Condensation and Decondensation of DNA by Cationic Surfactant

Article pubs.acs.org/Langmuir

Condensation and Decondensation of DNA by Cationic Surfactant, Spermine, or Cationic Surfactant−Cyclodextrin Mixtures: Macroscopic Phase Behavior, Aggregate Properties, and Dissolution Mechanisms Jonas Carlstedt,*,†,§ Dan Lundberg,*,†,‡,⊥ Rita S. Dias,‡ and Björn Lindman†,‡ †

Physical Chemistry, Center for Chemistry and Chemical Engineering, Lund University, POB 124, 221 00 Lund, Sweden Department of Chemistry, University of Coimbra, Rua Larga, 3004-535 Coimbra, Portugal

‡

S Supporting Information *

ABSTRACT: The macroscopic phase behavior and other physicochemical properties of dilute aqueous mixtures of DNA and the cationic surfactant hexadecyltrimethylammounium bromide (CTAB), DNA and the polyamine spermine, or DNA, CTAB, and (2-hydroxypropyl)-β-cyclodextrin (2HPβCD) were investigated. When DNA is mixed with CTAB we found, with increasing surfactant concentration, (1) free DNA coexisting with surfactant unimers, (2) free DNA coexisting with aggregates of condensed DNA and CTAB, (3) a miscibility gap where macroscopic phase separation is observed, and (4) positively overcharged aggregates of condensed DNA and CTAB. The presence of a clear solution beyond the miscibility gap cannot be ascribed to self-screening by the charges from the DNA and/or the surfactant; instead, hydrophobic interactions among the surfactants are instrumental for the observed behavior. It is difficult to judge whether the overcharged mixed aggregates represent an equilibrium situation or not. If the excess surfactant was not initially present, but added to a preformed precipitate, redissolution was, in consistency with previous reports, not observed; thus, kinetic effects have major influence on the behavior. Mixtures of DNA and spermine also displayed a miscibility gap; however, positively overcharged aggregates were not identified, and redissolution with excess spermine can be explained by electrostatics. When 2HPβCD was added to a DNA−CTAB precipitate, redissolution was observed, and when it was added to the overcharged aggregates, the behavior was essentially a reversal of that of the DNA− CTAB system. This is attributed to an effectively quantitative formation of 1:1 2HPβCD−surfactant inclusion complexes, which results in a gradual decrease in the concentration of effectively available surfactant with increasing 2HPβCD concentration.

1. INTRODUCTION Aqueous mixtures of DNA and cationic amphiphiles or polymers have, due to potential usefulness in various biotechnological applications, received much attention.1 Important examples of where such mixtures are involved are the successful use of cationic lipids in vectors for nonviral gene transfection2 as well as protocols for DNA purification.3 Furthermore, these systems constitute valuable model systems for a fundamental understanding of the ubiquitous interactions of DNA with cationic entities in biological systems. There is a strong attraction between DNA and oppositely charged amphiphile aggregates or polyions carrying more than three positive charges.4 This attraction is largely driven by ion correlation effects5,6 and the gain in translational entropy that is related with the release of the small counterions,7 which gives a strong tendency for association of the components. For the case of DNA−amphiphile mixtures, it should be stressed that, since single amphiphile molecules (unimers) typically have too low charge, it is effectively amphiphile aggregates, rather than amphiphile unimers, that associate to DNA. In this context, an © 2012 American Chemical Society

important factor is that the presence of DNA induces surfactant self-assembly far below the critical micelle concentration (CMC) in pure water.8 Depending on the conditions, the association of DNA and surfactant aggregates may be manifested as compaction, i.e., a conformational change of single DNA chains from an extended coil to a compact globule,8,9 or macroscopic phase separation.10 (In the following text, the term compaction refers to the conformational change of single DNA molecules, whereas the more general term condensation, although also covering compaction, is mainly used for cases involving multiple DNA molecules.) In the case of surfactant-induced compaction of single DNA molecules, a coil−globule coexistence is generally found at intermediate surfactant concentrations.8,11 The presence of the coexistence region has been ascribed to a strong cooperativity in the coil to globule transition.11 In Received: January 18, 2012 Revised: April 23, 2012 Published: April 30, 2012 7976

dx.doi.org/10.1021/la300266h | Langmuir 2012, 28, 7976−7989

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average Mw ∼ 1540 g mol−1), and NaBr (p.a. grade) were purchased from Sigma-Aldrich and used as received. The buffer solution was prepared with water purified using a Millipore Milli-Q purification system. 2.2. Sample Preparation. Stock solutions of each component were prepared in 10 mM Tris-HCl buffer with a pH of 7.6. For the DNA−CTAB and DNA−spermine mixtures, a fixed volume of aqueous cationic agent (CTAB or spermine) of varying concentration was added to DNA diluted in buffer to the desired concentration. The added volume of cationic agent represented 10% of the final volume; during the addition, particular care was taken to spread the solution evenly in the full sample volume by moving the pipet tip from the bottom to the top while ejecting. After the addition of cationic agent to the DNA solutions, the samples were gently agitated (tapped and turned end over end) and briefly centrifuged in order to collect the mixture at the bottom of the tubes. The samples were then left overnight at room temperature (21 °C) for equilibration. The majority of the DNA−CTAB−2HPβCD samples were prepared by first diluting DNA in buffer to 70% of the desired final volume followed by addition of a CTAB solution of the appropriate concentration and volume to end up with 80% of the desired final volume. Then the mixtures were gently agitated, briefly centrifuged, and left at room temperature for 1 h. Finally, a 2HPβCD solution of the appropriate concentration was added; the sample was then gently agitated, briefly centrifuged, and left overnight at room temperature for equilibration. For samples where the alternative mixing order was used, 90% of the desired final volume was present prior to addition of DNA; except for the mixing order, the preparation protocol was identical. Regardless of the experimental method employed, each sample was visually examined prior to analysis in order to verify its macroscopic phase behavior. 2.3. UV−vis Spectrophotometry. UV absorbance studies were performed at room temperature using a CARY 300 Bio UV−vis spectrophotometer (Varian). The samples were contained in a 10 mm quartz cuvette. The absorbance was recorded in the wavelength range of 800−200 nm with a scanning rate of 120 nm min−1. Data were collected every 0.2 nm. DNA has a distinct UV absorbance maximum at 260 nm, which was here utilized to estimate the assessable amount of DNA in the different samples. Superimposed on this maximum is an apparent contribution to the absorbance from scattering of the incident light by dissolved or dispersed molecules or aggregates. Since the character of the aggregates in the herein investigated samples varied with sample composition, the contribution from scattering did, in turn, vary among the samples and needed to be accounted for when evaluating the absorbance data. The scattering power of dissolved or dispersed molecules or particles scales with the wavelength, λ, by the power of −4.31 The contribution from scattering at 260 nm was thus estimated from the apparent absorbance at a wavelength where no absorbance is expected (320 nm) by a λ−4 scaling and subtracted from the measured absorbance value at 260 nm. An example of a corrected absorbance spectrum (together with the raw data and the estimated contribution from scattering) is shown in Figure S1 of the Supporting Information. In the phase separation regions, where large particles were formed, the λ−4 scaling is not valid, and the absorbance values thus not correct. In order to obtain an appropriate estimation of the contribution from scattering by large particles, one would need detailed knowledge of the character of the particles. An extensive investigation of the phaseseparating particles is beyond the scope of the present work, and no attempts were made to explain why the subtraction based on the λ−4 scaling typically gave an overcompensation of the contribution from scattering. 2.4. Dynamic Light Scattering and Electrophoretic Mobility. The dynamic light scattering (DLS) and electrophoretic mobility (EM) experiments were performed on a Zetasizer Nano ZS (Malvern Instruments Ltd.), equipped with a 4 mW He−Ne laser (632.8 nm) and an avalanche photodiode detector. This instrument measures, using the noninvasive backscatter (NIBS) technology, fluctuations in the intensity of the scattered light at a set angle of 173° and constructs an intensity autocorrelation function. The correlation functions were

contrast to the case of precipitates formed in many other mixtures of oppositely charged polyelectrolytes and surfactants,12 it appears not to be possible to redissolve DNA− cationic surfactant precipitates by the addition of excess surfactant or DNA.10,13 The lack of redissolution has been interpreted as a consequence of the high charge density of DNA.10 Furthermore, it has been observed that the behavior of DNA−cationic surfactant mixtures shows a significant dependence on the sample preparation procedure,10,13,14 which suggests an influence from kinetic effects. Cyclodextrins (CDs) are naturally occurring cyclic oligomers of glucose connected through α-(1,4)-glucosidic bonds.15 Their three-dimensional structure resembles a truncated hollow cone, which has an interior that is less polar than the exterior.16 As a consequence of their structure, CD molecules can form inclusion complexes with small hydrophobic molecules or hydrophobic moieties of larger molecules. The formation of inclusion complexes of CDs and surfactants has been extensively studied.17−26 We have previously shown that single, large DNA molecules (166 kbp), which were compacted by the cationic surfactant hexadecyltrimethylammonium bromide, CTAB, can be decompacted and released into solution by the addition of CDs.14,27 The proposed decompaction mechanism involves the formation of CD−surfactant inclusion complexes, which competes with the DNA−surfactant and surfactant−surfactant interactions. In this case no coil−globule coexistence region was found. Instead, the formation of large clusters including multiple DNA molecules was observed at concentrations of CD just below that required for decompaction. When the CD concentration was increased, clusters were not detected, and the DNA was instead found in either partially decompacted globules or fully released as coils; the former situation has been referred to as intrachain segregation.28,29 At even higher concentrations of CD, all DNA was found in the coil conformation. In the present work we investigated the phase behavior of dilute aqueous mixtures of DNA and CTAB as well as of mixtures of DNA, CTAB, and (2-hydroxypropyl)-β-cyclodextrin, 2HPβCD. 2HPβCD is a chemically modified βCD, i.e. a CD containing seven glucose units, with an aqueous solubility superior to that of the native βCD,30 which makes it useful in a larger range of compositions. A multimethod approach was employed to gain understanding of the cluster formation and the lack of a coil−globule coexistence region, which were previously observed in single-molecule studies, and to further improve the general understanding of DNA− surfactant and DNA−surfactant−CD systems. Furthermore, comparisons with the behavior of mixtures of DNA and the naturally occurring tetravalent polyamine spermine were made, and effects of the addition of simple salt were investigated.

2. EXPERIMENTAL SECTION 2.1. Materials. Salmon sperm DNA, received as a 10 mg mL−1 solution (which, if assuming an average molecular weight per nucleotide of 330 g mol−1, corresponds to 30 mM in nucleotides), was provided by Invitrogen (Carlsbad, CA). According to the supplier, ≥75% of the DNA is ≤2000 bp, as determined by gel electrophoresis (1% TAE agarose gel). Spermine (97%, used as received) and hexadecyltrimethylammonium bromide, CTAB (recrystallized twice from acetone), were obtained from Sigma. Trizma base (99.9% titration) and (2-hydroxypropyl)-β-cyclodextrin, 2HPβCD (1.0 M substitution, which signifies that, on average, one 2-hydroxypropyl group is substituted for one hydroxyl group on each glucose unit, 7977


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Figure 1. Visually determined phase map of the DNA−CTAB system presented as a function of (a) CTAB concentration and (b) CTAB:DNA molar ratio, in terms of charges, at four different DNA concentrations (in nucleotides, indicated to the right). Open circles correspond to clear solutions whereas filled circles correspond to turbid or macroscopically phase-separated samples. analyzed using the General Purpose inverse Laplace transformation algorithm, which was provided in the instrument software, in order to obtain z-average size distributions in terms of apparent hydrodynamic radii (RH,app); the values are apparent since extrapolation to infinite dilution was not performed. For samples where the inverse Laplace transformation gave monomodal size distributions or showed inseparable multiple modes (see Results and Discussion section), intensity-weighted mean apparent hydrodynamic radii were derived by cumulant analysis. The presented values of the mean hydrodynamic radii are averages from 10 measurements. The EM experiments were performed using the M3-PALS technique, where the polarity of the applied voltage is periodically reversed to eliminate the influence from electro-osmotic flow. The presented values of the EM are averages from 10 measurements. The DLS experiments were performed using 120 μL disposable cuvettes (Sarstedt) and the EM experiments using DTS 1060 folded capillary cells (Malvern). Stock solutions and samples for the DLS and EM measurements were prepared with buffer filtered through 0.20 μm Sartorius Minisart syringe filters to remove possible dust. 2.5. Circular Dichroism. All experiments were performed on a J815 CD spectrometer (Jasco) equipped with a Jasco PTC-423s/15 Peltier temperature controlling unit operating at 21 °C. The samples were contained in a 10 mm quartz cuvette. Scans were recorded in the range of 300−220 nm with a scanning rate of 50 nm min−1, a bandwidth of 1.00 nm, and a digital integration time (DIT) of 8 s.

concentrations, the double helix may spontaneously dissociate if the ionic strength is too low. Furthermore, the ionic strength from the buffer makes the relative increase in ionic strength with increasing concentrations of the polyionic solutes less pronounced. It can be noted that the contribution to the total ionic strength from the buffer, due to the fact that its base form is uncharged, is lower than its total concentration of 10 mM; the ionic strength was estimated to 7 mM.35 Finally, it should be mentioned that all final DNA concentrations were well below the overlap concentration of the herein used DNA, which is ∼170 μg mL−1.36 Thus, the influence from chain entanglements can be neglected, at least in the absence of condensing agents. 3.2. The DNA−CTAB System. Phase Behavior. The first step in this investigation was to screen the variation in macroscopic behavior with sample composition. Macroscopic phase behavior of dilute DNA−CTAB mixtures has previously been studied;10 however, since these systems are sensitive to variations in, e.g., ionic strength and, as discussed above, preparation protocol,10,13,14 we found it appropriate to determine the phase map for the conditions herein used. The resulting data, presented in two different ways, are found in Figure 1. For all investigated DNA concentrations, ranging from 15 to 120 μM in nucleotides, which corresponds to 5 to 40 μg mL−1, a similar variation in behavior was observed with increasing surfactant concentration: at the lowest surfactant concentrations samples were clear solutions, with increasing concentration macroscopic phase separation was found, and at the highest surfactant concentrations clear solutions were again observed. In the major part of the miscibility gap, i.e., the region of macroscopic phase separation, visible particles appeared immediately as the components were mixed; these particles remained dispersed for hours to days but agglomerated and sedimented with time. In some samples with compositions close to the boundaries of the miscibility gap, the particles were initially too small to be visually detectable, and phase separation was identified by a bluish appearance. In the latter samples, sedimentation was considerably slower. For all samples, the sediment could be partially redispersed by gentle agitation.

3. RESULTS AND DISCUSSION 3.1. Considerations on Sample Preparation. Because it has been observed that the preparation protocol may significantly influence the behavior of dilute aqueous mixtures of oppositely charged polyelectrolytes and surfactants,14,32 which can be attributed to kinetic entrapments, particular care was taken to mix each sample in an identical manner. In order to avoid excessive overshooting of the concentration of any component, these were diluted before mixing. Furthermore, to prevent pH changes as a consequence of dissolved CO2, which could lead to protonation of the DNA bases,33 samples were prepared in buffer. The presence of the buffer also promotes integrity of the DNA double helix. It should be remembered that the formation of double-stranded DNA from its complementary single strands is driven by hydrophobic interactions between the bases and counteracted by electrostatic repulsion between the phosphate groups of the polymer backbone.34 Thus, while working with very low DNA 7978


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indeed significantly influence the macroscopic behavior of dilute aqueous mixtures of DNA and oppositely charged amphiphiles. At elevated ionic strengths, precipitates formed in mixtures of oppositely charged polyelectrolyte and surfactant are often dissolved as a result of screening of the attractive electrostatic interaction.43 The screening can be a consequence of increased concentrations of either or both of the surfactant and the polyelectrolyte, then referred to as self-screening, or of the addition of simple salt. Because of the presence of buffer and the low concentrations of the solutes in the herein discussed samples, the relative change in ionic strength with variation in composition is rather limited, but can the herein observed dissolution at excess surfactant concentration nevertheless be explained by self-screening? In order to assess the sensitivity to an increased ionic strength, the influence of an increasing concentration of NaBr on the phase behavior of the DNA− CTAB mixtures was investigated. The results are found in Figure 2.

In Figure 1a, where the phase map is presented with absolute surfactant concentration, one can see that the boundaries of the miscibility gap are shifted to higher CTAB concentrations with increasing DNA concentration. On the other hand, Figure 1b, where the phase map is instead presented with CTAB:DNA charge ratio, shows that the number of surfactant molecules per nucleotide required for phase separation, as well as the formation of clear solution beyond the miscibility gap, actually decreases with increasing DNA concentration. For all DNA concentrations investigated, an excess, with respect to charge ratio, of surfactant is needed for phase separation to occur. As was discussed in the Introduction, only surfactant molecules residing in micelles are expected to be incorporated in the precipitate. The CMC of CTAB in pure water is 0.92 mM,37 while in the herein used buffer (in the absence of DNA) it is ∼0.5 mM (see surface tension data in Figure S2 of the Supporting Information). The presence of polymer can be expected to induce a significant decrease in the effective CMC; the effective CMC in the presence of polymer is often referred to as the critical association concentration (CAC).37,38 For the specific case of CTAB in the presence of DNA in aqueous solution at low salt content, the CAC has been found to be between 4 and 20 μM.9,39−42 In a binding isotherm study performed at solution conditions and DNA concentration similar to those herein used, the CAC was determined to be 8 μM.9 Thus, the expected CAC is about an order of magnitude lower than the CTAB concentration required for macroscopic phase separation. Indeed, it has been found, using fluorescence microscopy8 and dynamic light scattering,11 that, at low DNA concentrations and surfactant concentrations above the CAC, soluble individual DNA−surfactant aggregates (globules) are formed. At the point where macroscopic phase separation is observed, a vast majority of surfactant resides in micellar aggregates, from which follows that the micelle:DNA charge ratio is close to the global surfactant:DNA charge ratio. This shows that, although micellization induced by the presence of polyelectrolyte is a highly cooperative event,37 the influence from cooperativity on macroscopic phase separation is limited. The observation that the region of macroscopic phase separation is shifted toward higher CTAB:DNA charge ratios at decreasing DNA concentration (cf. Figure 1b) is in good agreement with previous phase diagram determinations in related oppositely charged polyelectrolyte−surfactant systems43 and can be attributed to a decreasing fraction of surfactant residing in micelles (which are, as discussed above, required for DNA neutralization and precipitation to occur) with decreasing total surfactant concentration. In light of results from previous work, showing that precipitates formed by DNA and CTAB cannot be dissolved by a presence of excess surfactant,10 the finding that clear solutions are found above a certain concentration of CTAB is striking. It can be noted that during the addition of surfactant to the DNA solution no indication of transient clouding was observed; i.e., the presence of clear solution above the miscibility gap seems not to be the result of precipitation and redissolution but rather a suppression of precipitation. In contrast, and in consistency with previous findings, no redissolution was observed in a sample where DNA (61 μM) was first precipitated by CTAB (0.20 mM), whereafter an additional aliquot of CTAB was added to yield a final surfactant concentration of 0.80 mM (i.e., to a concentration well into the upper clear region in Figure 1).44 Taken together, these findings confirm that the sample preparation protocol can

Figure 2. Visually determined phase map for the DNA−CTAB system at a fixed DNA concentration of 61 μM in nucleotides (20 μg mL−1) and varying concentrations of CTAB and NaBr. NaBr concentrations are indicated to the right. Open circles correspond to clear solutions, whereas filled circles correspond to turbid or macroscopically phaseseparated samples.

Along with the expectations the miscibility gap disappears in the presence of a high concentration (somewhere between 0.4 and 2 M) of NaBr.45 Thus, the concentration of simple salt required for dissolution of the precipitate is orders of magnitude higher than those contributed by the surfactant and DNA counterions, which strongly suggests that the dissolution at excess surfactant found in Figure 1 cannot be explained by self-screening. If only electrostatic screening had been at play, one would expect a monotonic narrowing of the miscibility gap with increasing salt concentration up to the point where it disappears. However, as can be seen in Figure 2, the region of phase separation is significantly increased at intermediate NaBr concentrations; whereas no major change in the position of the lower phase boundary was observed, there is either a widening of the miscibility gap (with 50 mM NaBr) or no upper phase boundary observed within the investigated range of CTAB concentrations (with 200 or 400 mM NaBr). 7979


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Figure 3. (a) Selected UV spectra for DNA−CTAB mixtures and (b) absorbance of DNA in DNA−CTAB mixtures at a fixed DNA concentration of 61 μM in nucleotide units (20 μg mL−1) and varying CTAB concentration. All values in (b) are normalized to the values for free DNA. A260,corr signifies that the contribution from scattering has been accounted for (cf. section 2.3). The shaded area indicates the miscibility gap, where visually observable precipitates are formed (cf. Figure 1).

function of sample composition. Figure 3a presents example spectra for DNA and DNA−CTAB mixtures with a constant DNA concentration of 61 μM and varying CTAB concentration, including samples taken from below, within, and above the miscibility gap. With a CTAB concentration of 0.0030 mM, which is below the expected CAC, the absorbance spectrum is practically identical to that in the absence of surfactant. A striking observation from these experiments is the dramatic reduction (relative to free DNA) in the recorded absorbance for the composition corresponding to the miscibility gap (0.20 mM CTAB; cf. Figure 1). Furthermore, there is a notable increase in absorbance for samples both just below and above the miscibility gap (0.060 and 0.80 mM CTAB, respectively). These samples also display a small shift in the position of the absorbance maximum toward a higher wavelength. For samples with CTAB concentrations of 0.060, 0.20, and 0.80 mM, an increased absorbance at higher wavelengths was also observed. CTAB alone, in the relevant concentration range, has only a limited influence on the UV−vis spectrum (cf. Figure S3 in Supporting Information); thus, the observed increase in measured absorbance at higher wavelengths can be ascribed to an increased scattering, which in turn gives an increase in turbidity. In order to further evaluate the variation in absorbance and turbidity with composition, a series of samples with a wide range of surfactant concentrations were investigated. The data are presented in Figure 3b where the phase separation region, as previously determined (cf. Figure 1), is indicated by a gray shade. The contribution from scattering to the measured absorbance at 260 nm was accounted for as described in section 2.3. One can see that with increasing CTAB concentration there is an initial slight decrease in the absorbance from DNA up to a CTAB concentration of 0.040 mM, above which there is a notable increase up to the point where macroscopic phase separation is observed; it can be noted that the concentration where the increase begins coincides reasonably well with reported CAC values for CTAB in the presence of DNA (cf.

The CAC of an ionic surfactant in the presence of an oppositely charged polyelectrolyte has been found to increase considerably in response to an increased salt concentration.46 One could, therefore, expect that also the boundary for macroscopic phase separation should change toward higher CTAB concentrations on addition of simple salt. The finding that the lower phase boundary is practically invariant, i.e. that the changes in CAC and macroscopic phase separation do not parallel, thus suggests that additional, herein not further discussed, factors are at play in controlling the lower boundary of the miscibility gap. Previous studies suggest that dissolution of stoichiometric CTA−DNA complexes by simple salt can be attributed to electrostatic screening of the attraction between the micelles and the DNA.13 The herein observed increase of the phase separation range at intermediate salt concentrations shows that, in the presence of excess surfactant, additional interactions are involved. In certain mixtures of oppositely charged polyelectrolyte and surfactant, binding of an excess of surfactant to the polyelectrolyte can result in overcharging of the mixed aggregates and possible redissolution; sometimes redissolution of such overcharged mixed aggregates can occur with a rather small excess of surfactant, i.e., at rather low net charge.47 Furthermore, weakly charged and not too hydrophilic polyelectrolytes are commonly precipitated by a salting-out mechanism at elevated ionic strengths.48 On the basis of the above facts, we suggest that, in the presence of excess surfactant, weakly overcharged net-cationic mixed DNA− CTAB aggregates are formed, which are salted out at intermediate NaBr concentrations. UV Absorbance and Circular Dichroism Spectroscopy. Double-stranded DNA shows a distinct UV absorbance maximum at 260 nm, a property commonly utilized for the determination of DNA concentration in solution.49 In this work, UV absorbance studies were undertaken in order to estimate variations in the amount of assessable DNA as a 7980


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turbidity observed around the miscibility gap to compaction and possible accompanying dehydration of the DNA. Dynamic Light Scattering and Electrophoretic Mobility. In order to gain information on the size and charge of the aggregates formed in solution, dynamic light scattering (DLS) and electrophoretic mobility (EM) measurements were performed. The signal-to-noise ratio from samples with a DNA concentration of 61 μM (20 μg mL−1) was rather low but was found to be considerably improved by doubling the DNA concentration (120 μM, 40 μg mL−1), and all DLS and EM experiments were performed with the latter concentration; the phase map for DNA−CTAB with 40 μg mL−1 DNA is found in the uppermost line of Figure 1. Compositions of samples characterized in the DLS and EM experiments are shown in Table 1.

above). Above the miscibility gap, the absorbance is approximately the same as just below it and does not change significantly with composition. In the clear solution regions, the variation in apparent absorbance at 320 nm, as a measure of turbidity, follows a similar trend as the variation in absorbance at 260 nm. It can be noted that the turbidity is higher close to the phase borders than in the middle of the precipitation region; the lower scattering in the middle of the miscibility gap can be ascribed to the fact that phase-separated particles have sedimented and thus do not contribute significantly to scattering (cf. above). From additional absorbance data (cf. Figure S4 in Supporting Information) we note a similar trend in absorbance and scattering with varying CTAB concentration at both lower (30 μM) and higher (120 μM) DNA concentrations. In a recent report, an elevated absorbance of DNA due to association with cationic dendrimers was observed and ascribed to reduced base stacking interactions in the DNA double helix.50 This, in turn, implies a conformational change of the DNA. Circular dichroism spectroscopy is extensively used to study changes in DNA conformation,51,52 and in order to identify possible conformational changes with increasing CTAB concentration, such experiments were performed. Circular dichroism spectra for a series of DNA−CTAB samples are found in Figure 4, with samples taken from both sides of the

Table 1. DLS and EM Data for Aqueous Mixtures of DNA and CTAB, with a Constant DNA Concentration of 120 μM in Nucleotides (40 μg mL−1) and Varying CTAB Concentration [CTAB]/ mM

CRCTAB:DNAa

RH,appb/ nm

μec/10−8 m2 V−1 s−1

0.020 0.040 0.080 0.15 0.25 0.40 0.55 0.70 0.80 1.0 1.4

0.17 0.33 0.66 1.2 2.1 3.3 4.5 5.8 6.6 8.3 12

48 63 73 −d −d −d 60 57 57 55 50

−2.4

comment

−3.0 precipitate precipitate precipitate

3.0 2.9

a

CRCTAB:DNA = CTAB:DNA charge ratio. bRH,app = apparent hydrodynamic radius, given as a mean of values from 10 measurements; the variation among measurements was within a few percent. c μe = electrophoretic mobility, given as a mean of values from 10 measurements; the variation among measurements was within a few percent. dDLS measurements were performed for these samples, and particle radii in the range of 2−3 μm were detected. However, since visible particles were formed in the mixtures, the DLS data do not reflect the whole size distribution and the measured values are not true means.

Figure 5 presents relaxation time distributions for DNA alone and selected samples with varying concentration of CTAB. DNA alone (Figure 5, bottom plot) shows a trimodal relaxation time distribution. From comparisons to previous DLS data on similar DNA, the intermediate mode can be ascribed to the translational diffusive motion of individual DNA molecules, whereas the fast and slow modes can be ascribed to mode coupling between translational diffusion and internal motion and to translational diffusion of domains containing several polymer molecules, respectively.54−56 The main translational diffusion mode corresponds to an apparent hydrodynamic radius, RH,app, of 37 nm. In previous DLS experiments on DNA with similar specifications as that used here (2000 ± 500 bp), but at half the concentration (62 μM, 20 μg mL−1), the hydrodynamic radius, RH, was determined to be 107 nm.36 The discrepancy between the value determined in ref 36 and the one herein obtained is mainly attributed to the fact that, in the latter case, extrapolation to infinite dilution was not made. Furthermore, the main translational mode cannot be fully

Figure 4. Circular dichroism spectra for DNA−CTAB mixtures at a DNA concentration of 61 μM in nucleotides (20 μg mL−1) and varying CTAB concentration (indicated in the upper left corner of the plot).

miscibility gap; the spectrum of DNA alone is included as a reference. Two significant differences are seen between the DNA and the DNA−CTAB samples: first, there is a small but consistent shift from approximately 258 to 260 nm in the crossover between negative and positive values of ellipticity, and second, there is a decrease in the magnitude of the long wavelength band. Our spectra for DNA in the presence of CTAB are very similar to circular dichroism spectra of compacted DNA in phage heads,53 suggesting that our observation is a consequence of DNA compaction. Similar changes have also been observed in spectra of DNA in 60% aqueous ethanol, which were attributed to dehydration of the DNA.52 We can thus relate the increase in UV absorbance and 7981


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sides of the miscibility gap and, importantly, that there is a shift in the sign of the effective charge of the aggregates: below the miscibility gap the effective net charge is negative, while above the miscibility gap it is positive. The finding that the mixed aggregates beyond the miscibility gap carry a net positive charge supports the above proposed overcharging and, consequently, the salting-out mechanism at intermediate NaBr concentrations. 3.3. The DNA−Spermine System. Phase Behavior and UV Absorbance. A commonly used DNA compacting/ condensing agent is the naturally occurring tetravalent cationic polyamine spermine.60 Most studies on DNA−spermine mixtures have had the emphasis on the influence of spermine on DNA conformation in single-molecule studies. However, investigations on bulk samples with DNA concentrations ranging from 0.01 to 1 mg mL−1 (corresponding to between 30 μM and 3 mM in nucleotides) have shown the occurrence of a miscibility gap.61 The action of spermine in its interaction with DNA is fundamentally different from that of CTAB. Spermine does not self-assemble but can nevertheless, due to its tetravalent cationic charge, induce compaction and phase separation of DNA. Therefore, it constitutes a relevant comparison when elucidating the behavior of the DNA− CTAB system. Absorbance data at 260 nm (with the contribution from scattering accounted for in the same way as above) and apparent absorbance values at 320 nm are presented in Figure 6; the visually determined phase separation

Figure 5. Relaxation time distributions from dynamic light scattering measurements at 173° on DNA and DNA−CTAB mixtures. The DNA concentration was fixed at 120 μM in nucleotides (40 μg mL−1), and the CTAB concentrations are indicated by the respective plots.

separated from the fast relaxation mode. Both factors are expected to contribute to a significant underestimation of the size. Slow modes arising from the concerted motion of multimolecular domains are commonly observed in polyelectrolyte solutions at low ionic strength.57−59 In ref 36, where, as discussed above, a lower DNA concentration was used, no slow mode was observed; this can be attributed to the fact that this lower concentration is further away from the overlap concentration. For samples with CTAB concentrations between 0.020 and 0.080 mM, which are all below the miscibility gap but above the expected CAC, the slow mode is absent. The disappearance of the slow mode can be understood from the partial neutralization and possible condensation of the DNA molecules upon the addition of surfactant. Indications of the fast mode are still observable although it is on the limit of being separable from the translational diffusion mode by the inverse Laplace transformation algorithm used; typically, it appears just as a shoulder toward faster relaxation times (see example in Figure 5, middle plot). When the translational and the fast relaxation modes are not separable, the inverse Laplace transformation is not expected to yield correct values. We thus used the cumulant method to evaluate the correlation functions and obtain mean hydrodynamic radii. The fact that the fast relaxation mode was found to prevail with increasing concentration of CTAB up to the point where phase separation was observed can be attributed to a coexistence of coils and globules where the noncompacted DNA retains its internal mobility.11 Above the miscibility gap, the relaxation time distribution profiles show single modes (see example in Figure 5, top plot), which were attributed to translational diffusion and evaluated using the cumulant method. The mean apparent hydrodynamic radii, RH,app, for mixtures of DNA and CTAB are found in Table 1. Data from EM measurements, shown together with DLS data in Table 1, were obtained for selected samples below and above the miscibility gap. Table 1 shows that the effective size, in terms of hydrodynamic radius, of the aggregates is similar on both

Figure 6. Absorbance of DNA in DNA−spermine mixtures at a fixed DNA concentration of 61 μM in nucleotides (20 μg mL−1) and varying spermine concentration. All values are normalized to the values for free DNA. A260,corr signifies that the contribution from scattering has been accounted for (cf. section 2.3). The shaded area indicates the miscibility gap, where visually observable precipitates are formed.

region is indicated by a gray shade. In order to allow for direct comparison, samples were prepared using a preparation protocol identical to that used for the DNA−CTAB samples. A miscibility gap was indeed found. We note that the lower boundary of the phase separation region occurs at a spermine:DNA charge ratio close to unity, which is comparable to that found for the DNA−CTAB system. However, a much larger excess of spermine, as compared to CTAB (more than an 7982


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Figure 7. Absorbance of DNA in aqueous mixtures of DNA, CTAB, and 2HPβCD at a fixed DNA concentration of 61 μM (20 μg mL−1), CTAB concentrations of either (a) 0.20 or (b) 0.80 mM, and a varying 2HPβCD concentration. All values are normalized to the values for free DNA. A260,corr signifies that the contribution from scattering has been accounted for (cf. section 2.3). The shaded areas indicate the miscibility gap, where visually observable precipitates are formed.

CTAB precipitate, whereas in the latter it is added to chargeinversed aggregates. As expected, addition of 2HPβCD to a sample where DNA has been precipitated by CTAB can lead to dissociation of the precipitate and redissolution of the DNA.14 However, as can be seen in Figure 7a, a significant molar excess of 2HPβCD compared to CTAB is needed for this to occur; for the herein used conditions a 2HPβCD:CTAB molar ratio of 5.5 was required for complete dissociation. Absorbance values at 260 nm as well as at 320 nm suggest that, at the point where the precipitate is dissociated, the DNA is fully released from the surfactant aggregates, a conclusion which is supported by fluorescence spectroscopy data reported in earlier work.14 The formation of inclusion complexes between CDs and surfactants is characterized by very high association constants. For instance, the association constant for the formation of 1:1 βCD:CTAB complexes is approximately 5 × 104 M−1; i.e., in a solution of CTAB and an excess of βCD, the fraction of free surfactant is negligibly small.20,62 The fact that the 2HPβCD:CTAB molar ratio necessary for DNA redissolution is significantly above unity thus indicates that the surfactant is strongly associated with the DNA in the precipitate; it can be noted that the phase boundary is unchanged over at least one month. It was difficult to obtain reproducible UV absorbance data close to the phase boundary; however, previous experiments (at significantly higher DNA concentration, but otherwise similar conditions) where the precipitate had been separated from the solution suggest a gradual release of DNA over a range of increasing CD concentrations as the phase border is approached.14 When the initial state is instead the charge-inversed DNA− surfactant aggregates, the addition of 2HPβCD (Figure 7b) results in a behavior that is essentially a mirror image of that found with an increasing amount of CTAB added to DNA (cf. Figure 3b). Strikingly, phase separation occurs at a 2HPβCD:CTAB molar ratio of 0.55. If assuming that every added 2HPβCD molecule forms an inclusion complex with one surfactant molecule, the remaining surfactant concentration is

order of magnitude in charge ratio), is required to arrive at the clear solution region beyond the miscibility gap. Contrary to the case of the DNA−CTAB system (cf. Figure 3) where an elevated absorbance of DNA was observed just below and beyond the phase separation region, the DNA absorbance in the DNA−spermine system is essentially retained at the value for DNA alone at all compositions except for those within the miscibility gap. Furthermore, for samples outside the miscibility gap there is no significant variation in turbidity. Taken together, these observations suggest that the DNA is not condensed by spermine at concentrations below and above those corresponding to macroscopic phase separation. Electrophoretic Mobility. EM measurements on DNA− spermine samples (at a DNA concentration of 120 μM, corresponding to 40 μg mL−1) did not indicate a charge inversion for samples at spermine concentrations above the miscibility gap. Instead, we found only a decreased electrophoretic mobility (from −1.2 × 10−8 m2 V−1 s−1 for a spermine:DNA charge ratio of 0.33 to −0.32 × 10−8 m2 V−1 s−1 for a spermine:DNA charge ratio of 500). The differences between the DNA−CTAB and DNA−spermine systems with respect to effective charge will be further elaborated on below. 3.4. The DNA−CTAB−2HPβCD System. Phase Behavior and UV Absorbance. The action of 2HPβCD on DNA−CTAB aggregates was investigated using a rigorous sample preparation protocol similar to that used in the experiments discussed above. Absorbance data at 260 nm (with the contribution from scattering accounted for in the same way as above) and apparent absorbance values at 320 nm are presented in Figure 7 for two series of DNA−CTAB−2HPβCD samples, where the DNA concentration was constant at 61 μM, the CTAB concentration was fixed at either 0.20 or 0.80 mM (Figure 7a and 7b, respectively), and the 2HPβCD concentration was varied over a wide range. As above, the gray shade indicates a visually determined phase separation region. The two surfactant concentrations used correspond to different initial states: in the former case the 2HPβCD is added to a macroscopic DNA− 7983


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Figure 8. Absorbance of DNA in aqueous mixtures of DNA, CTAB, and 2HPβCD at a fixed DNA concentration of 61 μM (20 μg mL−1), CTAB concentrations of either (a) 0.20 or (b) 0.80 mM, and a varying 2HPβCD concentration, with a different mixing order as compared to that used for the data underlying Figure 7 (see text). All values are normalized to the values for free DNA. A260,corr signifies that the contribution from scattering has been accounted for (cf. section 2.3). The shaded areas indicate the miscibility gap, where visually observable precipitates are formed.

0.36 mM, which almost perfectly coincides with the CTAB concentration at the upper phase boundary in Figure 3b. Similarly to the case in Figure 7a, the precipitate is fully dissociated at an excess of CD; however, here the excess is smaller, with a 2HPβCD:CTAB molar ratio of 3 (as compared to 5.5 in Figure 7a). An important point to note is that in samples with compositions corresponding to above the miscibility gap no transient clouding was observed on addition of 2HPβCD. Beyond the miscibility gap, the absorbance at 260 nm is, in correspondence to the observations in Figure 7a, very close to unity, which suggests that all DNA is here fully released. It can be noted that the surfactant concentration in the samples represented in Figure 7b, i.e. 0.80 mM, is above the CMC in the used buffer. However, due to the strong propensity for formation of CD−surfactant inclusion complexes, it is unlikely that free micelles are present at 2HPβCD concentrations where the DNA is completely released. The most obvious mixing order when studying DNA decondensation is the one used in the experiments discussed above, i.e., to first add the condensing agent to the DNA, equilibrate, and then add the decondensing agent. We have also used an alternative mixing order where CTAB and 2HPβCD were equilibrated before the addition of DNA; a change of the mixing order can be expected to give information on equilibrium vs kinetics. UV absorbance and phase behavior are presented in Figure 8, in an analogous way as in Figure 7. The general qualitative features of Figure 8 are the same as those in Figure 7, but there are some important quantitative differences. The main difference in Figure 8a as compared to Figure 7a is that dissociation occurs at a lower excess of 2HPβCD with the alternative mixing order. The fact that the phase boundary is different for the two mixing orders shows that kinetic effects are at play. It was found, indeed, that after three additional days of equilibration, the phase boundary in Figure 8a was shifted from a 2HPβCD concentration of ∼0.35 mM to ∼0.50 mM. It then remained unchanged for at least one month. This finding can be taken to suggest that the

equilibrium phase boundary lies somewhere in-between the phase boundaries found in Figures 7a and 8a. For the situation of higher CTAB concentration, 0.80 mM, the behavior is much less sensitive to mixing order, and Figures 7b and 8b are practically identical (note the difference in the xaxis scale). The only notable difference is that the lower phase boundary is slightly shifted toward a higher 2HPβCD concentration when the DNA is added to pre-equilibrated mixtures of CTAB and 2HPβCD. The difference, however, is small and may be a consequence of other factors than mixing order per se, such as differences in volume at each mixing step in the two sample preparation protocols (cf. Sample Preparation section). Dynamic Light Scattering and Electrophoretic Mobility. DLS and EM experiments were performed on mixtures of DNA, CTAB, and 2HPβCD, where the DNA concentration was fixed at 120 μM in nucleotides (40 μg mL−1), the CTAB concentration was fixed at 0.80 mM, and the 2HPβCD concentration was varied. (UV absorbance data for these compositions are found in Figure S5 of the Supporting Information; this figure is similar to Figure 7b but with the lower boundary of the miscibility gap shifted to a slightly higher 2HPβCD concentration.) The initial DNA−CTAB mixture (without 2HPβCD) gives overcharged DNA−CTAB aggregates (cf. Table 1). At 2HPβCD concentrations below the miscibility gap, the effective net charge was positive (3.5 × 10−8 and 2.6 × 10−8 m2 V−1 s−1 for 2HPβCD concentrations of 0.16 and 0.30 mM, respectively), and the DLS relaxation time distributions were monomodal (with RH,app of 56 and 52 nm for 2HPβCD concentrations of 0.16 and 0.30 mM, respectively). Above the miscibility gap, on the other hand, the effective net charge was negative (−3.3 × 10−8 and −2.2 × 10−8 m2 V−1 s−1 for 2HPβCD concentrations of 4.0 and 20 mM, respectively). DLS data from just above the miscibility gap showed both translational diffusion modes and fast modes (with the former corresponding to a RH,app of 73 nm for a 2HPβCD concentration of 4 mM). With higher 2HPβCD concentrations, 7984


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evaluation of the DLS data was complicated by the occurrence of intense signals with relaxation times corresponding to RH,app of 1 and 200 nm. These modes are ascribed to the 2HPβCD:CTAB inclusion complexes and unidentified aggregates (tentatively clusters of 2HPβCD), respectively (data not shown). These findings support the UV absorbance data presented above in that they suggest a full release of DNA from the DNA−CTAB aggregates as surfactant is extracted by 2HPβCD. 3.5. Comparisons of the Investigated Systems from a Molecular Perspective. In the DNA−CTAB system we observed a miscibility gap at CTAB:DNA charge ratios between approximately 1 and 10, with the exact values varying with DNA concentration. Below the miscibility gap, the mixed aggregates were found to carry a negative net charge whereas above this the net charge was positive. Although positive overcharging of DNA has been found in related DNA−cationic gemini surfactant mixtures at much lower DNA concentration,63 the occurrence of a miscibility gap in a DNA− surfactant system has not, to our knowledge, previously been presented. However, it appears to be possible to obtain the positive overcharging only when applying a mixing protocol where the excess surfactant is initially present; if the excess surfactant is added to preexisting precipitates, redissolution does not occur, in accordance with previous findings. From the finding that the final state of the sample is different for the two mixing protocols follows that at least one of these states represents a nonequilibrium situation. If one of the situations does represent the equilibrium state, which of them does? For dilute aqueous mixtures of oppositely charged polyelectrolyte and surfactant it is difficult to judge whether reentrant dissolution is an equilibrium or a kinetically stabilized state; it has been found that, in certain cases, apparently redissolved mixed aggregates are in fact nonequilibrium dispersions of particles of the precipitate, electrostatically stabilized by adsorbed surfactant.47,64 In the following, arguments for equilibrium and kinetic stabilization will be considered. It is found that true redissolution of surfactant−polyion complexes, on addition of excess surfactant, is promoted by the presence of hydrophobic moieties on the polyion.47 Indeed, hydrophobic molecules or molecules carrying hydrophobic groups can be solubilized in the DNA double helix and/or influence the DNA melting temperature.65 On the other hand, results from investigations on aqueous mixtures of DNA and nonionic surfactant suggest that the extent of the hydrophobic interaction between these components is limited.66 Stoichiometric complexes formed by DNA and CTAB have been found to likely be arranged in a 2D hexagonal structure of elongated surfactant micelles each surrounded by six DNA chains; a schematic representation of the proposed structure is shown in Figure 9.67 Fully hydrated CTA−DNA complexes contain a significant amount of water, 13−14 water molecules per surfactant ion.67 From the illustration in Figure 9 it can be understood that the electroneutral stoichiometric unit cell (as indicated by the red hexagon) has a significant exposure of hydrophobic surfactant tails to the surrounding. From this we can understand that there is a strong propensity for the formation of large aggregates, which is indeed manifested as macroscopic phase separation under conditions close to a global CTAB:DNA charge ratio of unity.

Figure 9. Schematic idealized representation of the fully hydrated CTA−DNA hexagonal arrangement; redrawn from ref 67.

With the above discussion in mind, the finding that no transient clouding was observed when DNA is mixed with excess CTAB according to our protocol, i.e., that the excess surfactant appears to cause suppression of precipitation rather than redissolution of a precipitate, could be taken to suggest that the positively overcharged aggregates are kinetically stabilized. On the other hand, it should be remembered that kinetic effects can play a major role in the dissolution of macromolecules, and even the dissolution of polymers in a good solvent may be a considerably time-consuming process.37 Furthermore, NMR studies have shown that, although the surfactant ions in CTA−DNA complexes are locally highly mobile, their long-range translational displacement is slow (selfdiffusion coefficient, D < 10−13 m2 s−1).68 These factors can reasonably render the approach to the equilibrium state impossible on a practical time scale and, if the overcharged aggregates do in fact represent the equilibrium state, hinder penetration of excess surfactant into the precipitate. Taken together, the above discussion clearly points out the difficulty to unambiguously judge whether the soluble aggregates or the precipitate is, or is closest to, an equilibrium situation. As was discussed above, several fluorescence microscopy studies have addressed the coexistence of DNA coils and globules in the presence of cationic surfactant. These findings can be directly linked to our results from the absorbance and DLS studies, which are consistent with a region of coexistence of noncompacted and compacted DNA in a composition range between the CAC and the onset of macroscopic phase separation. In this context, it is valuable to consider a singlemolecule study where the conformational behavior of DNA was investigated within a very large range of CTAB concentrations.8 With an excess of CTAB corresponding to a CTAB:DNA charge ratio of 33, all DNA existed as single globular entities, each containing one DNA molecule. Notably, at a further increased surfactant concentration, corresponding to a charge ratio of 270, a clustering of the globules was observed. Thus, it appears that not only the compaction, but also the macroscopic phase separation observed in the herein presented experiments, shows direct parallels to the behavior observed at singlemolecule conditions. 7985


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are prone to aggregation into larger structures, in direct parallel to the macroscopic phase separation observed at the herein used higher concentration of DNA (cf. Figure 7b). An alternative explanation for the cluster formation was recently presented, where it is suggested to arise from stacking of CTAB−CD inclusion complexes between the surfaces of individual DNA−surfactant globules.73 However, in this case native βCD, which has an aqueous solubility much lower than that of 2HPβCD, was used as the decompaction agent. Furthermore, βCD has been shown to aggregate into large structures in aqueous solution74 as well as to readily cocrystallize with the cationic surfactant dodecyltrimethylammonium bromide (DTAB).75 Therefore, the behavior of the system discussed in ref 73 may not be directly comparable to the behavior of the herein investigated one. Coil−globule coexistence regions are typically observed on compaction of DNA with cationic surfactant as well as when DNA−cationic surfactant aggregates are decompacted using, for instance, nonionic or anionic surfactant.76,77 On the other hand, as was discussed in the Introduction, when DNA−CTAB globules are decompacted by CDs, no such region has been detected.14,27 Instead, there is a narrow CD concentration range, just above the concentrations at which clusters involving multiple DNA molecules are observed, where partially decompacted DNA globules are abundant. It is a fair assumption that the formation of 1:1 CD−surfactant inclusion complexes is less influenced by cooperativity than the formation of mixed micelles. Thus, although kinetic effects may potentially also have an influence, the intrachain segregation observed on decompaction using CDs can reasonably be regarded as a lower cooperativity variant of coil−globule coexistence.

Similarly to the case for mixtures of DNA and CTAB, mixtures of DNA and spermine show a miscibility gap. Important differences between the systems were the width of the miscibility gap and that the sign of the electrophoretic mobility beyond it was different in the two cases. Since spermine does not have a distinct hydrophobic part, redissolution of the DNA−spermine precipitate cannot be explained by a similar mechanism as that discussed for the DNA−CTAB system; indeed, the electrophoretic mobility data did not indicate overcharging in the latter system. This result is supported by theoretical work on mixtures of polyelectrolyte and multivalent ions, where it has been found that reentrant dissolution on increasing concentration of the multivalent ions can occur without overcharging of the polyelectrolyte.69 Another fundamental difference between the behaviors of the DNA−CTAB and DNA−spermine systems is that phase separation in the latter has been shown to be completely reversible; i.e., a precipitate of DNA and spermine is redissolved by a later added excess of spermine.61 The most likely dissolution mechanism is simply an electrostatic screening of the DNA−spermine attraction as the total ionic strength increases with the spermine concentration. This suggestion finds further support in previous reports showing that the phase separation region decreases in width or completely vanishes61 and that compacted single DNA chains are unfolded,6 on the addition of simple salt. It should be noted that other theoretical work predicts that multivalent counterions will overcompensate the DNA charge when present at high concentration,70 which has also been observed experimentally for certain conditions not directly comparable to those in the present work.71 Nevertheless, whether charge inversion occurs at some point or not, the mechanism underlying redissolution should be fundamentally electrostatic in nature.72 Turning our attention to the mechanism underlying dissociation of DNA−CTAB precipitates by the addition of CD, the basis of which is fundamentally different from that in the two previous cases. The most likely action of CD is to extract the surfactants from the bulk solution, molecule by molecule, causing a depletion of accessible surfactant unimers and thus a shift in the distribution of surfactant between the bulk (free unimers and unimers in CD−surfactant inclusion complexes) and the DNA−surfactant aggregates. The fact that the 2HPβCD:CTAB molar ratio where dissociation is observed is different depending on the order of mixing shows that kinetic effects are at play. As noted in the Introduction, cluster formation has previously been observed in fluorescence microscopy studies on CD-induced decompaction of single-molecule DNA−CTAB globules.14,27 In these reports, a very large excess of CTAB was used for compaction of the DNA; in terms of the CTAB:DNA charge ratio an almost 500 times excess was used. We recall that, in other single-molecule studies performed at very similar conditions, clustering of multiple DNA−CTAB globules, which we correlate to the herein observed miscibility gap (cf. Figure 1), was found at a CTAB:DNA charge ratio of 270 (in the absence of CD; cf. above).8 In the light of our new findings (i.e., miscibility gap and overcharging), we can conclude that the single globules observed at a CTAB:DNA charge ratio of 500 are net-cationic DNA−CTAB aggregates. On addition of CD, the surfactant molecules are extracted from the globules, a process which, at some point, can be expected to lead to a situation where the net charge of the globules is zero, and they

4. CONCLUSIONS We have investigated the macroscopic phase behavior and other physicochemical properties of dilute aqueous mixtures of DNA−CTAB, DNA−spermine, and DNA−CTAB−2HPβCD, with the main objective to understand phenomena previously observed in single-molecule experiments. In order to allow for optimal comparability of the results from the different techniques applied, a consistent sample preparation protocol was used. With an increasing concentration of CTAB, condensation of the DNA precedes macroscopic phase separation where a charge neutral precipitate forms. Furthermore, we found that when mixing DNA with an excess of CTAB, positively overcharged mixed aggregates were formed. It is difficult to judge whether these aggregates represent an equilibrium situation or not. Redissolution of a DNA−spermine precipitate is most likely a consequence of electrostatic screening as the ionic strength increases with the spermine concentration. We conclude that, in contrast to what was observed for DNA−CTAB mixtures, no DNA condensation occurred at spermine concentrations lower than that necessary for macroscopic phase separation, in other words, that compaction of DNA by spermine observed in single-molecule studies directly parallels the herein observed macroscopic phase separation. We argue that the main underlying mechanism for dissolution of DNA−CTAB precipitate by addition of a sufficient concentration of 2HPβCD is effectively an extraction of “accessible” surfactant from the solution. This notion is supported by the fact that addition of 2HPβCD to overcharged 7986


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(6) Khan, M. O.; Jönsson, B. Electrostatic Correlations Fold DNA. Biopolymers 1999, 49, 121−125. (7) Wagner, K.; Harries, D.; May, S.; Kahl, V.; Rädler, J. O.; BenShaul, A. Direct Evidence for Counterion Release Upon Cationic Lipid-DNA Condensation. Langmuir 2000, 16, 303−306. (8) Mel’nikov, S. M.; Sergeyev, V. G.; Yoshikawa, K. Discrete CoilGlobule Transition of Large DNA Induced by Cationic Surfactant. J. Am. Chem. Soc. 1995, 117, 2401−2408. (9) Mel’nikov, S. M.; Sergeyev, V. G.; Yoshikawa, K. Transition of Double-Stranded DNA Chains between Random Coil and Compact Globule States Induced by Cooperative Binding of Cationic Surfactant. J. Am. Chem. Soc. 1995, 117, 9951−9956. (10) Dias, R. S.; Mel’nikov, S.; Lindman, B.; Miguel, M. G. DNA Phase Behavior in the Presence of Oppositely Charged Surfactants. Langmuir 2000, 16, 9577−9583. (11) Dias, R. S.; Innerlohinger, J.; Glatter, O.; Miguel, M. G.; Lindman, B. Coil-Globule Transition of DNA Molecules Induced by Cationic Surfactants: A Dynamic Light Scattering Study. J. Phys. Chem. B 2005, 109, 10458−10463. (12) Goddard, E. D.; Hannan, R. B. Polymer-Surfactant Interactions. J. Am. Oil Chem. Soc. 1977, 54, 561−566. (13) Rosa, M.; Dias, R. S.; Miguel, M. G. Lindman, B. DNA-Cationic Surfactant Interactions Are Different for Double- and Single-Stranded DNA. Biomacromolecules 2005, 6, 2164−2171. (14) Carlstedt, J.; González-Pérez, A.; Alatorre-Meda, M.; Dias, R. S.; Lindman, B. Release of DNA from Surfactant Complexes Induced by 2-Hydroxypropyl-Beta-Cyclodextrin. Int. J. Biol. Macromol. 2010, 46, 153−158. (15) Larsen, K. L. Large Cyclodextrins. J. Inclusion Phenom. Macrocyclic Chem. 2002, 43, 1−13. (16) Szejtli, J. Introduction and General Overview of Cyclodextrin Chemistry. Chem. Rev. 1998, 98, 1743−1754. (17) Junquera, E.; Tardajos, G.; Aicart, E. Effect of the Presence of Beta-Cyclodextrin on the Micellization Process of Sodium DodecylSulfate or Sodium Perfluorooctanoate in Water. Langmuir 1993, 9, 1213−1219. (18) Mehta, S. K.; Bhasin, K. K.; Dham, S.; Singla, M. L. Micellar Behavior of Aqueous Solutions of Dodecyldimethylethylammonium Bromide, Dodecyltrimethylammonium Chloride and Tetradecyltrimethylammonium Chloride in the Presence of Alpha-, Beta-, HP-Betaand Gamma-Cyclodextrins. J. Colloid Interface Sci. 2008, 321, 442− 451. (19) Cepeda, M.; Daviña, R.; García-Río, L.; Parajó, M. CyclodextrinSurfactant Binding Constant as Driven Force for Uncomplexed Cyclodextrin in Equilibrium with Micellar Systems. Chem. Phys. Lett. 2010, 499, 70−74. (20) Cabaleiro-Lago, C.; Nilsson, M.; Söderman, O. Self-Diffusion NMR Studies of the Host-Guest Interaction between Beta-Cyclodextrin and Alkyltrimethylammonium Bromide Surfactants. Langmuir 2005, 21, 11637−11644. (21) Valente, A. J. M.; Nilsson, M.; Söderman, O. Interactions between n-Octyl and n-Nonyl Beta-D-Glucosides and Alpha- and Beta-Cyclodextrins as Seen by Self-Diffusion NMR. J. Colloid Interface Sci. 2005, 281, 218−224. (22) De Lisi, R.; Lazzara, G.; Milioto, S.; Muratore, N.; Terekhova, I. V. Heat Capacity Study to Evidence the Interactions between Cyclodextrin and Surfactant in the Monomeric and Micellized States. Langmuir 2003, 19, 7188−7195. (23) Sehgal, P.; Sharma, M.; Wimmer, R.; Larsen, K. L.; Otzen, D. E. Interactions between Anionic Mixed Micelles and Alpha-Cyclodextrin and Their Inclusion Complexes: Conductivity, NMR and Fluorescence Study. Colloid Polym. Sci. 2006, 284, 916−926. (24) Bendazzoli, C.; Mileo, E.; Lucarini, M.; Olmo, S.; Cavrini, V.; Gotti, R. Capillary Electrophoretic Study on the Interaction between Sodium Dodecyl Sulfate and Neutral Cyclodextrins. Microchim. Acta 2010, 171, 23−31. (25) Bai, Y.; Xu, G. Y.; Xin, X.; Sun, H. Y.; Zhang, H. X.; Hao, A. Y.; Yang, X. D.; Yao, L. Interaction between Cetyltrimethylammonium Bromide and Beta-Cyclodextrin: Surface Tension and Interfacial

DNA−CTAB aggregates essentially results in a reversal of the behavior observed on addition of CTAB to DNA. Furthermore, our findings suggest that the cluster formation observed in CDinduced decompaction of DNA in single-molecule experiments is a result of stripping overcharged DNA−CTAB aggregates from surfactant up to a point where charge-neutral aggregates are obtained, which are prone to agglomeration and macroscopic phase separation. From experiments on DNA−CTAB− 2HPβCD mixtures where the mixing order was altered, we conclude that there is a larger barrier for extraction of CTAB with 2HPβCD from the DNA−CTAB precipitate than there is for the expulsion of CTAB from the 2HPβCD−CTAB inclusion complex to allow formation of DNA−CTAB precipitate. The occurrence of intrachain segregation, rather than coil−globule coexistence, observed in single-molecule investigations of CD-induced DNA−CTAB decompaction is rationalized by a low cooperativity in the formation of 1:1 CD− surfactant inclusion complexes.

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ASSOCIATED CONTENT

* Supporting Information S

Figures S1−S5. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (J.C.); [email protected] (D.L.). Present Addresses §

Biomedical Science, Faculty of Health and Society, Malmö University, 205 06 Malmö, Sweden. ⊥ CR Competence AB, Center for Chemistry and Chemical Engineering, Lund University, POB 124, 221 00 Lund, Sweden. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Karin Schillén and John Janiak for valuable input on interpretation of the dynamic light scattering data. Peter Schurtenberger is acknowledged for input on correction of the absorbance data. Financial support by the Swedish Research Council (VR) through the Linnaeus grant Organizing Molecular Matter (OMM) Center of Excellence (239-20096794) is gratefully acknowledged. D.L. thanks the Portuguese Science Council (Fundacão para a Ciência e a Tecnologia, FCT) grant SFRH/BPD/48522/2008, for funding. R.S.D. is also thankful to FCT for financial support through the program Ciência 2007.

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REFERENCES

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