DNA conformational changes induced by cationic gemini surfactants: the key to switching DNA compact structures into elongated forms

DNA conformational changes induced by cationic gemini surfactants: the key to switching DNA compact structures into elongated forms† Elia Grueso,* a Edyta Kuliszewska, b Emilio Roldan, a Pilar Perez-Tejeda, a Rafael Prado- Gotor a and Lothar Brecker c The DNA conformational changes induced by different members of the N,N0-bis(dimethyldodecyl)-a-u- alkanediammonium dibromide series (m-s-m,m¼12, s¼3 and 6) and the analogous series of hexadecyl gemini surfactants (m¼16, s¼3 and 6) were investigated in aqueous media by means of circular dichroism (CD), zeta potential, dynamic light scattering (DLS), viscometry, and atomic force microscopy (AFM) methods. The measurements were carried out by varying the gemini surfactant–DNA molar ratio, R¼C m-s-m /C DNA . For the conditions investigated two significantly different conformational changes were observed, the second of them being worth noting. At low molar ratios, all methods concurred by showing that gemini surfactants were able to form ordered aggregates which precedes DNA compaction. The second effect observed, at high molar ratios, corresponds to the transition from the compact state to a new more extended conformation. The degree of decompaction and the morphologies of the visualized structures are different not only depending on the surfactant tail's length, but also on the spacer's length. The results obtained for the 16-3-16/DNA and 16-6-16/DNA systems point out that the compaction/decompaction processes are somewhat different to those previously visualized for the analogous monoquaternary chain surfactant CTAB. 1. Introduction The understanding of the interaction between double stranded (ds) DNA and cationic gemini surfactants will have a broad bearing on many important applications from drug delivery 1 to the translocation of DNA across the cell for the purposeofgenetherapy. 2 These applications require that genetic material is placed inside the cell in order to produce an effective medical treatment. However, the entry of naked DNA into the cell nucleus would need to resolve two fundamental problems: (1) the polyelectrolyte character of DNA polynucleotide gives rise to electrostatic repulsion when the DNA is approximated to the negatively charged cell membrane, 3 (2) the steric restrictions inside the cell which are able to hinder the DNA transportation to the nucleous. 4 To achieve these requirements, DNA rst has to form a positively charged complex and then, needs to modify its conformation from an extended coil state to a compact globular state in order to adapt its structure to tine specic sites in the cell. The collapse process by which extended polynucleotide chains modify their structures into compact particles is called condensation, which is a common process for native genomes. 5 The controlled DNA condensation has been demonstrated to be possible with a variety of reagents such as enzymes, 6 multivalent ions, 7,8 alcohols, 9,10 polyamines, 11 metal complexes, 12 nanoparticles, 13 and cationic surfactants. 14–18 In this regard, the gemini surfactants have received a lot of attention recently due to its unique physicochemical properties, low cytotoxicity and potential bactericidal properties. 19–21 Due to their positive charge, low toxicity and the strong capacity as DNA complexing agents, the cationic gemini surfactant–DNA systems are suitable to be properly transferred to living cells. Accordingly, motivated by the promising application of these cationic surfactants in gene delivery, several groups have explored the efficiency of these vectors on DNA compaction. 16,17,20,21 Among a variety of gemini surfactants, those that possess a small spacer (s#3) present better DNA compaction efficiency. 22–24 In spite of some progress having been made in understanding the DNA compaction processes induced by gemini surfactants, these are not yet well understood. In particular, the interactions of DNA with the 16-s-16 series have been less explored. Hence, a a Department of Physical Chemistry, Faculty of Chemistry, University of Seville, C/ Profesor Garc´ ıa Gonz´ alez, s/n, 41012, Sevilla, Spain. E-mail: [email protected]; Fax: +34- 954557174; Tel: +34-954557175 ext. 215 b Institute of Heavy Organic Synthesis-Ul, Energetykow 9, Kedzierzyn-Kozle 47-225, Poland c Institute of Organic Chemistry, University of Vienna, W¨ ahringerstrasse 38, A-1090 Wien, Austria †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra03944d Cite this: RSC Adv.,2015,5, 29433 Received 5th March 2015 Accepted 20th March 2015 DOI: 10.1039/c5ra03944d www.rsc.org/advances This journal is © The Royal Society of Chemistry 2015 RSC Adv.,2015,5, 29433–29446 | 29433 RSC Advances PAPER Published on 20 March 2015. Downloaded by Gral Universidad Sevilla on 6/18/2019 9:09:02 AM. View Article Online View Journal | View Issue thorough study of the effect of the tail and spacer length on DNA compaction seems to be pertinent. Even though the compaction of DNA is relevant in drug delivery and gene therapy, the study of the dissociation of DNA/gemini surfactants complexes in the appropriate place is also crucial to ensure the nal transfection. 25 Accordingly, once the compact DNA molecule arrives in the nucleus, it needs to be accessible to the cell enzymatic machinery. However, this nalstepisonlypossibleifdecompaction occurs or if the degree of compaction of the DNA is not too high. 26,27 Therefore, from an application viewpoint, controlling the balance between compacted/decompacted DNA structures with different gemini surfactant concentrations could be a matter of critical interest. In this regard, recent investigations have demonstrated that reversible compaction is possible in DNA–CTAB systems, without using additional chemical agents, by using a high surfactant content. 15 The mechanism of DNA decompaction induced by CTAB was explained by postulating the existence of sphere-rod transitions in CTAB micelles, the latter being favored by an increase of bromide anions in solution. 28 However, the factors that control the DNA–CTAB decompaction remain unclear. Different from conventional monomeric surfactants, the aggregation behavior of gemini surfactants in solution is controlled by both inter-molecular and intra-molecular interactions of these surfactants, along with their interactions with solvents. 29 In comparison with their conventional counterparts, gemini surfactants, composed of two hydrophobic chains and two hydrophilic head groups covalently attached through a spacer, present strong self-aggregation ability. 29 In addition, variety in spacerlengthgeneratesagreattendencytoformavarietyof aggregate structures, such as tubule aggregates, 29,30 elongated vesicles 31 and rod-like or worm-like micelles 32 in aqueous solutions. Atkin et al. postulated that in the gemini surfactants 12-s-12 with a short spacer (s#3) the head groups are close in proximity and they can assemble, forming attened ellipsoidal aggregates structures in aqueous solution, when the surfactant concentration is increased. 33 These aggregate structures, however, are strikingly different from those observed in gemini surfactants with longer spacers (12-s-12 with s$6). Due to the increase in the head group area, the surfactant chains interact with each other promoting the formation of more attened aggregates of higher curvature. We believe that these aggregation properties can be applied to effectively inuence the conformational behavior of m-s-m/DNA systems. Therefore, the purpose of this study was to investigate whether gemini surfactants are able to induce reversible DNA compaction/decompaction phenomena in aqueous solutions at different molar ratios. The choice of 12-3-12, 12-6-12, 16-3- 16 and 16-6-16 gemini surfactants (see Fig. S1†)wasmadeto explore the effectofvaryingthesizeofthetailtogetherwith the spacer's length on the conformational changes of the polynucleotide. The results obtained here show that switching DNA conformation is possible using the appropriate gemini structure (as a complexing agent), as well as a suitable surfactant concentration to achieve the critical C m-s-m /C DNA molar ratio. 2. Experimental section 2.1. Materials Calf thymus DNA was purchased from Pharmacia and used without further purication since preliminary experiments showed that purication did not make a difference in the experimental results. An agarose gel electrophoresis test using ethidium bromide indicated that the average number of base pairs per DNA molecule was greater than 10 000 bp. 34 Polynucleotide concentrations, given in phosphate groups, were determined spectrophotometrically from the molar absorptivity (6600 M 1 cm 1 at 260 nm). 35 Sodium cacodylate and 3-ami- nopropyltriethoxilane (APTES) were purchased from Sigma- Aldrich. All reagents were used as purchased. The water used in the preparation of solutions had a conductivity of less than 10 6 Sm 1 . All measurements were performed at pH 7.0 (0.01 M NaCac buffer where NaCac ¼sodium cacodylate ¼(CH 3 ) 2 - AsO 2 Na) and 298.2 0.1 K. The total concentrations of the polynucleotide and the gemini surfactants in a working solution will now be referred to as C DNA and C m-s-m , respectively; the molar ratio C m-s-m /C DNA will be designated as R. 2.2. Synthesis and characterization of gemini surfactants For the synthesis of the gemini surfactant compounds a mixture of a,u-dibromoalkane (0.05 mol) and N,N-dimethylalkylamine (0.12 mol) in acetonitrile (100 mL) was stirred under reux for 20 h. Upon cooling a white solid was recovered by ltration. All products were recrystallized from ethyl acetate up to ve times and dried under vacuum. 36 Characterization of the gemini surfactants was based on mass spectrometry results with detection of fragments in the third quadrupole, 37 nuclear magnetic resonance spectroscopy experiments, elemental analysis and surface tension experiments. The critical micelle concentrations (CMC) of the gemini surfactants were measured by the surface tension technique. The measured CMC values were collected in Table S1,†which are in good agreement with the previous values registered by Zana et al. 38 The characterization techniques used for gemini surfactants are described in the ESI†section of the paper. 2.3. Methods 2.3.1. Viscosity measurements. Viscosity measurements were carried out employing an Ostwald viscosimeter, immersed in a thermostatic water bath. Each value of the viscosity was the average of 10 measurements. The ow time of the samples was measured aer a thermal equilibrium time of 15 min. The viscosimetric measurements were done at C DNA ¼110 4 M and varying the C m-s-m concentration. For the 12-s-12 (s¼3, 6) series a Rratio was explored in the range of 0.0025–5.0. Owing to the low CMC values which the 16-s-16 series of gemini surfactants present (see Table S1†), it was determined that it was not feasible to explore the viscosity changes in the high Rrange (only the Rranges were explored from 0.0025 to 0.25). Data have been presented as (h/h 0 ) 1/3 versus the Rratio, 29434 |RSC Adv.,2015,5, 29433–29446 This journal is © The Royal Society of Chemistry 2015 RSC Advances Paper Published on 20 March 2015. Downloaded by Gral Universidad Sevilla on 6/18/2019 9:09:02 AM. View Article Online h h0 ¼ttsolv tDNA tsolv (1) where t,t solv and t DNA denote respectively the ow time of the sample (m-s-m–DNA mixture), the ow time of the solvent and the ow time of 1 10 4 M DNA. his the viscosity of DNA in the presence of the surfactants, while h 0 is the viscosity of DNA in the absence of gemini surfactants. 2.3.2. CD measurements. CD spectra were recorded in a Biologic Mos-450 spectropolarimeter. The measurements were performed in a standard quartz cell of 1 cm path length in the 220-320 nm range. For each spectrum 5-10 runs were averaged with a 5 min equilibration interval before each scan. All the spectra were recorded using a xed concentration of DNA in the absence and in the presence of varying amounts of gemini surfactants in order to obtain the appropriate molar ratio R. Concentration of DNA used for the 12-s-12 (s¼3, 6) series of gemini surfactants was 5 10 5 M, while for the 16-s-16 (s¼3, 6) series a DNA concentration of 1 10 5 M was employed. Different polymer concentrations were chosen to ensure, on the basis of the data of Table S1,†the work below CMC of the surfactants. The spectra were expressed in terms of molar ellipticity, [q]. 2.3.3. Dynamic light scattering (DLS) measurements. The size and distribution of DNA and gemini surfactants–DNA complexes were characterized by means of the DLS technique using a Zetasizer Model ZS-90 (Malvern Instrument, Ltd., UK). The sample was illuminated with a laser with a xed detection arrangement of 90to the center of the cell area to analyze the intensity uctuation in the scattered light. At least 5 size measurements were taken for each sample and the relative error for hydrodynamic diameter was calculated to be <5%. A DNA concentration of 5 10 6 M was used and the gemini surfactant concentration was varied so as to keep the Rratio in the range of 0.03–5.0. All the samples were analyzed before 60 min of equilibration. The results were obtained in terms of average hydrodynamic diameters, giving the percentage of different complexes obtained in solution. 2.3.4. Zeta potential measurements. Zeta potential experiments were carried out with a Zetasizer Nano ZS Malvern Instrument Ltd. (UK), which measured the electrophoretic mobility of the sample from the velocity of the particles using a laser doppler velocimeter (LDV). A DTS1060 polycarbonate capillary cell was used. All the measurements were done at a xed C DNA ¼510 6 M and the gemini surfactant concentration was varied so as to keep the Rratio in the range of 0.03– 5.0. 2.3.5. Atomic force microscopy measurements (AFM). The AFM images were obtained with a Molecular Imaging Picoscan 2500 (Agilent technologies). Silicon cantilevers (Model Pointprobe, Nanoworld) with a resonance frequency of around 240 kHz and a nominal force constant of 42 N m 1 were used. All AFM imaging was recording in air and in tapping mode, with scan speeds of about 0.5 Hz and data collection at 256 256 pixels. Due to the large size of DNA molecules, in order to obtain images of isolated DNA molecules, it was necessary to use dilute solutions (3 10 7 M). For solution modication of the mica surface, 0.1% (v/v) APTES solution was dropped onto a freshly cleaved mica surface, incubated for 20 min, the surface washed with ultrapure water, and then air-dried. Subsequently, a total of 30 mL of DNA (or DNA/gemini surfactant) solution was dropped onto this modied surface, adsorbed for 60 min, washed with doubly distilled water, and air dried for AFM imaging. For some of the samples in which DNA–surfactant complexes had a positive charge (see below), the mica surface did not have to be modied for the species present in the solution to be adsorbed. To do this, 30 mL of DNA–surfactant solution was directly dropped onto a freshly cleaved mica surface and incubated for 60 min. Then, the same procedure described above was followed. 3. Results 3.1. Viscosity measurements In Fig. 1A, the viscosity ratio, (h/h 0 ) 1/3 , was plotted as a function of the Rmolar ratio for the 12-3-12/DNA and 12-6-12/DNA systems. The complexity of the analysed systems is conrmed by the viscosity trend that changes its features depending on the reagents ratio. In such a way that the viscosity trend at rst is ascending (C m-s-m /C DNA < 0.025), subsequently it descends until reaching a minimum (C m-s-m /C DNA ¼0.35–0.50), and nally increases again for the higher Rratios. Importantly, as displayed in Fig. 1B, the position of the (h/h 0 ) 1/3 maxima for the 16- 3-16/DNA and the 16-6-16/DNA systems correlate well with those obtained from the dodecyl series of surfactants. 3.2. CD measurements The CD technique was used to investigate the inuence of gemini surfactants on the DNA conformation. As is known, the backbone conformation of DNA shows a characteristic CD spectrum of the right-handed B form in the far UV region (220– 320 nm). Structural alterations of the DNA caused by its interaction with ligands are reected in changes in this intrinsic CD spectrum. 39,40 Fig. 2 shows that the secondary structure of DNA is perturbed by the four surfactants studied here, this perturbation being different for the 12-s-12 series (Fig. 2A) and the 16- s-16 series (Fig. 2B). Besides, the addition of different surfactant concentrations to the DNA solution registers complex behavior according to the value of the molar ratio R. For the 12-s-12 series, the measured dichroic bands are found to be dramatically different (with respect to sign, position, shape and intensity) exhibiting two inection points: one of them at C 12-s-12 / C DNA 0.03 (red curves of Fig. 3B and S2B†) and the other at C 12-s-12 /C DNA z0.5–0.6 (red curves of Fig. 3C and S2C†). On the other hand, the 16-s-16 series also display characteristic changes in CD bands upon DNA binding, showing two inection points. The rst maximum is coincident in position with the one obtained for the 12-s-12 series, C 16-s-16 /C DNA 0.03 (red curves of Fig. 3E and S2E†); while, the position of the minimum is slightly displaced to low Rratios, C 16-s-16 /C DNA 0.4 (red curves of Fig. 3F and S2F†). This journal is © The Royal Society of Chemistry 2015 RSC Adv.,2015,5, 29433–29446 | 29435 Paper RSC Advances Published on 20 March 2015. Downloaded by Gral Universidad Sevilla on 6/18/2019 9:09:02 AM. View Article Online 3.3. Dynamic light scattering (DLS) measurements Fig. S3 and S4†show the intensity-weighted size distributions of a DNA solution in the presence and absence of the 12-s-12 and 16-s-16 series of gemini surfactants, working at different molar ratios. Besides, the hydrodynamic diameters of the DNA molecules, taken from the position of the peaks of the intensity distribution function, are collected in Table 1. Results reect two different trends in the systems studied varying the Rratios. At the low Rratios, in all the four surfactants systems, the hydrodynamic diameter decreases reaching a minimum, between a Rratio of 0.4 and 0.6, depending on the surfactant studied. The positions of the minimum correlate well with the minimum observed by viscosity and CD measurements (see Fig. 1A and 2). Then, as the Rratio increases aer passing the minimum, a drastic change in the hydrodynamic diameter occurs. However, a closer examination of Fig. S3 and S4† highlighted that this behavior is different for the 12-s-12 and the 16-s-16 series of gemini surfactants. 3.4. Zeta potential measurements In an applied electric eld, charged species are attracted to the electrode of the opposite polarity resulting in an electrostatic Fig. 1 (A) Viscosity (h/h 0 ) 1/3 dependence on C m-s-m /C DNA molar ratios for the 12-s-12/DNA and 16-s-16/DNA systems. (A) ( ) 12-3-12/DNA system; ( ) 12-6-12/DNA system. The inset shows an enlarged view of the maximum position at low molar ratios. (B) ( ) 16-3-16/DNA system; ( ) 16-6-16/DNA system. Fig. 2 (A) CD dependence of 12-s-12/DNA systems on the C 12-s-12 /C DNA molar ratios. ( ) 12-3-12/DNA system; ( ) 12-6-12/DNA system. The inset shows an enlarged view of the maximum position at low Rratios. (B) CD dependence of 16-s-16/DNA systems on the C 16-s-16 /C DNA molar ratios. ( ) 16-3-16/DNA system; ( ) 16-6-16/DNA system. The inset shows an enlarged view of the maximum position at low Rratios. 29436 |RSC Adv.,2015,5, 29433–29446 This journal is © The Royal Society of Chemistry 2015 RSC Advances Paper Published on 20 March 2015. Downloaded by Gral Universidad Sevilla on 6/18/2019 9:09:02 AM. View Article Online Fig. 3 Intrinsic CD spectra of 12-6-12/DNA and 16-6-16/DNA systems. Arrows indicate direction of change during surfactant titration. (A) Curves correspond to 0, 0.5, 1.0 and 1.5 mM of 12-6-12, C DNA ¼50 mM; (B) curves correspond to 1.5, 5.0, 12.5, 15.0, 20.0, 25.0 and 30.0 mM of 12-6-12, C DNA ¼50 mM; (C) 30, 50, 100, 150, 200 and 250 mM of 12-6-12, C DNA ¼50 mM; (D) curves correspond to 0, 0.05, 0.1, 0.2 and 0.35 mM of 16-6-16, C DNA ¼10 mM; (E) curves correspond to 0.35, 0.50, 1.0, 2.5, 2.8, 3.0 and 4.0 mM of 16-6-16, C DNA ¼10 mM; (F) 4.0, 5.0, 7.5, 10.0, 15.0, 22.5, 25.0, 30.0 and 40.0 mM of 16-6-16; C DNA ¼10 mM. This journal is © The Royal Society of Chemistry 2015 RSC Adv.,2015,5, 29433–29446 | 29437 Paper RSC Advances Published on 20 March 2015. Downloaded by Gral Universidad Sevilla on 6/18/2019 9:09:02 AM. View Article Online potential called zeta potential. The zeta potential value of the 12-s-12/DNA and 16-s-16/DNA complexes is determined by the charge ratio between the gemini surfactants and the DNA. Fig. 4A and B show the zeta potential values obtained for the interaction of DNA with the 12-s-12 and 16-s-16 series of gemini surfactants, respectively. Two inection points are observed from the gures, whose position is in agreement with that observed from the rest of the techniques. 3.5. AFM measurements The AFM images show the existence of a series of structures which depend on the Rmolar ratio for each surfactant studied. The morphology of these structures depends notably on the alkyl chain length and, in a less signicant way, on the spacer length. These structures are stable in a time scale of hours and some of them appear to be early intermediates in a compaction/ decompaction pathway. Fig. 5 shows AFM topographic images of CT–DNA, adsorbed onto an APTES modify mica surface, in cacodylate buffer at pH ¼7.0. As can be observed in the gure, the double stranded DNA is in extended-coil conformation in the absence of any added surfactant. When gemini surfactants were added to the DNA, at low Rratios, a compaction process from an elongated coil to a compact globule was observed. In particular, at a molar ratio of R¼0.03, Fig. 6 shows the formation of different aggregates of DNA. The morphology of these aggregates are different depending on the spacer length of the surfactant, longer in size for surfactants with spacers s¼3. Multiple intramolecular and intermolecular loops are formed, specially, for the gemini surfactants with spacers s¼3, only some random parts of the DNA chains being condensed. Subsequently, when the Rratio increases to 0.1, Fig. S5,†several intermediates of the compaction process with well-dened characteristics are seen. A partially condensed phase was observed with the formation of different globules protruding from the DNA chain where loops are increased in number. Another feature of these intermediates is the formation of some crossover points (see Fig. S5B and C†). With further addition of gemini surfactants, molar ratio was close to 0.6 for the 12-s-12 series and near 0.4 for the 16-s-16 series; only compact globule structures appear, pointing out the completion of the compaction process (Fig. 7). A closer examination of the structures reveals that globules of a larger size are formed for surfactants with a longer spacer length. Table 1 Values of the hydrodynamic diameters (d 1 and d 2 )ofdifferent DNA–gemini surfactants structures 12-3-12 12-6-12 Rd 1 (nm) d 2 (nm) Rd 1 (nm) d 2 (nm) 0.00 951 (100%) 0.00 951 (100%) 0.03 566 (81%) 84 (19%) 0.03 505 (64%) 90 (36%) 0.10 476 (78%) 78 (22%) 0.05 434 (88%) 48 (18%) 0.25 203 (100%) 0.40 252 (53%) 61 (47%) 0.60 82 (100%) 0.60 156 (100%) 1.00 239 (100%) 1.50 920 (100%) 2.50 789 (100%) 4.00 1221 (100%) 4.00 1179 (100%) 5.00 1531 (100%) 5.00 1526 (100%) 16-3-16 16-6-16 Rd 1 (nm) d 2 (nm) Rd 1 (nm) d 2 (nm) 0.03 615 (93%) 35 (7%) 0.03 534 (85%) 52 (15%) 0.05 330 (92%) 35 (8%) 0.14 294 (27%) 74 (73%) 0.25 255 (88%) 45 (12%) 0.25 282 (48%) 52 (52%) 0.50 120 (100%) 0.40 182 (100%) 3.50 157 (100%) 0.60 222 (100%) 4.00 293 (31%) 60 (69%) 2.50 637 (2%) 88 (98%) 5.00 460 (21%) 90 (79%) 5.00 615 (39%) 130 (61%) Fig. 4 Plot of zeta potential values, x,vs. the molar ratio, C 12-s-12 /C DNA (or C 16-s-16 /C DNA )ofdifferent gemini surfactant/DNA complexes. (A) ( ) 12-3-12/DNA, and ( ) 12-6-12/DNA systems; (B) ( ) 16-3-16/DNA, and ( ) 16-6-16/DNA systems. 29438 |RSC Adv.,2015,5, 29433–29446 This journal is © The Royal Society of Chemistry 2015 RSC Advances Paper Published on 20 March 2015. Downloaded by Gral Universidad Sevilla on 6/18/2019 9:09:02 AM. View Article Online Once the compaction process has been reached, the further addition of gemini surfactant entails a increment in the size of the complexes (see Fig. S6 and S7†). This behavior points out that a strictly different conformational change is beginning to be observed, which is consistent with the above mentioned results of viscosity and DLS. It is a remarkable fact that different stages of DNA decompaction phenomena are observed when it goes from molar ratio R¼1toR¼5.0, for both series of gemini surfactants. Considering rst the 12-s-12/DNA systems, at R¼ 1.0, the DNA globules interact with each other to form larger DNA/surfactant aggregates (see Fig. S6A and S6D†). Then, at molar ratio R¼2.5, extended DNA chains emerge from DNA globules (Fig. S6B and S6E†); and nally, at the 4.5 Rratio, the structures are almost fully decompacted (Fig. 6SC and 5SF†). We next examined the intermediates of decompaction for the 16-s- 16 series. At rst, starting from the molar ratio R¼1, DNA bers emerging from the extreme of DNA globules can be seen (Fig. S7A and B and S7D†). Then, at the molar ratio R¼2.5, characteristic features called “pearling”structures, in which spherical small globules are generated on a single polymer chain, are seen (see Fig. S7C and S7E and F†). 41 It is important to mention here that the “pearling”structures were not detected as intermediates in the compaction process. Consequently, for the 16-s-16/DNA systems, the decondensation process is not simply the reverse of the previously described condensation process. Importantly, the number of protruding DNA chains from the Fig. 5 (A) and (B) show AFM topographic images of CT–DNA adsorbed on APTES-modified mica surface in cacodylate buffer, pH ¼7.0 and C DNA ¼310 7 M. DNA molecules are fully extended and the detailed morphology of DNA can be observed. Fig. 6 AFM topographic images of CT–DNA and gemini surfactants in intermediate DNA condensation state, C DNA ¼310 7 M, and R¼0.03. (A) 12-3-12/DNA; (B) 12-6-12/DNA; (C) 16-3-16/DNA; and (D) 16-6-16/DNA. The images show the formation of different aggregates. This journal is © The Royal Society of Chemistry 2015 RSC Adv.,2015,5, 29433–29446 | 29439 Paper RSC Advances Published on 20 March 2015. Downloaded by Gral Universidad Sevilla on 6/18/2019 9:09:02 AM. View Article Online compact aggregates when the Rratio increases is less signicant for the 16-s-16 series in comparison with the 12-s-12 series of gemini surfactants. This situation gives rise to such a diverse gradation in the decompaction process for both the 12-s-12 and 16-s-16 series. In such a way that, at the higher Rvalues (R¼5), the situation is thoroughly different for both series (Fig. 8). That is, at high Rratios, the 12-s-12/DNA systems are fully decompacted (Fig. 8A and B), thus adopting a structure similar to the free DNA in solution (Fig. 5). However, for the 16-s-16/DNA systems, the complexes are not able to adopt fully decompacted structures, exhibiting a partially decompacted state (Fig. 8C and D). Fig. S8†shows an enlarged view for the 16-3-16/ DNA and 16-6-16/DNA systems at the higher Rratio. In such images extended DNA chains together with compact globules structures are seen. Furthermore, from these images, it is noteworthy that the appearance of compact globules is much less frequent for the 16-6-16/DNA system than for the 16-3-16/ DNA system, which reveals that the latter system is the worst one to be used as the delivery vehicle. Note that these results are in good agreement with DLS experiments, in which the presence of a signicant percentage of compact structures is relevant for the 16-s-16 systems, while no condensed structures were seen for the 12-s-12 series at this stage of the interaction. 4. Discussion The principal result stemming from the analysis of the data provided by the different experimental techniques considered in the present investigation is that, once the compaction of the system was accomplished, DNA decompaction was induced by gemini surfactants at high molar ratios. This behavior is particularly of interest, since even though the capacity of diverse gemini surfactants to compact DNA polymers was already recognized, 16–18 such an ability to decompact DNA structures so far was not observed. Firstly, the viscosity results were examined. As is known, the viscosimetry is a well-established and simple methodology which is extremely sensitive to the length change of a biopolymer and it provides, perhaps, the least ambiguous criteria to identify the changes in extension of the DNA helix in solution upon ligand binding. 14 The change in contour length (from L 0 to L) has been related to the change in intrinsic viscosities (from h 0 to h) of the free DNA and the DNA/gemini surfactant complexes by: 42 L L0 ¼"hfðpÞ0 h0fðpÞ 1=3#z1þr(2) where, ris the ratio of the bound gemini surfactant to DNA base-pairs and f(p) 0 ,f(p) are, respectively, functions of the paxial ratio of free and DNA complexes, respectively. The extent of compaction–decompaction processes identied here depends on the C m-s-m /C DNA ratio, and on the nature of the gemini surfactant which differ in the length of the spacers and extension of the hydrocarbon tails. The effects of the gemini surfactants on the viscosity of DNA are summarized in Fig. 1. The viscometric data collected in the gure conrm the presence of multiple DNA conformational changes as the Rratio varies. First, for low values of C m-s-m /C DNA (R< 0.03), the relative Fig. 7 AFM topographic images of CT–DNA and gemini surfactants forming DNA compacted structures, C DNA ¼310 7 M. (A) 12-3-12/DNA, R ¼0.6; (B) 12-6-12/DNA, R¼0.5; (C) 16-3-16/DNA, R¼0.3; and (D) 16-6-16/DNA, R¼0.4. 29440 |RSC Adv.,2015,5, 29433–29446 This journal is © The Royal Society of Chemistry 2015 RSC Advances Paper Published on 20 March 2015. Downloaded by Gral Universidad Sevilla on 6/18/2019 9:09:02 AM. View Article Online viscosity of the systems increases, but, then begins to decrease if the surfactant concentration exceeds the above mentioned value. The initial increase, observed for the four surfactants studied here, is unlikely in agreement with DNA compaction. A possible explanation could be that in the presence of lower surfactants concentrations, the formation of DNA–surfactants complexes contribute to a local lengthening of the DNA helix, meanwhile the base pairs are separated to bind the ligand. In connection with the initial increase of the relative viscosity, the subsequent decrease registered can be reasonably attributed to a conformational change characterized by a smaller DNA radius of gyration, which is in agreement with DNA compaction phenomena. 13 In other words, a hypothesis can be made assuming that the behavior observed corresponds to the surfactant partial intercalation into DNA bases. On this basis, the portion of the gemini surfactant protruding out of the polymer backbone could act as a bridge, thus promoting the interaction between other DNA helix or with base pairs of the same DNA molecule. The latter interaction can stabilize the less elongated polynucleotide conformation, giving rise to a decrease in the viscosity. 43 In contrast, the behavior is strikingly different at high Rratios (R> 0.5) for 12-s-12 series. Note that for the 16-s-16 the high Rratios cannot be explored due to the fact that these surfactants show much lower CMC values compared to the 12-s-12 series (see Table S1†). That is, the relative viscosity shows a marked increase with the surfactant concentration reaching a plateau at the higher Rratio; thus, aer passing the minimum in Fig. 1A; surprisingly, the higher surfactant concentrations provide greater DNA gyration radii. This nding suggests that at a particular surfactant concentration the systems change their conformation to a more elongated form. To verify the nature of the conformational changes that occur upon binding of gemini surfactants to the DNA, circular dichroism and dynamic light scattering techniques were employed. In the following, for the sake of comprehension, we will describe in detail the behavior of the two series of gemini surfactants, separately. 4.1. 12-s-12 effect on DNA structure Fig. 3A shows that the CD spectrum of free DNA has a positive peak at approximately 279 nm and a negative peak at 249 nm which corresponds to B-DNA structure. These bands are caused by stacking interactions between the bases and the helical suprastructure of the polynucleotide that provides an asymmetric environment for the bases. 44 In the same gure the CD changes induced by the 12-6-12 surfactant clearly demonstrates that the helical conformation is not maintained. Gradual addition of each of the two 12-s-12 gemini surfactants leads to remarkable perturbations on both positive and negative bands (Fig. 3A and C and S2A–C†). At low Rratios, Fig. 3A shows that the bands intensity are enhanced without appreciable shiin their position. These changes are indicative of a destabilization of the DNA helix and a disruption of staking contact of the bases, which could indicate that the gemini surfactant–DNA binding was via an intercalation mode. 45 It is important to note Fig. 8 AFM topographic images of CT–DNA and gemini surfactants showing full decompacted (series 12-s-12) and partially decompacted (series 16-s-16) structures, C DNA ¼310 7 M, and R¼5.0. (A) 12-3-12/DNA; (B) 12-6-12/DNA; (C) 16-3-16/DNA; and (D) 16-6-16/DNA. This journal is © The Royal Society of Chemistry 2015 RSC Adv.,2015,5, 29433–29446 | 29441 Paper RSC Advances Published on 20 March 2015. Downloaded by Gral Universidad Sevilla on 6/18/2019 9:09:02 AM. View Article Online