Association of DNA with Multivalent Organic Counterions: From

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Biomacromolecules 2009, 10, 530–540

Association of DNA with Multivalent Organic Counterions: From Flowers to Rods and Toroids Yi Li, Umit Hakan Yildiz, Klaus Mu¨llen, and Franziska Gro¨hn* Max Planck Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germany Received October 18, 2008; Revised Manuscript Received December 17, 2008

In this study, complexes of DNA with different organic di- and tetravalent counterions were systematically investigated in solution and on a surface. The complexation behavior was studied by dynamic light scattering, static light scattering, atomic force microscopy, analytical ultracentrifugation, and UV-vis spectroscopy. Results show that both divalent and tetravalent counterions can induce the formation of DNA complexes. Divalent counterions cause aggregation only at high counterion excess, with charge ratios of 50:1 for supercoiled DNA and 200:1 for linear DNA, while for different tetravalent counterions aggregation is already observed for charge ratios of about 1:1. Flower-like aggregates are observed with divalent counterions. For a tetravalent perylene based counterion, a transition from flower-like aggregates at low charge ratios to toroids and rods at high charge ratios is observed. A transition regime for intermediate charge ratios is found. The influence of concentration, added salt, and preparation method is also discussed. It is concluded that it is the interplay of electrostatics and component architectures that directs the structure formation.

Introduction Association of DNA with different counterions has attracted a lot of attention, both with the aim to develop an understanding of DNA packing inside viruses and cells and for possible applications in gene delivery systems.1-3 With these goals, complexation with various molecules has been studied: Complexes of DNA with multivalent ions were first observed with the natural polyamine spermidine: a well-defined toroid structure was observed in transmission electron microscopy.4 Later toroid and rodlike structures were also found for other counterions, for example, spermidine derivatives,5,6 spermine,7 hexamine cobalt,8 chitosan,9 and poly-L-lysine.10,11 Although rodlike and toroid structures are well-known, the formation process is not fully understood yet. Fang et al. reported flower-like and disklike aggregates with spermidine and suggested they represent intermediate stages of DNA complexes on the pathway to the final rod or toroid structure, however, this could not be proven.12 Krauβ et al. investigated the morphology of DNA complexes induced by a perylene bisimid with 16 positive charges and observed flower-like aggregates by atomic force microscopy (AFM).13 In difference to multivalent ions, it was predicted theoretically that divalent counterions cannot induce aggregation of DNA.14,15 Meanwhile, however, a few studies exist on the interaction behavior of divalent ruthenium coordination compounds with the DNA,16 on a coil-globule transition of DNA molecules complexed with diaminoalkanes,17,18 and on other topographic changes induced by divalent metal ions.19 A further branch of intensively investigated DNA-probe structures are DNA-dye complexes, chromophores being porphyrin,20,21 acridine orange,22,23 and many others. These are of particular interest as they can be used for DNA detection. Mostly the focus of these studies was on spectroscopic properties. Thus, the behavior of DNA with different small ionic molecules can result in different structures and a variety of results have been reported, but it is difficult to relate them. It is therefore of interest to * To whom correspondence should be addressed. Fax: 49-6131-379100. E-mail: [email protected].

compare counterions with different valences and structures to systematically investigate whether or when toroids and rodlike structures, and when flower-like structures are obtained. The aim is to develop basic knowledge on the formation of DNA aggregates with small counterions as basis for (other) studies on more complicated biological or pharmaceutical systems. Usually three main binding modes in the interaction of small molecules with the DNA double helix are distinguished.24-27 In external binding, it interacts with the phosphate group via electrostatic interaction staying outside the DNA strand at its surface. In groove binding, the probe molecule is either in the deep major or the shallow minor groove of the DNA helix, interacting with nucleic acid bases due to hydrophobicity and/ or with the phosphate group via hydrogen bonds. In the intercalation mode, the molecule causes the double helix to unwind and slides between the base pairs to interact with them by π-π stacking. A planar probe molecule structure is a requirement for this binding mode. In this study we are interested in nonplanar and thus nonintercalating counterions. The focus is on external binding of the counterion to the DNA strand by electrostatic interaction and investigating the capability of di- and multivalent counterions to induce aggregation of multiple DNA molecules. We apply different organic counterions to deduce a systematic behavior. In particular, this includes stiff counterions because we recently found that multivalent stiff counterions allow for the formation of defined complexes via “electrostatic selfassembly” with other macroions.28-31 The water soluble tetravalent perylene based ion PSPDI (Chart 1)32 has a threedimensional molecular structure that suggests it cannot intercalate into the DNA helix due to steric hindrance arising from the groups surrounding the aromatic system. Thus, it is expected to bind outside of the DNA helix and potentially connect multiple DNA strands. Moreover, exhibiting two different charge distances offers more possibilities to arrange in the complex formation. In particular, the smaller charge distance corresponds to the distance of phosphate groups on the DNA double strand; thus, a different behavior as with classical porphyrins is

10.1021/bm8011852 CCC: $40.75  2009 American Chemical Society Published on Web 02/09/2009

Association of DNA with Multivalent Counterions Chart 1. Structural Formulae of Organic Counterions Used in this Study

Biomacromolecules, Vol. 10, No. 3, 2009

Table 1. Light Scattering Characterization of DNA Samples Used in this Studya DNA

D0 (m2 s-1)

RH (nm)

RG (nm)

RG/RH

supercoiled pUC19 linear pUC19

4.6 × 10-12 3.3 × 10-12

46 66

77 126

1.7 1.9

a

expected. The solubility in aqueous solution and its fluorescence properties and photostability may lead to applications in the biological area.13,33 Methyl viologen MV2+ (Chart 1) is an excellent electron acceptor with which photoinduced electron transfer can be achieved.34-37 Additional potential may thus lie in the PSPDI and MV2+ counterion apart from serving as model probes. Divalent and tetravalent organic counterions used in this study are shown in Chart 1. In addition to varying the counterion, studies on counterion/DNA ratio and two types of DNA, linear and supercoiled DNA with the same molar mass, were performed. Complexes were investigated in solution by static and dynamic light scattering and after deposition on a surface by AFM.

Experimental Section Chemicals. Methyl viologen dichloride hydrate (Aldrich) and TE buffer (50×, GE healthcare) were used as received. Counterions 1,1′(hexane -1,6- diyl)bis(4 - aza- 1- azoniabicyclo [2.2.2]octane) (C6D2+) and 4,4′-(hexane-1,6-diyl)bis(1-ethyl-1,4-diazoniabicyclo[2.2.2]octane) (C6T4+) were synthesized as part of this study: a mixture of 1,4diazabicyclo(2.2.2)octane (DABCO) in DMF/MeOH and 1,6-dibromohexane or ethyl bromide was stirred at 100 °C for 96 h. The product was precipitated in acetone. The white precipitate was filtered, dissolved in MilliQ water, and extracted with ether multiple times. The solution was freeze-dried and the product was obtained as white powder. The synthesis of pyridinium-substituted perylene-3,4,9,10-tetracarboxylic acid diimide chromophore (PSPDI) was described elsewhere.32 Supercoiled pUC19 DNA. The pUC19 supercoiled DNA (ElimBiopharmaceuticals) was used as received. The degree of supercoiling was higher than 90% as specified by the supplier. pUC19 DNA contains 2686 base pairs and has a theoretical molar mass of 1.74 × 106 g mol-1. Linear pUC19 DNA. The pUC19-linear DNA was obtained from the same supercoiled DNA by incubation with restriction enzyme BamHI (Fermentas) at 37 °C for 1 h. The digestion mixture was characterized by 1% agarose gel electrophoresis. The DNA solution was then purified by phenol/chloroform extraction and precipitation with ethanol. To check for protein contamination, we measured the UV absorbance ratio A260/A280. Values between 1.84-1.88 indicated essentially protein-free DNA. The number of base pairs and the molecular weight correspond to the ones of the supercoiled DNA. Complex Preparation. DNA and the respective counterions were dissolved in 1 x TE buffer (10 mM tris(hydroxymethyl)aminomethane(Tris)-HCl, 1 mM ethylene diamine tetra acetic acid (EDTA), pH ) 7.5) or MQ-water. A counterion stock solution was added dropwise into the DNA solution to result in a final DNA concentration of c(DNA) ) 0.01 g L-1 and desired charge ratios. The mixed solution was stirred overnight. The opposite mixing order was also tested. Dynamic and Static Light Scattering. Light scattering experiments were performed with an ALV set up with goniometer and an ALV

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c (DNA) ) 0.01 g L-1 in 1× TE buffer, pH ≈ 7.5.

5000 full digital correlator. A Uniphase He-Ne laser (22 mW output power, 632.8 nm wavelength) or an infrared laser (80 mW output power, 831.5 nm wavelength) was used.31 The scattered intensity was divided by a beam splitter (approximately 55:45), each portion of which was detected by a photomultiplier. The two signals were cross-correlated to eliminate nonrandom electronic noise. All measurements were carried out at a temperature T ) 20 °C and measured in a scattering angle range 30° e θ e 150° in steps of 10° (50° e θ e 110° for the IR laser). For light scattering the complexes were prepared in dust-free 20 mm diameter quartz cuvettes (Hellma) by either filtering the sample solutions with 0.45 µm LCR filters (Millipore) or, in the case of the PSPDI complexes, by mixing the prefiltered stock solutions. UV-vis absorption measurements were used to check for sample loss upon filtration. Dynamic light scattering data were analyzed by inverse Laplace Transformation of the electric field autocorrelation function using a constrained regularization method (CONTIN). AFM Imaging. AFM images were recorded on a MultiMode Nanoscope IIIa Atomic Force Microscope (Veeco Instruments, California) in tapping mode at room temperature. A silicon cantilever (OMCLAC 160 TS-W, Olympus, Japan) with 42 Nm-1 spring constant and nominal tip radius 1.7 and increases again for l < 1.7, while no isosbestic point is observed. This indicates that the system cannot be

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Figure 5. (a) Hydrodynamic radius, (b) radius of gyration, and (c) RG/RH for complexes formed by supercoiled (b) and linear DNA (O) with tetravalent counterion C6T4+; horizontal lines: pure supercoiled DNA (lower line) and linear DNA (upper line) (all: c (DNA) ) 0.01 g L-1 in TE buffer).

Figure 6. AFM images of complexes induced by (a) supercoiled DNA and C6T4+ and (b) linear DNA and C6T4+ at charge ratio l ) 3; c (DNA) ) 0.01 g L-1 in TE buffer.

described by a simple equilibrium of two spectroscopically distinguishable species, as it is often the case for dye molecules binding to polyelectrolytes where monomeric and (bound) stacked dye molecules are in equilibrium.58,59 The result is also in difference to UV-vis properties of meso-tetrakis(4-Nmethylpyridiumyl) porphyrin (TMPyP) that interacts with DNA by intercalation,60,61 but rather in some similarity with mesotetrakis(4-(N-trimethylammonium)-phenyl)porphyrin (TAPP)62,63 or meso-tetrakis(4-N-ethylpyridiumyl)-porphyrin (TEPyP)64 that bind outside at the DNA surface. No mutual π-π interaction of PSPDI is induced upon binding to DNA, as expected for a molecular geometry with bulky groups surrounding the aromatic system. This steric hindrance should also prevent the dye

molecules to intercalate into DNA helix. The decrease in absorption may be simply due to the interaction of PSPDI with DNA, that is, thereby caused changes in the polarity surrounding the chromophore. Light scattering was carried out for further characterization, due to the absorption in the visible range using an IR laser with a wavelength of λ ) 831.5 nm.31 As evident from Figure 8, both for supercoiled and linear DNA, for a charge ratio l e 1, one diffusion process was detected, while for l > 1, two processes were observed: one corresponding to a particle size of 20 nm < RH < 30 nm and independent of charge ratio, the other one corresponding to an increasing RH with increasing

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Figure 7. UV-vis spectra of PSPDI with supercoiled DNA, c (PSPDI) ) 3.1 µM, l ) charge ratio.

charge ratio up to RH ≈ 130 nm. Because the two processes are close, it is difficult to determine reliable radii values in some cases, but it is clearly proven that two diffusive processes occur. Usually two processes are observed in polyelectrolyte systems due to domain formation and special polyelectrolyte diffusion behavior,65-70 but these effects disappear with increasing amount of added low molecular mass salt. In the present case, samples are in ionic buffer solution so that no polyelectrolyte effects are expected (c (PO4-)/c (buffer) ) 0.0031). In addition, varying the concentration of NaCl from 0.01 mM to 10 mM always yielded two processes. Thus, there are in fact two species present. For l ) 1.5, the relative contribution of the smaller species was 17% for supercoiled and 6% for linear DNA. As these are intensity weighted values, the number weight of the smaller species is much higher, that is, much more small particles are present. Figure 8c shows the total scattering intensity (extrapolated to zero angle) as function of charge ratio. A strong increase is observed starting at l ) 1, which is an indication of increase in molecular weight. More DNA molecules become connected into an aggregate for l > 1. This however is a rough consideration as DLS elucidated two processes and the total intensity only represents an average value. AFM provides further insight, although complex structures may change upon deposition on the surface. Figure 9 presents a series of AFM images for complexes induced by PSPDI at different charge ratios for the two types of DNA. At low charge ratio (l ) 0.5, left images) where only one process is obtained in light scattering, only flower-like aggregates are found. This is similar to other complexes in this study. When the charge ratio increases to l ) 1 (middle), rodlike and flower-like aggregates and few toroids are observed in coexistence. At a charge ratio of l ) 1.4-1.5, where two processes are observed in LS, only rod and toroid structures are found and no flowerlike aggregates (right images). This is similar for both DNA types, only that the complexes formed with supercoiled DNA show more toroidal aggregates as compared to linear DNA. The rod lengths of 190 ( 50 nm for supercoiled and 200 ( 80 nm for linear DNA and toroid diameters of 55 ( 8 nm for supercoiled and 60 ( 10 nm for linear DNA are consistent with the RH and the bimodal distribution resulting from DLS. Rods and toroids have a defined height that again is larger than for

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DNA only indicating the association of multiple DNA molecules: Rodlike structures yield an average height of 2.9 ( 0.3 for supercoiled DNA and 2.0 ( 0.4 nm for linear DNA, toroids a height of 3.4 ( 0.4 nm for supercoiled DNA and 2.3 ( 0.5 nm for linear DNA, as compared to 1.1 and 0.7 nm for the DNA only. Thus, with increasing charge ratio, that is, with an increasing amount of PSPDI, the complex morphology changes from flower-like to rod- and toroid-like with mixtures of these morphologies as intermediates. Adding more PSPDI to DNA, the repulsive force between like-charged DNA molecules may be reduced due to progressing counterion condensation and neutralization of DNA chains. It has the consequence that different DNA strands can approach in parallel and come closer, forming strand-strand stabilization. Images in the transition range clearly show that rodlike aggregates and toroids are composed of several DNA strands (middle, marked with arrows in Figure 9b,h). Again, as far as resolutions allow, AFM observations correspond to light scattering results. Yet, obviously, one has to bear in mind that further interactions play a role for the surface structure and make the electrostatically assembled system even more complex; plus, the structure is a two-dimensional one. Reason for the correspondence may be that the assemblies are quite stable due to the multivalent nature of the counterions, while the surface is only weakly charged. In addition, aggregates with a composition in the range of charge stoichiometry are expected to be only weakly charged, so that again interaction with the surface is not too strong and assemblies formed in solution stay intact upon deposition on the surface. The influence of concentration is shown in Figure 10 based on light scattering results. As the DNA concentration increases, splitting into two processes is observed. Accordingly, AFM yields for example for l ) 0.5 only flower-like aggregates at c (DNA) ) 0.01 g L-1 but flower-like aggregates coexisting with rods and toroids at c (DNA) ) 0.1 g L-1. The splitting point depends on charge ratio. Hence, the structure of the complexes formed by DNA and PSPDI is not only directed by the counterion/DNA ratio, but also by the total concentration. This is in contrast to the finding for divalent counterions, where the overall concentration did not show any significant influence. This result may again be assigned to the stronger “connection” tendency of the tetravalent counterions. Results are consistent with the suggestion by Fang et al. that flower-like structures might be intermediates on the pathway to toroids and rods.12 Here we provide evidence for this assumption by showing a complete complexation range starting from flower-like aggregates to the final rods and toroids. It should also be noted that in comparison experiments a tetravalent porphyrin (TAPP) did not induce formation of rod or toroid structures when combined with supercoiled DNA at l ) 1.5 but flower-like aggregates (as C6T4+). This again demonstrates the importance of the counterion structure. It may be that the smaller charge distance of positive charges in the PSPDI molecule (0.7 nm), comparable to the distance of the DNA phosphate groups, plays a key role. Formation of rods with well-defined diameter is in analogy to other results with DNA8-10 and other biomacromolecules.71-73 It is in agreement with theoretical considerations on counterion induced bundle formation by Pincus et al.74 As he pointed out, bundle sizes diverge with point-like counterions due to complete charge neutralization, but equilibrium bundles with defined size can form with larger counterions where steric hindrance prevents full charge neutralization. This is due to



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Figure 8. (a) Normalized electric field autocorrelation function and relaxation time distribution at a scattering angle θ ) 50° for the supercoiled DNA/PSPDI sample with l ) 1.5, (b) hydrodynamic radius of complexes formed by supercoiled (b) and linear DNA (O) with tetravalent PSPDI; horizontal lines: pure supercoiled DNA (lower line) and linear DNA (upper line), (c) relative scattering intensity of complexes formed by supercoiled DNA (b) and linear DNA (O) with PSPDI; all: c (DNA) ) 0.01 g L-1 in TE buffer.

the self-energy of the aggregate caused by its net charge in analogy to the Rayleigh instability of a charged oil droplet.75 In accordance, computer simulations of semiflexible polyelectrolytes with trivalent counterions based on the coupling of surface tension, self-energy, and entropic degrees of freedom showed that thermodynamically stable bundles of finite size can form under certain conditions.76 The general result of finite-size assemblies formed by a combination of electrostatics and geometric constraints corresponds to synthetic systems recently obtained by “electrostatic self-assembly”28-31,77,78 and natural systems such as, for example, DNA histone complexes.The formation of toroids may occur to avoid end-cap effects, as described by Fo¨rster et al. for polyelectrolyte block copolymer systems.79 E. Complexation in Salt-Free Solution. Results described so far were for buffer solutions with pH ≈ 7.5, that is a concentration of low molecular mass salt of c ) 8 mM. The complexation of supercoiled DNA with different counterions was therefore also carried out in salt-free solution to investigate possible effects of the buffer ions. Light scattering results are shown in Figure 11. For both divalent and tetravalent counterions, the hydrodynamic radius goes through a minimum with increasing charge ratio. In this case, the counterions act like low molecular mass salt first. (Interaction effects play a role for pure DNA in salt-free solution.) When the amount of counterions is further increased, repulsive forces between different DNA molecules are screened and larger aggregates can form. The location of the minimum at smaller charge ratio for the tetravalent counterions is consistent with both screening and “connection” being stronger for higher valence ions.

In addition, the complexes induced by sufficient divalent counterions (l ) 500) have similar morphologies as those in TE buffer. The absence of low molecular weight salt does not influence the complex formation substantially. This is as expected in this case, as the excess of free counterions that became evident from analytical ultracentrifugation (section B) should cause a similar screening effect as added salt. In contrast, for the tetravalent counterion C6T4+, RG, and RG/RH (not shown) reach a minimum value at l ≈ 3. When the tetravalent counterion reduces the size of DNA assemblies by screening the negative phosphate groups, it may also force the DNA molecules into a more compact structure. F. Thermodynamic versus Kinetic Structure Control. It is of fundamental interest whether the observed assemblies are kinetically controlled and depend on the preparation procedure or represent equilibrium structures, which usually means the same structure can be obtained via different preparation routes. Complexes of two oppositely charged polyelectrolytes usually represent kinetically trapped structures due to the high number of charges.55,80 In contrast, recently described dendrimer-dye assemblies were equilibrium structures.28-30,77,78 We therefore prepared samples using two different mixing orders, that is, adding DNA into counterion solution or adding counterion solution into DNA solution. For the divalent counterions and for C6T4+, both in TE buffer and MQ-water, changing the order did not give different results. Thus, the structures formed for these counterions can be regarded as equilibrium structures. These assemblies are stable in solution over weeks. In contrast, a difference is found for complexes with PSPDI in TE buffer. Adding PSPDI to DNA solution (as usually applied

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Figure 9. AFM images of complexes induced by PSPDI with supercoiled DNA (a-f), and linear DNA (g-l). Charge ratio l ) 0.5 (a, d, g, i), l ) 1 (b, e, h, k), l ) 1.4 (c, f), and l ) 1.5 (i, l); all: c (DNA) ) 0.01 g L-1 in TE buffer.

in this study) yields two diffusion modes and AFM shows rod and toroid structures. The opposite procedure gives only one relatively broad distribution and rod-like structures with few flower-like aggregates in AFM. This corresponds to the structure “usually” observed for lower charge ratios, that is, less PSPDI per DNA. This may be due to temporary concentrations in the mixing process causing different charge ratios in different solution regions and complexes once formed not redissolving

later. Thus, in total, we conclude that the aggregate structures induced by the divalent counterions and by C6T4+ represent equilibrium structures. In contrast, for PSPDI kinetic aspects play an additional role. This may be because of the formation of more compact aggregates due to a better molecular fit of DNA and counterion. This can directly lead to more connection points causing the structure to be (somewhat) kinetically trapped. In either structure, equilibrium or kinetically controlled, geo-



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formation with small synthetic counterions but may also have an impact on applications of ionic DNA complexes, for example, in gene therapy. In addition to the solution structures studied by light scattering, AFM yielded corresponding results for the complexes deposited on surfaces. Such composite assemblies of DNA and synthetic counterions representing defined entities on a surface may be of interest for further kinds of applications, for example, taking advantage of optical and electrical properties of the counterions.

Figure 10. Hydrodynamic radius of complexes formed by supercoiled DNA and PSPDI for l ) 0.5 (b) and l ) 1.4 (O) in dependence of concentration (in TE buffer).

Acknowledgment. We thank Dr. R. Berger and U. Rietzler for help with AFM measurements, Dr. H. Co¨lfen and A. Vo¨lkel (MPI Golm) for AUZ measurements, K. Peneva for the synthesis of the PSPDI counterion, and C. Ruthard for help with the IRlight scattering setup and comments on this manuscript. We are thankful to Prof. Dr. G. Wegner and Prof. Dr. M. Schmidt for fruitful discussions. Financial support of the Max Planck Society, Deutsche Forschungsgemeinschaft (SFB 625), and the POLYMAT Graduate School of Excellence (University of Mainz) is gratefully acknowledged.

References and Notes

Figure 11. Hydrodynamic radius of supercoiled DNA complexed with C6D2+ (b) and C6T4+ (O) in MQ-water; c (DNA) ) 0.01 g L-1.

metric constraints may cause the complex to be charged and thus stable in aqueous solution, as discussed above.

Conclusions In this study we presented the association of double-strand DNA with small organic counterions. It was found that in aqueous solution and under ambient temperature divalent counterions induce assembly of DNA molecules into flowerlike aggregates at high counterion excess, that is at charge ratios as high as 500:1. Here the separation of the two charges in the organic molecule was essential and calcium or magnesium ions did not induce formation of flower-like aggregates. Different tetravalent counterions caused aggregation at much lower charge ratios, which is around 1:1. For the exact onset of aggregation and aggregate size, it played a role whether supercoiled or linear double-strand DNA of the same molecular mass was used. Further, the tetravalent chromophore PSPDI yielded different aggregate types depending on charge ratio: While flower-like structures were observed for low charge ratios, a bimodal distribution and the existence of well-defined rods and toroids were found for higher charge ratios. Rods and toroids consist of multiple DNA molecules. The stiff structure of this counterion and its distance and position of charged groups may cause this behavior. Thus, in accordance with theoretical considerations and recent experimental results on other systems, it is the counterplay of electrostatics and geometric factors that directs the structure formation. Thereby, even small counterions can cause essential differences in the resulting morphologies via valence, structure, and mixing ratio. Findings of this study are thus of importance for a basic understanding of DNA complex

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