition coefficientb,c,d

volume ratioe,f

dsDNA Tris buffer 34.5 2.5 0.62 ( 0.32 0.4 ssDNA Tris buffer 34.5 2.5 0.54 ( 0.22 0.4 dsDNA Tris buffer 38.3 10 0.09 ( 0.07 0.6 ssDNA Tris buffer 38.3 10 0.23 ( 0.09 0.6 dsDNA water 34.5 2.5 0.46 ( 0.18 0.4 ssDNA water 34.5 2.5 0.64 ( 0.00 0.4 dsDNA water 38.3 10 0.20 ( 0.11 0.6 ssDNA water 38.3 10 0.12 ( 0.12 0.6 a The total DNA concentration was 2.8 mM (in terms of phosphate groups) in all samples. b The partition coefficient is defined as the ratio of the DNA concentrations in the top, surfactant-rich phase, and in the bottom, surfactant-poor phase, respectively. c The results are represented as the mean value ( standard deviation (n = 2). d The experimental error associated with the determination of phosphorus by ICP-MS is within 5%. e The ratio of the volume of the top phase to the volume of the bottom phase. f The experimental error associated with the determination of the volume by measuring the height was estimated to be within 10%.

where [DNA]t and [DNA]b denote the concentrations of DNA in the top, surfactant-rich, and in the bottom, surfactant-poor phases, respectively. The coefficients were determined in 2 mM Tris buffer as well as in pure water, and the results are shown in Table 1. For all conditions tested, the partition coefficients obtained were clearly below 1, indicating that DNA is preferably located in the bottom, surfactant-poor phase. The coefficient was lower when the surfactant concentration was higher. These results suggest that the repulsive component of the interaction between DNA and C12E5 micelles is dominant in all cases. The absence of significant differences between ds and ssDNA show that, even though there might be a small hydrophobic interaction in the case of ssDNA, this is overshadowed by the repulsive interactions. It can be noted that there were no significant differences between the results obtained in water or in Tris buffer. The net repulsive character of the interaction may be ascribed to excluded volume in the top phase by the large volume fraction of surfactant aggregates and to the steric effect from the large surfactant headgroups. These findings are consistent with those previously obtained for samples containing DNA and 7.8 wt % Triton X-114 (which carries 9-10 EO units as its headgroup) in phosphate-buffered saline (PBS),45 where the clear preference of DNA for the surfactant-poor phase was explained by repulsive, steric, excluded volume interactions. In Table 1, it can also be noted that the presence of DNA in the system induced a shift in the relative volumes of the phases, with the volume of the bottom, surfactant-poor phase, increasing at the expense of the top, surfactant-rich phase, for all conditions tested. The deviation is roughly of the same order for both investigated surfactant concentrations. Consequently, the final surfactant concentrations in the top phases are higher in the presence of DNA than in the binary samples. Presuming that the bottom phase is free from surfactant after separation, a surfactant concentration of approximately 9 wt % in the top phase was obtained for the samples containing in total 2.5 wt % of surfactant (at 34.5 °C), whereas the corresponding value was 27 wt % for the samples with a total surfactant concentration of 10 wt % (at 38.3 °C). These values should be compared to those reported for the binary system of C12E5-H2O by Strey et al.,30 where final surfactant concentrations of 7 and 16 wt % in the top phase are found for the corresponding samples kept at 34.5 and 38.3 °C, respectively. The differences in relative volumes in samples with or without DNA can be explained by the increase of the osmotic pressure exerted by the DNA in the surfactant-poor phase. The increase in the volume of the bottom phase is more pronounced (45) Mashayekhi, F.; Meyer, A. S.; Shiigi, S. A.; Nguyen, V.; Kamei, D. T. Biotechnol. Bioeng. 2009, 102, 1613–1623.

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when the partitioning coefficient is low, as more DNA is confined in the bottom phase. The fact that DNA is excluded from the phase concentrated in surfactant is consistent with results from previous studies performed with EO-based surfactants and polymers. Using the method of single-molecule observation with fluorescence microscopy, it was observed that at high concentrations of Triton X-100 (50-90%)46 or poly(ethylene glycol) (PEG, 230 mg/mL)47 compaction of DNA macromolecules through a discrete coil-globule transition could be induced. This finding was explained by the increase in osmotic pressure in the surfactant solution and depletion interactions. Since the DNA used in these experiments is polydisperse, it is conceivable that there is an uneven distribution of DNA sizes between the two phases. Particularly, one might imagine that smaller fragments could be accumulated in the top phase. This possible effect was assessed for samples prepared with 2 mM Tris buffer by performing agarose gel electrophoresis. The DNA size distributions in the separated surfactant-rich and surfactant-poor phases from the samples prepared in 2 mM Tris buffer are shown in Figure 3. It can be seen that there are no notable differences in the sizes of the DNA between the top and bottom phases of each sample, with neither ds nor ssDNA. Thus, the distribution of DNA between the two phases is not significantly influenced by a molecular size selectivity. The slight broadening of the bands of the separated samples relative to the starting DNA solutions might be ascribed to a minor extent of degradation due to storage at elevated temperatures. Surface Tension Measurements in the Presence of ds or ssDNA. The presence of polymers in aqueous surfactant solutions might influence the aggregation into micelles. If the interaction between the surfactant and the polymer is attractive, the polymer can induce surfactant micellization at concentrations significantly lower than the critical micelle concentration (CMC) in a binary solution.23,48,49 In addition, a noncooperative binding starting at very low surfactant concentrations can also occur, as, for instance, in the presence of HMP.50-52 A convenient way to assess the effect of a polymer on the CMC of a surfactant is to determine the dependence of the surface tension on surfactant concentration, as the formation of aggregates in the bulk is reflected by changes in the surface tension. (46) Mel’nikov, S. M.; Yoshikawa, K. Biochem. Biophys. Res. Commun. 1997, 230, 514–517. (47) Kojima, M.; Kubo, K.; Yoshikawa, K. J. Chem. Phys. 2006, 124, 024902. (48) Goddard, E. D. J. Colloid Interface Sci. 2002, 256, 228–235. (49) Taylor, D. J. F.; Thomas, R. K.; Penfold, J. Adv. Colloid Interface Sci. 2007, 132, 69–110. (50) Chang, Y.; Lochhead, R. Y.; McCormick, C. L. Macromolecules 1994, 27, 2145–2150. (51) Johnson, K. M.; Fevola, M. J.; Lochhead, R. Y.; McCormick, C. L. J. Appl. Polym. Sci. 2004, 92, 658–671. (52) Deo, P.; Somasundaran, P. Langmuir 2005, 21, 3950–3956.

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Figure 3. Electrophoresis gel showing the DNA size distributions in the two phases of separated samples prepared in 2 mM Tris buffer, for the two surfactant concentrations used, i.e., 2.5 wt % (lanes 4-7) and 10 wt % (lanes 8-11). The gel also shows the differences between the two forms of DNA, dsDNA top (lanes 4 and 8) and bottom (lanes 5 and 9) phases, and ssDNA top (lanes 6 and 10) and bottom (lanes 7 and 11) phases. For reference, results from a DNA ladder (lane 1) as well as from solution of pure dsDNA (lane 2) or pure ssDNA (lane 3) are also shown.

Figure 4. Surface tension as a function of surfactant concentration for two concentrations, 1 mM (squares) and 5 mM (diamonds) of native (closed symbols) and denatured (open symbols) DNA. The curve for the pure C12E5 in 2 mM Tris buffer (crosses) is also shown. DNA concentration is expressed in terms of phophate groups. The standard deviation is below 2% (n = 3).

Figure 4 shows the surface tension as a function of surfactant concentration in the presence of 1 or 5 mM of ds or ssDNA. The CMC of C12E5 in 2 mM Tris buffer (pH 8) (i.e., in the absence of DNA) was determined to be 76 μM, which is close to the aforementioned literature values for the CMC of C12E5 in water.23,31 The surface tension of the pure DNA solutions in 2 mM Tris buffer was also assessed. In comparison to the surface tension of water (72.2 mN/m) and the Tris buffer (71.2 mN/m), there was a 13108 DOI: 10.1021/la101403j

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small decrease of the surface tension values to 69.8 and 69.0 mN/m for 1 and 5 mM dsDNA and 68.0 and 66.8 mN/m for 1 and 5 mM ssDNA, respectively. The decrease in surface tension with dsDNA is somehow unexpected, as it has been previously shown that DNA does not exhibit surface activity and thus does not affect the surface tension of water.53 The denatured DNA is expected to be more surface active, due to its more amphiphilic character. Our results indicate that ssDNA does indeed induce a decrease of the surface tension that is slightly larger than that observed with dsDNA. However, the observed decrease is very small in comparison to that promoted by, e.g., the anionic hydrophobically modified poly(maleic acid/octyl vinyl ether) (PMAOVE), which has been shown to decrease the surface tension values by ∼25%.52 The very small decrease in surface tension induced by ssDNA can be explained by the same arguments discussed in connection to the cloud point experiments, by the ssDNA retaining a helical structure where the hydrophobic domains are largely hidden. It can be seen in Figure 4 that, despite a slight decrease of the surface tension values for lower C12E5 concentrations in the presence of DNA, which can be attributed to the lower surface tension values of the pure DNA solutions, the addition of 1 or 5 mM of either ss or dsDNA did not significantly affect the CMC of the surfactant. Thus, neither of the polymers induced surfactant micellization. This behavior differs from, e.g., that reported by Deo et al. with PMAOVE.52 In mixtures of this polymer with C12E5, clear changes were observed in the surface tension plot due to the incorporation of C12E5 into the hydrophobic domains of the polymer.52 The results from the surface tension experiments are in accordance with the findings discussed in previous sections and the weak interaction between ss or dsDNA with the surfactant monomers, as the one with the surfactant aggregates, can be ascribed to the influence of the large headgroup that prevents the interaction between the surfactant tail and the hydrophobic domains of DNA. Effect of Surfactant on the Melting Temperature of DNA. All the previous experiments were performed to identify the interactions between DNA and surfactant by assessing the effects of the presence of ds or ssDNA on the surfactant behavior. In this last section, the possible influence of the surfactant on DNA stability will be addressed. The determination of the melting temperature is a straightforward way to assess the relative stability of the different DNA conformations in the presence of cosolutes. It is known that cationic surfactants can have either a stabilizing or destabilizing effect on DNA, depending on several factors, for instance, the surfactant alkyl chain length.2 It is also known that phospholipids can stabilize or destabilize the double helix under different conditions.54,55 As a general rule, compounds that are able to stabilize dsDNA are expected to induce an increase of the melting point, whereas the opposite is expected for substances that stabilize ssDNA. The influence of surfactant on the DNA melting temperature was, as mentioned above, investigated using C12E8, instead of C12E5. Due to the higher hydrophilicity associated with the additional ethylene oxide groups, C12E8 has a higher cloud point (∼80 °C), which is above the range of expected DNA melting temperatures. (53) McLoughlin, D.; Langevin, D. Colloids Surf., A: Physicochem. Eng. Aspects 2004, 250, 79–87. (54) Manzoli, F. A.; Muchmore, J. H.; Bonora, B.; Sabioni, A.; Stefoni, S. Biochim. Biophys. Acta 1972, 277, 251–255. (55) Manzoli, F. A.; Muchmore, J. H.; Bonora, B.; Capitani, S.; Bartoli, S. Biochim. Biophys. Acta 1974, 340, 1–15.

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presence of DNA did not affect the surfactant cloud point, which is consistent with the previously discussed experiments. These results give further support to the notion that DNA does not adsorb to nonionic EO surfactant micelles.

Conclusions

Figure 5. Effect of 1 wt % C12E8 (red) and 2 wt % C12E8 (blue) on the melting temperature of dsDNA (solid line). For reference, the DNA melting curve obtained in the absence of surfactant (black, solid line), the surfactant cloud point curve (dotted line), and the curve resulting from the addition of the two (dash dotted line) are also presented.

Figure 5 shows the melting curve for DNA in the presence of 1 or 2 wt % (18.6 and 37.1 mM, respectively) C12E8, as well as reference data for solutions of DNA or C12E8. The results for DNA in the presence of 1 wt % C12E8 are practically identical to the curve obtained by adding the melting curve for DNA in the absence of surfactant and the surfactant cloud point curve. Experiments performed with 2 wt % of the same surfactant also led to similar results. Additionally, it could be observed that the

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All of the results discussed herein show that the net interactions between DNA and nonionic surfactants of the EO type are weak, but effectively repulsive. The weak repulsion can probably be attributed mainly to a combination of steric effects associated with the bulky surfactant headgroup, which can hinder effective contact between the DNA and the surfactant tails, and excluded volume interactions. The results were practically identical for the ds and ss forms of DNA, except in the cloud point experiments, where there were indications on an attractive component to the ss DNA-surfactant interaction. This can reasonably be ascribed to hydrophobic effects. Acknowledgment. The authors thank the Swedish Research Council (VR) and the Fundac-~ao para a Ci^encia e a Tecnologia, Portugal, for their financial support (SFRH/BD/41424/2007, A.H.E.M., and SFRH/BPD/48522/2008, D.L.). Tommy Olsson is acknowledged for performing the ICP-MS experiments. Bruno Medronho and Wei Wang are acknowledged for valuable input on this work. Supporting Information Available: Representative plots of cloud point and DNA melting temperature determination are included in this section. This material is available free of charge via the Internet at http://pubs.acs.org.

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