Cyclodextrin−Surfactant Complex: A New Route in DNA Decompaction

772 Biomacromolecules 2008, 9, 772–775 Cyclodextrin-Surfactant Complex: A New Route in DNA Decompaction Alfredo González-Pérez,* Rita S. Dias, Tommy Nylander, and Björn Lindman Physical Chemistry 1, Centre for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, 22100 Lund, Sweden Received November 22, 2007; Revised Manuscript Received January 21, 2008 In the present work, we show a new approach for decompaction of DNA-cationic surfactant complexes, e.g., lipoplexes, by using β-cyclodextrin (β-CD). The DNA decompaction was achieved by dissolving the surfactant aggregates in the complex by making use of the high affinity between the β-CD and the free surfactant in solution. The results from fluorescence microscopy and adiabatic compressibility measurements indicate that coils and globules do not coexist. The reported procedure using β-CD is an efficient way to decompact DNA surfactant complexes because the association constant of surfactants with β-CD is large. The surfactant’s interaction with β-CD is specific and the nonspecific interaction between β-CD and biological interfaces is small. Introduction concentration at which the surfactants form micelles in the absence of DNA, the so-called critical micelle concentration The possibility to treat diseases, in particular hereditary (cmc). The structure of the surfactant aggregates on the DNA diseases, by the insertion of genes into human cells and tissues, is expected to depend on the type of surfactant. For instance, so-called gene therapy, has encouraged many studies in the last for CTAB, we have shown by SANS that is likely to be few years.1 This promising new technology is still in its infancy, elongated aggregates.16 and therapeutic objective gene delivery requires a precise control Some pioneering work has been done in order to promote of both the uptake into the cell nucleus (transfection) and after the redissolution or decompaction of DNA, but there is still a the replication into RNA (transcription). need for rational approaches to control the decompaction Both DNA compaction and decompaction are needed for process. In the case of multivalent cations, decompaction can successful delivery. First, DNA must be taken up by the natural be achieved by increasing the salt concentration.17 In the case cell membrane barriers. At this level, DNA compaction or of cationic surfactants, anionic surfactants can be used to condensation is essential to protect DNA from the nucleases decompact DNA by forming catanionic aggregates that has and to allow it to reach the nucleus. In the second step when significantly lower affinity to DNA.13,18 Chen et al. reported DNA arrives in the nucleus, it should be decompacted so it is specific formation of bends-on-a-chain on giant DNA by using accessible to the enzymatic machinery responsible for transcrip- polyamide derivatives.19 More recently, Le Ny et al.20 were able tion into RNA. In living cells, transcription is accompanied by to control the condensation by using light-responsive surfactants. the alteration of chromatin structure in transcripted regions of Cyclodextrins have been used in many applications due to their the genome. Highly compacted structures of DNA and histones ability to form inclusion complexes with different molecules.21 are inherently repressive of all DNA dependent processes. In In the present work, we show a new approach for decompaction general, regions of the genome that are actively transcripted of DNA-cationic surfactant complexes, e.g., lipoplexes, by have a more open and accessible chromatin.2,3 Consequently, using β-cyclodextrin (β-CD). The DNA decompaction, studied it was recently suggested that agents that bend DNA can be using fluorescence microscopy, density, and sound velocity used to control gene expression.4 It is clear that precise control measurements, was achieved by dissolving the surfactant of DNA decompaction is needed to efficiently control gene aggregates in the complex by making use of the high affinity expression. between the β-CD and the free surfactant in solution. DNA compaction can be achieved by different complexing Despite all the efforts in the synthesis of novel cationic lipids agents such as multivalent ions, cationic surfactants, lipids, and surfactants, these amphiphilic molecules still pose a problem neutral, and cationic polymers or alcohols.5–12 In general, the of toxicity in vivo and therefore their effective use is still not choice of the complexing agent will determine the compaction possible. However, we believe that the mechanism for decom- mechanism and the structure of the complex. The most studied paction presented in this study using a model system will be complexing agents are polycations, multivalent cations, or the same independently of the chemical structure of the surfactants. Compaction with multivalent ions is achieved by surfactant that is used. ion correlation effects that induce effective attractions between different parts of the DNA molecule.13 The compaction induced by surfactants is a cooperative process associated with the self- Experimental Section assembly of the surfactant.14,15 The self-assembly of the Materials. Coliphage T4DNA 166kbp was supplied by Wako surfactant in the presence of DNA starts at the critical association Nippon Gene. Hexadecyltrimethylammonium bromide (CTAB) was concentration (cac), which is much lower than the critical obtained from Sigma and recrystallized twice in acetone. Fluorescent dyes 4′,6-diamidinio-2-phenyl-indole (DAPI) and GelStar nucleic acid * To whom correspondence should be addressed. E-mail: gel stain were from Sigma and Cambrex, respectively. The latter is [email protected]. supplied as a 10000× concentrated stock solution in DMSO. The 10.1021/bm7012907 CCC: $40.75  2008 American Chemical Society Published on Web 02/08/2008 Communications Biomacromolecules, Vol. 9, No. 3, 2008 773 antioxidant ascorbic acid was purchased from Sigma. β-Cyclodextrin (β-CD) was obtained from Sigma Sample Preparation. All stock solutions were prepared in a 10 mM Tris-Cl buffer (pH 7.6). DNA molecules were diluted in the 10 mM Tris-Cl buffer containing 4% of ascorbic acid and fluorescent dye. The final concentration of DNA was 0.5 µM in nucleotide units. DNA was compacted by using a CTAB concentration of 2.42 × 10-4 M. At this concentration, all the DNA molecules were compacted and no coexist- ence between coils and globules was found.10 The concentration of β-CD was varied in the decompaction studies and all the other parameters were kept constant. The experiments were made with GelStar and DAPI (the former gave a higher contrast). The density and sound velocity experiments have been carried out without fluorescent dye and antioxidant. Fluorescent Microscopy. The fluorescence microscopy study was conducted by placing a drop of the sample on a thoroughly cleaned microscope slide and then placing a coverslip on top of it. The samples were illuminated with a UV-mercury lamp, the fluorescence images of single DNA molecules were observed using a Zeiss Axioplan micro- scope, equipped with a 100U oil-immersed objective lens, and then Figure 1. (a) Fluorescent images and the corresponding fluorescent digitized on a personal computer through a highly sensitive SIT Cvideo intensity profiles of individual T4 DNA molecules in the globular and camera and an image processor, Argus-20 (Hamamatsu Photonics, coil state. (b) Conformation of single DNA molecules compacted with CTAB as a function of log β-CD concentration at constant CTAB Japan). The apparent long-axis length of the DNA molecules, L, was concentration. Filled circles correspond to the globular sate and open deemed as the longest distance in the outline of the fluorescence image to the coil state. The experiments were performed using both 4, of single DNA. Images of the dynamic motion of single DNA-lipid 6-diamidino-2-phenylindole (DAPI), 0.5 µM, and Gelstar (Cambrex), complexes in solution were recorded by using the C-image software 1×, as fluorescent dyes. The results were equivalents using both obtained form Hamamatsu. The observations were carried out at room dyes. temperature. Adiabatic Compressibility Measurements. The adiabatic com- pressibility, defined by, an elongated coil state (L ) 4–6 µm). Unlike decompaction obtained by anionic surfactants,22 these results show no coexist- βad ) (-1 ⁄ V)(∂V ⁄ ∂P)S (1) ence region between coil and compact states. In Figure 2b, we where V is the volume of the sample and P is the pressure and can be evaluated the amount of β-CD-CTAB complexes present in calculated directly from the density and sound velocity using the solution. The values are expressed in fractions of β-CD-CTAB expression, complexes. This quantity was estimated using the association constants for CTAB and β-CD23 and assuming that all the βad ) 1 ⁄ u2F (2) surfactant is accessible to the β-CD and that the CTAB has a much higher affinity for β-CD than for DNA. If we compare where F is the density and u is the sound velocity. The changes in density and sound velocities due to the compaction decompaction parts a and b of Figure 2, the results suggest that the DNA process were followed continuously using an Anton-Paar DSA 5000 decompaction coincides with 98% or more of CTAB bound to densitometer and sound velocity analyzer. Because both speed of sound β-CD (see hatched region). At lower concentration of β-CD, and density are extremely sensitive to temperature, the temperature was DNA remains in the compact state DNA, i.e., the surfactant kept constant to within (10-2 °C using the Peltier method. Furthermore, concentration available to DNA is above the cac. the samples were equilibrated in the instrument for 30 min at 25 °C. To obtain additional information about the conformational The reproducibility of densities and sound measurements was better state of DNA as a function of β-CD concentration, we performed than (5 × 10-6 g cm-3 and (10-2 m s-1, respectively. adiabatic compressibility measurements at 25 °C. Here we note that the samples were prepared by following the same protocol as used in the fluorescence microscopy experiments, however, Results and Discussion these measurements do not require a probe. The adiabatic The conformational state of T4DNA was studied by fluores- compressibility has previously been used to investigate the cence microscopy (FM) following the protocol described in the conformational state of proteins in solution,24 but this is the Experimental Section. By increasing the concentration of β-CD, first time to our knowledge that this approach has been applied the DNA was decompacted. Typical fluorescence images and to study the compaction/decompaction of DNA in solution. the corresponding intensity profiles are shown in Figure 1a, Some of the obtained data are presented in Figure 3, where we and the compact and coil states regions as a function of β-CD show the adiabatic compressibility as a function of the logarithm concentration are indicated in Figure 1b. These images indicate of the β-CD concentration. The curve shows two well-defined a very sharp transition between the globules and coils with linear regions, and the dramatic change in the slope of the curve increasing of β-CD concentration. suggests changes in the DNA conformation. We also note that To quantify the compaction/decompaction DNA process, the break point coincides with the changes in size reported in further FM studies were performed and the results in terms of Figure 2a. In the globular conformation region, the compress- the average length of T4DNA as a function of β-CD concentra- ibility is high and almost constant with a low decrease when tion are shown in Figure 2a. Every data point is the result of the β-CD concentration is rising. This is in agreement with the the average of at least 100 images of DNA molecules. At low compact state conformation in which we expect to have a higher- β-CD concentration, T4DNA exhibits a narrow unimodal size order structure formed by T4DNA decorated with some sur- distribution characteristic of the compact state (L < 1 µm). At factant aggregates. This cluster is likely to be quite hydrated concentrations higher than 0.01 M, all the molecules exist in and therefore compressible as well as the surfactant aggregates 774 Biomacromolecules, Vol. 9, No. 3, 2008 Communications Figure 3. Adiabatic compressibility, obtained from density and sound velocity measurements at 25 °C for T4DNA compacted using 2.42 × 10-4 M of CTAB at pH ) 7.6 as a function of β-CD concentration. DNA complex is in thermodynamic equilibrium with monomers in solution.10,27 We have shown that, by using an appropriate cyclodextrin concentration, free surfactant in the bulk will form an inclusion complex. The decrease in the free surfactant concentration will affect the thermodynamic equilibrium in such a way that micelles associated with DNA will be disrupted. This process, which can be regarded as opposite to micelle formation, will eventually lead to a critical concentration where no surface micelles are present and DNA is decompacted. This process can be regarded as a non-first-order transition from globule to coil conformation. The reported procedure using β-CD is a convenient way to decompact DNA when this has been previously compacted using surfactants because the association constant of surfactants with β-CD is large. The surfactant is also removed from the solution by complexation without introducing new ionic species in the bulk that can potentially affect the transcription process in a hypothetical transcription test. The observed decompaction phenomenon was consistently studied by using fluorescence microscopy and adiabatic com- pressibility measurements. The latter were found to be a Figure 2. (a) Long axis length L of T4DNA molecules vs β-CD concentration. Error bars indicate the statistical error in size distribu- promising tool to determine changes of the conformational state tion. (b) Fraction of β-CD-CTAB complex as a function of β-CD of DNA because they do not require probes and can be used concentration estimated from the binding constant given in ref 23. for DNA that is too small to be visualized. Furthermore, the adiabatic compressibility can tell us something about the density formed within the complex. It is known that the adiabatic of the aggregate, i.e., it is a rather convenient way to screen compressibility of CTAB micelles is higher than that of the how different compacting agents affect the structure of the monomers in solution.25 The slightly decrease in compressibility DNA-compacting agent complex. can be directly related to the increase on β-CD concentration, Additionally, other types of cyclodextrins can be used to which has been reported previously when increasing the β-CD decompact DNA. Preliminary results in our laboratory show concentration at low CTAB concentration.26 that R-cyclodextrin is also efficient in DNA decompaction. We After decompaction, DNA adopts a coil conformation that is expect that this simple approach to DNA decompaction will expected to be much less compressible and hence a dramatic encourage many studies of ways to decompact DNA by drop in compressibility is observed. The reason why compress- unbinding cationic surfactant compaction agents. ibility levels out in this region is probably due to the fact that decompaction of DNA occurs before all of the surfactant is Acknowledgment. A.G.-P. is thankful to the EU Research removed from the DNA. As discussed above, decompaction Training Network, CIPSNAC (contract no. MRTN-CT-2003- occurs when 98% or more of CTAB is bound to β-CD. 504932) and to Dr. Cabaleiro-Lago for invaluable discussion on β-CD-CTAB binding. R.S.D. acknowledges the Fundação para a Ciência e Tecnologia (FCT), Portugal (SFRH/BPD/ Conclusion 24203/2005). Financial support was also obtained from the It is well-known that DNA can be compacted with cationic Swedish Research Council and its Linnaeus grant “Organised surfactant and that the surfactant aggregates in the surfactant- Molecular Matter”, OMM. Communications Biomacromolecules, Vol. 9, No. 3, 2008 775 Note Added after ASAP Publication. There was a typo- (13) Khan, M. O.; Jönsson, B. 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