Interaction Btw Dna and Surf Act Ant

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Introduction

Mixtures of water-soluble polymers and surfactants arepresent in a large number of systems in nature andindustrial applications, such as in foods, pharmaceuticalformulations, cosmetics, detergents, paints, etc., andthese systems have been the subject of a large number of studies [1, 2]. The interactions between amphiphilicmolecules and biologically active polyelectrolytes havereceived particular attention not only from the physicalchemistry viewpoint, but also were specially investigatedin biomedical studies. Within this group we considerDNA±cationic surfactants systems. These systems have anumber of applications, such as the puri®cation of DNA

by condensation and precipitation [3], DNA renatur-ation and ligation [4], and selective separation of DNA inthe presence of RNA [5]. Surfactants can also be used forpositive charging of neutral liposomes for gene deliverypurposes [6]. Owing to the growing interest in this ®eldand numerous applications of these systems severalstudies have been presented in the literature concerningDNA interaction with cationic amphiphiles [7±13]. Inspite of this, a number of facts are still not quite clear inthese systems. The mechanism of surfactant binding and

the structure of the complex are some examples. In our

work an attempt is made to understand these and otherpoints. The surfactants used are cetyltrimethyl-ammonium bromide (CTAB), tetradecyltrimethylammo-nium bromide (TTAB), and dodecyltrimethylammoniumbromide (DTAB).

DNA association with cationic surfactants

Some applications of the DNA±cationic surfactantsystems are based in the fact that DNA phase-separatesassociatively with the amphiphiles. The formation of aprecipitate, or phase concentrated in both components,

has been reported for several systems of polyelectrolytesand oppositely charged surfactants [14±16]. The electro-static interactions between them are obviously strongand lead to strong association. Surfactant aggregatesinduced by the polymer will act as counterions, therebyreducing the charge of the complex and the entropicdriving force for mixing and the interpolymer repulsions[2]. However, in contrast to other polyelectrolyte± surfactant systems reported [15, 16] the precipitate doesnot redissolve with an excess of surfactant. Other

Progr Colloid Polym Sci (2001) 118: 163±167Ó Springer-Verlag 2001 COLLOIDS IN PHARMACEUTICAL AND BIOLOGICAL APPLICATIONS

R. S. DiasB. LindmanM. G. Miguel

Interactions between DNA and surfactants

R. S. Dias á B. LindmanM. G. Miguel (&)Chemistry Department, CoimbraUniversity, 3004-535 Coimbra, Portugale-mail: [email protected]

R. S. Dias á B. LindmanPhysical Chemistry 1, Center for Chemistryand Chemical EngineeringP.O. Box 124, 221 00 Lund, Sweden

Invited lecture, 14th Conference of theEuropean Colloid and Interface SciencePatras, Greece, 2000

Abstract The interaction betweenDNA and alkyltrimethylammoniumbromides of dierent chain lengths is

studied, both on a macroscopic andon a single-molecule level. The phasemaps for aqueous DNA±surfactantsystems as well as some interestingsalt eects are presented. Somepreliminary results on the structureof DNA±surfactant complexes arealso given. Studies involving DNAand surfactant mixtures are alsoconducted.

Key words DNA±surfactantsystems á Catanionic mixtures á

DNA±amphiphiles complexe á

Phase behavior



information obtained is that the precipitate is formed atvery low amounts of DNA and minor surfactantconcentrations, far below the surfactant critical micelleconcentration (cmc). Polyelectrolyte±oppositely chargedsurfactant systems are known to have a critical aggrega-tion concentration (cac) lower than the cmc of freesurfactants, often by orders of magnitude. The fact that

the cationic surfactant binding occurs preferentially toanionic polyelectrolytes of high charge density furtherenhances this behavior. These results are presentedschematically in Fig. 1. These phase maps, studied byturbidimetry, are presented in a simpli®ed two-dimen-sional representation. Since the amount of water in thesesystems is extremely high, this type of representationprovides a better visualization.

The salt effect

The same systems were studied with the addition of salt[17]. The results are presented in Fig. 2. It is a commonlyaccepted viewpoint that the cac of the polyelectrolyte± oppositely charged surfactant systems increases onaddition of salt [18]. This is due to a weakened

interaction between the polymer and the surfactantinduced by the stabilization of free micelles and ascreening of the electrostatic interactions. With this wewould expect a decrease in the two-phase region and notthe observed broadening of it. By looking at the DNA± TTAB phase map, for example, we observe that the linesof precipitation of the system with and without salt crossinstead of being dislocated one in relation to the other(Fig. 3); this is unexpected behavior. We believe that theknowledge of the DNA±surfactant complex structurecan help us in understanding this trend.

Fig. 1 a Schematic representation of the isothermal pseudoternaryphase diagram for the DNA±dodecyltrimethylammonium bromide(DTAB)±water system. There is a phase separation into two phases inalmost the entire region considered. b Expanded view of the water-richcorner of the system (diamonds), including the DNA±tetradecyltrim-ethylammonium bromide (TTAB)±water (triangles), and DNA± cetyltrimethylammonium bromide (CTAB)±water (circles) systemsfor comparison. The open symbols refer to the clear one-phasesolutions and the ®lled symbols to two-phase samples. The dashed lineindicates the charge neutralization. T 25 °C. From Ref. [17]

Fig. 2 a Same as Fig. 1b, but the phase map is represented on alogarithmic scale for better visualization of the eect of surfactantchain length on the phase transition. b Phase map for the threesystems in the presence of 0.1 M NaBr. The circles represent sampleswithout salt and the triangles samples in the presence of salt. The opensymbols refer to the clear one-phase solution and the ®lled symbols totwo-phase samples. T 25 °C. From Ref. [17]

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DNA±surfactant complexes

In spite of all studies made with these systems [8±13], thestructure of the complex is not fully known. It is agreed[9, 19] that the complexes formed have a loosely packedhexagonal structure in which the DNA induces andstabilizes the formation of rodlike micelles. In fact, ourpreliminary results point in that way (Fig. 4a). We canalso see that the structure is more ordered for the longer-

chain surfactant. The same experiments were performedwith the addition of salt and the results are very similar(Fig. 4b). The only dierence appears to be that with saltthe hexagonal lattice becomes less loose. This could bedue to the salt osmotic pressure.

Studies were also conducted within the two-phaseregion to investigate the dependence of the amount of theprecipitate and water in the supernatant on the variationof the DTAB/DNA mixing concentration ratio [17]. Weobtained some interesting results. The amount of pre-cipitate reaches the maximum amount for a mixing ratioof approximately 1.0 and remains constant with furtheraddition of surfactant, at least for the region studied.

This indicates that the excess of surfactant added to thesamples remains in the supernatant, probably as freemicelles, and can give an explanation for the nonredis-solution of the DNA±surfactant complexes. Since there isno binding of the surfactant to the complex after itsneutralization, an inversion of the complex charge is notobserved, as happens with many similar systems [15, 16].The same studies were made with the addition of salt, butagain there were no major dierences between the results.The precipitation starts for higher amounts of surfactant,

as expected, and there is a slight decrease in the amountof water in the samples, which corresponds to the saltcontribution to the weight of the sample and means,probably, that the major fraction of salt is dissolved inthe supernatant. Further studies are being conducted onthis.

DNA compaction in the presence of cationic surfactants

Fluorescence microscopy is a technique that has recentlybeen used in the study of DNA conformational behaviorin the presence of several cosolutes, having as its mainadvantage the visualization of single molecules insolution. DNA molecules in aqueous solution presentan extended conformation, ¯owing in the solutionand exhibiting a relatively slow wormlike motion,the designated unfolded coil conformation. With the

Fig. 3 Eect of the addition of NaBr on the phase behavior of theDNA±TTAB aqueous system. The circles represent samples withoutsalt and the triangles samples in the presence of salt. As before, theopen symbols refer to the clear one-phase solution and the ®lled symbols to two-phase samples. The dashed line represents the expected

direction of change for the system with salt. T

25 °C. Redrawnfrom Ref. [17]

Fig. 4 Small-angle X-ray scattering diractograms of DNA±surfac-tant complexes a in the absence and b in the presence of NaBr. Thesepreliminary results show a loosely packed hexagonal structure. The

®lled diamonds correspond to the DNA±CTAB complexes, the opencircles to DNA±TTAB, and the ®lled triangles to the DNA±DTABsystem. T 25 °C

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addition of cationic surfactants, for example, the DNAmolecules undergo compaction into a globular state. Theinteresting point in these systems is that the DNAmolecules undergo a discrete, or a ®rst-order, phasetransition [20, 21]. Associative phase behavior is welldocumented for mixed aqueous systems of a polyelec-trolyte and oppositely charged surfactants [14, 15, 18]

and so is the eect of surfactant on the polyionconformation [15, 16]. However the ®ndings for ¯exiblepolyions of low charge density dier qualitatively fromwhat we ®nd for DNA. In these cases it can be inferredthat while surfactant binding is cooperative, in that itresults in surfactant self-assembly aggregates and ischaracterized by a well-de®ned cac, there is an essentiallyuniform distribution of surfactant aggregates among thedierent polyions. Phase separation takes place when thenet charge of the polyion±surfactant aggregates attains alow value and the surfactant distribution explains whyrather high amounts of surfactant are needed for phaseseparation in these cases. For DNA, the very low values

of surfactant concentration at which phase separationstarts demonstrate a dierent binding situation andeither complete or no binding: as binding to DNA starts,further binding is facilitated and one DNA double helixmolecule is saturated before binding starts at another, i.e.there is double cooperativity. This is directly con®rmedby the ¯uorescence microscopy results, demonstratingthe coexistence of original extended coils and globulescompacted to the ®nal state. Our results for the DNA± CTAB, DNA±TTAB, and DNA±DTAB systems showthat this coexistence interval is narrower for DNA-CTAB aqueous mixtures and becomes wider for theshorter-chain surfactant. It is shown in Fig. 5 that a

larger amount of DTAB is needed to induce thecompaction of the DNA molecules, which is in agree-ment with our previous results.

Forthcoming work

Since many details in these systems are not wellunderstood more experiments are being conducted inour groups. One of them is the study of the DNAinteractions with alkyltrimethylammonium surfactantsof even shorter chain length, such as decyltrimethylam-

monium bromide and octyltrimethylammonium bro-mide. Some preliminary phase diagrams are presentedin Fig. 6. Hexyltrimethylammonium bromide was foundnot to precipitate DNA. As expected the area of the two-phase region decreases with the decrease in the surfactanthydrophobicity. For the short-chain surfactant theassociation is no longer observed.

One problem with these cationic systems is the toxicityof the surfactants, which reduces substantially theirapplications. An eort is being made to use morebiocompatible amphiphiles. The use of surfactant mix-tures is another way of dealing with this problem. Byreducing the amount of cationic amphiphile we decrease

the toxicity of the systems. Cationic liposomes, forexample, made of neutral and cationic lipids, are wellknown as gene delivery vehicles. Owing to the undeniableimportance of these systems, the interaction betweenDNA and surfactant mixtures is being carefully studied.

Interactions between DNA and surfactant mixtures

We studied the DNA conformational behavior in thepresence of nonstoichiometric mixtures of two oppositelycharged surfactants, CTAB and sodium octyl sulfate [22].The aqueous mixtures of these surfactants exhibit a rich

Fig. 5 DNA conformational behavior in the presence of cationicsurfactants CTAB, TTAB, and DTAB. The DNA charge concentra-tion was maintained at 0.5 lM. The open circles correspond to the coilconformational state of DNA and the ®lled circles to the presence of globular molecules. The shaded circles represent the coexistencebetween elongated coils and compacted globules. T 25 °C. FromRef. [17]

Fig. 6 Same as Fig. 2a with some preliminary results on the DNA± decyltrimethylammonium bromide±water (squares) and DNA±octyl-trimethylammonium bromide±water (crosses) systems. As before, theopen symbols correspond to one-phase solution and the ®lled symbolsto two-phase samples. T 25 °C

166



phase behavior, with the formation of surfactant aggre-gates with dierent charges and geometries [23, 24]. Wewere mainly interested in the phases of the vesicles. Weobserved that the presence of anionic-rich vesicles inaqueous DNA solutions does not lead to any associativeinteraction and does not aect the DNA conformationalbehavior; however, in the cationic-rich phase, DNA is inthe globular state and it adsorbs onto the surface of the

vesicles [22]. Moreover, we observed that the change inthe molar ratio between cationic and anionic surfactantsin solution leads to DNA unfolding. Owing to theinteresting results and promising use in several applica-tions other studies are being conducted on this system,such as phase diagrams and the complex structuredeterminations.

Another interesting part in the study of the DNA± cationic surfactant interactions is the addition of anonionic surfactant. We observed that the addition of octaethyleneglycol mono-n-dodecyl ether induces aweakening in the interaction between the polyion andthe oppositely charged surfactant and is capable of preventing the precipitation of the systems at highenough concentrations. Some preliminary results arepresented in Fig. 7. During this project other amphi-philes will be used.

Acknowledgements This work was supported by grants fromJNICT and Praxis XXI (PRAXIS/BD/21227/99), the CaloustGulbenkian Foundation, the Swedish Research Council forEngineering Sciences, and the Center for Amphiphilic Polymersin Lund. Sergey Mel'nikov is thanked for valuable participation inthe initiation of this project and for useful discussions. We aregrateful to Filipe Antunes and Stina Lindman for technicalassistance.

Fig. 7 The addition of a nonionic surfactant prevents the precipita-tion of DNA±cationic surfactant. A simpli®ed two-dimensionalrepresentation of the preliminary results is given for the aqueoussystem of DNA±octaethyleneglycol mono-n-dodecyl ether±CTAB.The ®lled symbols refer to two-phase samples and the open symbols toclear one-phase solution. T 25 °C

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