Langmuir 2006, 22, 6293-6301
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DNA Condensation and Interaction with Zwitterionic Phospholipids Mediated by Divalent Cations Sandra Gromelski and Gerald Brezesinski* Max Planck Institute of Colloids and Interfaces, Research Campus Golm, 14476 Potsdam, Germany ReceiVed NoVember 23, 2005. In Final Form: April 21, 2006 Artificial viruses are considered to be a promising tool in gene therapy. To find lipid-DNA complexes with high transfection efficiency but without toxicity is a fundamental aim. Although cationic lipids are frequently toxic for cells, neutral lipids are completely nontoxic. Zwitterionic lipids do not interact with DNA directly; however, the interaction can be mediated by divalent cations. Langmuir monolayers represent a well-defined model system to study the DNA-lipid complexes at the air/water interface (quasi-2D systems). In this work, isotherms, infrared reflection absorption spectroscopy (IRRAS), X-ray reflectivity (XR), grazing incidence X-ray diffraction (GIXD), and Brewster angle microscopy (BAM) measurements are used to study the interaction of calf thymus DNA with DMPE (1,2dimyristoyl-phosphatidylethanolamine) monolayers mediated by Ca2+ or Mg2+ ions. DNA adsorption is observed only in the presence of divalent cations. At low lateral pressure, the DNA partially penetrates into the lipid monolayer but is squeezed out at high pressure. The adsorption layer has a thickness of 18-19 Å. Additionally, GIXD provides information about a one-dimensional ordering of adsorbed DNA. The periodic distance between DNA strands depends on the type of the divalent cation.
1. Introduction Transfer of genetic material into mammalian cells can be performed by various techniques. One important technique is gene delivery by viruses. Two different types of viruses have been tested as drug delivery systems: natural viruses and artificial viruses. Natural viruses are cell parasites. They usually display the highest efficiency of all transfection systems as they are specialized for delivering their own genetic information (DNA or RNA) into their host cells, to replicate, and survive. However, viral vectors exhibit various disadvantages, such as induction of strong immune responses, virus particle associated toxicity, limited target cell specificity, genomic instability, and potential mutagenicity. Also the production of natural viruses used in gene therapy is very expensive. Therefore, nonviral gene therapy is considered to be a very important tool for the future.1 Many attempts have been made to create effective artificial viruses. Their use as drug delivery vessels for “good” genetic material is aimed at treating a variety of diseases. They should exhibit the ability to pass through cell barriers without the drawbacks of natural viruses. Lipid-DNA systems are unlimited in size, easy to produce, and very flexible concerning the composition of the coverage. A simplified model of an artificial virus is a hollow capsule produced by adsorbing layer-wise polyelectrolytes on a soluble template.2,3 For application, such capsules have to be covered by a lipid bilayer with integrated virus membrane components. In a first step, the molecular basis of polyelectrolyte-lipid interactions has to be understood. Biomembranes are composed of a phospholipid bilayer, in which the lipids are in a disordered state. Many physiologically important interactions take place at the interface between the membrane and the aqueous compartment. Therefore, Langmuir monolayers of charged lipids coupled to oppositely charged * To whom correspondence should be addressed. (1) Mahato, R. I.; Smith, L. C.; Rolland, A. AdV. Genet. 1999, 41, 95. (2) Lvov, Y.; Decher, G.; Mo¨hwald, H. Langmuir 1993, 9, 481. (3) Sukhorukov, G. B.; Donath, E.; Lichtenfeld, H.; Knippel, E.; Knippel, M.; Budde, A.; Mo¨hwald, H. Colloid Surf., A 1998, 137, 253.
polyelectrolytes represent well-defined model systems to study complex formation at the air/water-interface.4 Structural data can be obtained from such systems using a variety of methods, including grazing incidence X-ray diffraction (GIXD).5,6 Beside the investigation of the coupling of negatively charged phospholipids to positively charged polyelectrolytes,7 the interaction of positively charged phospholipids with negatively charged DNA8 was studied. Safinya and co-workers 9 investigated cationic liposomes complexed with DNA as nonviral carriers of DNA vectors for gene therapy using SAXS (small-angle X-ray diffraction) and optical microscopy.9-11 They observed a multilamellar structure with alternating cationic DOTAP (dioleoyl trimethylammonium propane) lipid bilayers and λ-DNA monolayers above, below, and at the isoelectric point.9 Around the isoelectric point, an increase of interaxial spacing from 24.5 to 57.1 Å as a function of lipid/DNA ratio was observed. In 2000, Safinya and co-workers examined complexes composed of DOPC/DOTAP (1,2-dioleoylphosphatidylcholine/1,2-dioleoyl-trimethylammonium propane) mixtures and DNA.12 The spatial dimension available to DNA plays obviously a key role in the interactions between DNA chains. DNA adsorbed onto a cationic membrane undergoes a collapse transition in the presence of simple divalent cationic biological ions, such as Ca2+, Mg2+, and Mn2+. Synchrotron X-ray diffraction experiments showed that the collapsed phase consists of DNA chains electrostatically tethered by divalent counterions. Symietz et al.8 showed, that calf thymus DNA binds in an ordered way to a positively charged TODAB (trioctadecyl(4) Brezesinski, G.; Mo¨hwald, H. AdV. Colloid Interface Sci. 2003, 100, 563. (5) Als-Nielsen, J.; Jacquemain, D.; Kjaer, K.; Leveiller, F.; Lahav, M.; Leiserowitz, L. Phys. Rep.-ReV. Sect. Phys. Lett. 1994, 246, 252. (6) Jensen, T. R.; Kjaer, K. In NoVel Methods to Study Interfacial Layers, Studies in Interface Science; Moebius, D., Miller, R., Eds.; ElseVier Sci. B. V. 2001, 11, 205. (7) de Meijere, K.; Brezesinski, G.; Kjaer, K.; Mo¨hwald, H. Langmuir 1998, 14, 4204. (8) Symietz, C.; Schneider, M.; Brezesinski, G.; Mo¨hwald, H. Macromolecules 2004, 37, 3865. (9) Ra¨dler, J. O.; Koltover, I.; Salditt, T.; Safinya, C. R. Science 1997, 275, 810.
10.1021/la0531796 CCC: $33.50 © 2006 American Chemical Society Published on Web 06/09/2006
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methylammonium bromide) monolayer. The interaxial DNA spacings decrease from 49 Å to 32 Å during the monolayer compression. These experiments showed that cationic lipids can easily form stable complexes with DNA. Unfortunately, cationic lipids are frequently toxic for the cells. Complexes composed of neutral lipids offer an alternative to cationic lipids as they are completely nontoxic. Turbidity measurements revealed an interaction of radioactive oligo- and polynucleotides with zwitterionic liposomes only in the presence of magnesium.13 Huster et al.14,15 showed that there is a calcium-mediated interaction between DMPC and the negatively charged dextran sulfate. De Meijere et al.16 observed that dextran sulfate can couple to the noncharged 1,2-dipalmitoyl-phosphatidylethanolamine (DPPE) monolayer at the air-water interface via ionic bridges of calcium, which influences the lateral order of the lipid monolayer. Zwitterionic lipids do not interact with the DNA directly, but the interaction can be mediated by divalent cations.13,17 McLoughlin et al.18 observed that the interaction of DNA with phospholipid monolayers is ion specific: The presence of calcium leads to a stronger interaction than magnesium and barium. The liquid-condensed domains adopt an elongated morphology in the presence of DNA, especially in the presence of calcium. It was also reported that DNA lowers the charge density of the monolayer as a consequence of charge neutralization, as evidenced by a change of the surface potential. In the present work, the coupling of DNA to the zwitterionic lipid DMPE mediated by divalent cations (calcium or magnesium) was examined using a film balance coupled with infrared spectroscopy (IRRAS), Brewster angle microscopy (BAM), X-ray reflectivity, and grazing incidence X-ray diffraction (GIXD). An ordered coupling of DNA was observed only in the presence of divalent cations. This coupling of DNA leads to a smaller tilt angle of the lipid hydrocarbon chains compared to DMPE on water. 2. Experimental Section 2.1. Materials. Calf thymus DNA and DMPE (1,2-dimyristoylphosphatidylethanolamine)werepurchasedfromSigma(Taufkirchen, Germany) and used as received. DNA was dissolved in Milli-Q deionized water with a specific resistance of 18.2 MΩ‚cm containing 1 mM NaCl (Merck) for stabilization. The sodium chloride was heated to 600 °C to remove any content of potential organic impurities. 1 mM NaCl has no influence on the DMPE monolayer structure. CaCl2 and MgCl2 were purchased from Fluka and used in a concentration of 5 mM. The DNA concentration (0.1 mM) refers to a monomer containing one charge per phosphate moiety. DMPE was spread from 1 mM chloroform (Merck, Germany) solution onto the corresponding subphase. 2.2. Methods. 2.2.1. Pressure-Area Isotherms. Chloroform was used as the spreading solvent because of the high spreading coefficient. The lateral surface pressure π of the monolayer is the difference between the surface tension of pure water (72.8 mN‚m-1) and water covered with a monolayer: (10) Zantl, R.; Baicu, L.; Artzner, F.; Sprenger, I.; Rapp, G.; Ra¨dler, J. O. J. Phys. Chem. B 1999, 103, 10300. (11) Safinya, C. R. Curr. Opin. Struct. Biol. 2001, 11, 440. (12) Koltover, I.; Wagner, K.; Safinya, C. R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 14046-14051. (13) Budker, V. G.; Godovikov, A. A.; Naumova, L. P.; Slepneva, I. A. Nucleic Acids Res. 1980, 8, 2499. (14) Huster, D.; Paasche, G.; Dietrich, U.; Zscho¨rnig, O.; Gutberlet, T.; Gawrisch, K.; Arnold, K. Biophys. J. 1999, 77, 879. (15) Huster, D.; Arnold, K. Biophys. J. 1998, 75, 909. (16) de Meijere, K.; Brezesinski, G.; Zscho¨rnig, O.; Arnold, K.; Mo¨hwald, H. Physica B 1998, 248, 269. (17) Bailey, A. L.; Sullivan, S. M. Biochim. Biophys. Acta 2000, 1468, 239. (18) McLoughlin, D.; Dias, R.; Lindman, B.; Cardenas, M.; Nylander, T.; Dawson, K.; Miguel, M.; Langevin, D. Langmuir 2005, 21, 1900.
Gromelski and Brezesinski π ) γsolvent - γsolution
(1)
The surface tension was recorded with a continuous Wilhelmy-type pressure measuring system using a filter paper as plate. By means of movable barriers the lipid monolayer was compressed and surface pressure/area isotherms were recorded continuously at a given temperature (20 °C). After spreading DMPE on subphases that contained DNA, an adsorption time of 1 h followed, after which equilibrium conditions were observed. Compression of the DMPE film with a speed of 5 Å2/molecule/min leads to an increase of the lateral pressure and to a change of the monolayer phase state. 2.2.2. IRRAS. Infrared reflection absorption spectroscopy (IRRAS) detects changes in conformation and orientation of lipid chains as well as interactions of the lipid headgroup via changes in vibrational frequencies. IRRA spectra can be directly correlated to molecular structures and conformations by measuring simultaneously pressure/ area (π/A) isotherms. IRRA spectra have been recorded using the IFS 66 FT-IR spectrometer (Bruker, Germany), equipped with a liquid-nitrogencooled MCT detector and coupled to a Langmuir film balance, which was placed in a sealed container to guarantee a constant vapor atmosphere. Using a KRS-5 (thallium bromide and iodide mixed crystal) wire grid polarizer, the IR-beam was polarized vertically (s) and focused on the fluid subphase at an angle of incidence of 40°. A computer controlled “trough shuttle system”19 enables us to choose between the compartment with the sample (subphase with spread monolayer) and a reference compartment (pure subphase). The two compartments are connected by little pipes to guarantee an equal filling height. The single-beam reflectance spectrum from the reference trough was taken as background for the single-beam reflectance spectrum of the monolayer in the sample trough to calculate the reflection absorption spectrum as -lg(R/R0) in order to eliminate the water vapor signal. FTIR spectra were collected at a resolution of 8 cm-1 using 200 scans. Table 1 shows the Mid-IR group frequencies of phospholipids and DNA (B-form).20,21 2.2.3. GIXD. Grazing incidence X-ray diffraction (GIXD) and X-ray reflectivity (XR) measurements were carried out at the undulator beamline BW1 using the liquid surface diffractometer at HASYLAB, DESY (Hamburg, Germany). The experimental setup and evaluation procedures are described in detail elsewhere.5,6,22-24 The diffractometer is equipped with a temperature controlled Langmuir trough (R&K, Potsdam, Germany), which is enclosed in a sealed, helium-filled container. The synchrotron X-ray beam was monochromated to a wavelength of 1.304 Å by a beryllium (002) crystal and was adjusted to strike the monolayer on the water surface at an angle of incidence Ri ) 0.85Rc, where Rc is the critical angle of total external reflection (0.13°), to maximize the surface sensitivity.25 The lipid monolayer on a fluid subphase is a two-dimensional (2D) powder. The intensity of the diffracted radiation is detected by a position-sensitive detector (PSD) (OED-100-M, Braun, Garching, Germany) as a function of the vertical scattering angle Rf. The resolution of the horizontal scattering angle 2θxy is given by a Soller collimator located in front of the PSD, where 2θxy is the angle between the incident and diffracted beam. By scanning the horizontal angle 2θxy, the horizontal (Qxy ≈ (4π/λ) sin(2θxy/2)) and the vertical (Qz ≈ (2π/λ) sin(Rf)) components of the scattering vector Q can be detected simultaneously. The accumulated position-resolved scans were corrected for polarization, effective area and Lorentz factor. Model peaks taken as Lorentzian in the in-plane direction and as Gaussian in the out-of-plane direction were fitted to the corrected intensities. (19) Flach, C. R.; Brauner, J. W.; Mendelsohn, R. Biophys. J. 1994, 66, A373. (20) Blume, A. Curr. Opin. Colloid Interface Sci. 1996, 1, 64. (21) Banyay, M.; Sarkar, M.; Graslund, A. Biophys. Chem. 2003, 104, 477. (22) Kjaer, K.; Als-Nielsen, J.; Helm, C. A.; Tippmann-Krayer, P.; Mo¨hwald, H. J. Phys. Chem. 1989, 93, 3200. (23) Frahm, R.; Weigelt, J.; Meyer, G.; Materlik, G. ReV. Sci. Instrum. 1995, 66, 1677. (24) Kjaer, K. Physica B 1994, 198, 100. (25) Eisenberger, P.; Marra, W. C. Phys. ReV. Lett. 1981, 46, 1081.
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Table 1. Mid-IR Group Frequencies of Phospholipids and DNA (B Form)20,21 wave number (cm-1)
assignment
symbol
2956 2925 2918 2845 2849 1740 1645 1470 1250-1220 1170 1086-1072 1085 1070 970
Mid-IR Group Frequencies of Phospholipids asymmetric CH3-valence vibration asymmetric CH2-valence vibration (liquid phase) asymmetric CH2-valence vibration (condensed phase) symmetric CH2-valence vibration (liquid phase) symmetric CH2-valence vibration (condensed phase) ester-carbonyl-valence vibration HOH-strech vibration CH2-strech vibration asymmetric phosphate-diestcr-valence vibration asymmetric ester CO-O-C -valence vibration symmetric phosphate-diester-valence vibration symmetric ester CO-O-C -valence vibration phosphate-ester-valence vibration asymmetric choline-valence vibration
vas(CH3) vas(CH2) vs(CH2) vs (CH2) vs(CH2) v (CdO) 5 (HOH) 5 (CH2) vas(PO2-) vas(CO-O-C) vs (PO2) vs(CO-O-C) v (CO-O-PO2) vas(C-N+-C)
1250-1220 970 938
Mid-IR Group Frequencies of DNA (B Form) asymmetric phosphate-diester-valence vibration DNA backbone AT base pairs in B-from helices
The lattice spacings are obtained from the in-plane diffraction data dhk )
2π λ ) 2 sin θ Qhk xy
(2)
from which the lattice parameters can be calculated. Tilt angle t of the hydrocarbon chains and the tilt azimuth ψ can be obtained by Qmax ) Qmax z xy cos ψhk tan (t)
(3)
Assuming that the monolayer consists of 2D crystallites that are perfect and have a finite size, the fwhm (full width at half maximum) of the peaks gives information about the position correlation using the Scherrer formula6,22,24 ξ)
0.88‚2‚π ∆
(4)
where ∆ ) x∆peak2 - ∆det2 and ∆det ) 0.009 Å-1
(5)
2.2.4. X-ray Reflectivity. Biological membranes are generally highly disordered systems. Therefore, diffraction methods are often not suited to characterize the physiologically relevant states of such systems. In distinction from diffraction, reflectivity measurements are not limited to ordered systems. Specular reflectivity measurements reveal information about the electron density distribution F along the surface normal z. The reflectivity R can be calculated kinematically using the so-called “master formula for reflectivity” R(Qz) RF(Qz)
1 )| Fw
exp(iQ ‚z) dz| ∫ dF dz z
2
(6)
where RF(Qz) is the Fresnel reflectivity calculated from standard optics for a perfectly sharp interface between air and the pure subphase (of average electron density Fw). Qz )
4π sin R λ
(7)
is the vertical component of the scattering vector Q, λ the wavelength, and R the incidence angle with respect to the surface. The specular X-ray reflectivity (XR) data collection was performed by using a NaI scintillation detector. The X-ray reflectivity was measured with the geometry, Ri ) Rf ) R, where Ri is the vertical incidence angle and Rf is the vertical exit angle of the reflected
vas(PO2-)
X-rays. An X-ray wavelength of λ ) 1.304 Å was used. XR data were collected as a function of the incidence angle, Ri, varied in the range of 0.05°-5°, corresponding to a range of 0.01-0.85 Å-1 of the vertical scattering vector component Qz. The background scattering from the subphase was measured at 2θxy ) 0.7° and subtracted from the signal measured at 2θxy ) 0. The X-ray footprint area on the sample is inversely proportional to the incident angle of the X-rays. The direct Fourier inversion of the reflectivity into the electron density distribution is not possible, because one measures the absolute square of a complex number and not the phase. For the interpretation of the reflectivity data, two models can be used, both described by Schalke et al.26 In this work, the “box model” is used, which describes the submolecular organization of phospholipids as molecularly homogeneous slabs.27 A model density profile with a certain number of adjustable parameters was fitted to the measured reflectivity curve using the Parratt program.28 2.2.5. BAM. Brewster angle microscopy (BAM) allows direct observation of ultrathin organic films on transparent dielectric substrates. For a Fresnel interface (an interface where the refractive index changes steeply) and for a polarization where the electric field is in the plane of incidence, the reflectivity vanishes at the Brewster angle. For a real interface, the reflected light intensity has a minimum at the Brewster angle (53.1° for water) but does not completely vanish. However, the reflected intensity at the Brewster angle is strongly dependent on the interfacial properties and is particularly sensitive to monolayers at the interface. The reflectivity of a real interface at the Brewster angle for a distinct polarization has three origins: (a) the thickness of the interface, (b) the roughness of real interfaces, and (c) the anisotropy of monolayers. The morphology of the films was imaged using a computer interfaced KSV 2000 Langmuir balance combined with a Brewster angle microscope (KSV Optrel BAM 300, Helsinki). p-polarized light from a 10 mW HeNe laser (633 nm) was directed at the Brewster angle onto the pure water surface giving zero reflectivity. The presence of a monolayer gives rise to reflected light registered by a CCD camera. Optical anisotropy of the monolayer, due to specific molecular structure or collective molecular tilt, can be detected via an analyzer in the pathway of the reflected beam. During compression of the monolayer, the reflected beam was recorded digitally with frames of 400 × 300 µm2. The π/A isotherm was simultaneously registered to ascribe each image to the corresponding surface pressure and molecular area. (26) Schalke, M.; Lo¨sche, M. AdV. Colloid Interface Sci. 2000, 88, 243. (27) Helm, C. A.; Mo¨hwald, H.; Kjaer, K.; Als-Nielsen, J. Europhys. Lett. 1987, 4, 697. (28) Parratt, L. G. Phys. ReV. 1954, 95, 359.
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Figure 1. Pressure/area isotherms of DMPE monolayers on water (A), 1 mM NaCl + 5 mM MgCl2 + 0.1 mM calf thymus DNA (B), and 1 mM NaCl + 5 mM CaCl2 + 0.1 mM calf thymus DNA (C).
3. Results 3.1. Isotherms. Figure 1 shows π/A isotherms of a DMPE monolayer on water as well as on DNA solutions containing 1 mM NaCl and 5 mM MgCl2 or 1 mM NaCl and 5 mM CaCl2. Upon compression, the area per lipid molecule decreases. At very low surface pressures the liquid-expanded (LE) phase is present. Compression leads to a plateau at 5 mN‚m-1 indicating a first-order transition from the disordered LE phase to an ordered condensed phase. In the LE/LC phase transition region, regions with disordered and ordered lipids coexist. Further decrease of the molecular area forces all lipid molecules into the ordered state. At ca. 32 mN‚m-1 a kink appears at which the compressibility of the monolayer decreases indicating a second-order phase transition from the tilted into the untilted state. The presence of salts such as 1 mM NaCl, 5mM MgCl2, or 5 mM CaCl2 does not influence the shape of the DMPE isotherm. Similar results were observed for DMPE on 0.1 mM calf thymus DNA + 1 mM NaCl. Only the presence of DNA and divalent cations as calcium or magnesium in the subphase effects the isotherm conspicuously. At low pressures, the isotherms are shifted to larger areas per molecule compared to those on pure water. This can be explained by partial penetration of DNA. However, the LE/LC transition pressure is not markedly influenced. At a surface pressure of ca. 28 mN‚m-1, the isotherms of DMPE on DNA in the presence of calcium or magnesium ions show a kink, which possibly also marks the second-order phase transition from the tilted to the nontilted state of the lipid molecules. Above this pressure, the molecular area corresponds to that of DMPE on water. 3.2. IRRAS. Infrared reflection absorption measurements confirm the presence of DNA at the interface. The spectral region of 1800-700 cm-1 is rich with so-called DNA marker bands. They give important information, such as helix conformation or base pairing. Figure 2 (right) shows IRRA spectra of DMPE on DNA plus calcium, respectively magnesium, from which the IRRA spectra of DMPE on calcium, respectively magnesium, were subtracted. For both experiments, the band at 970 cm-1, caused by the 2′endo deoxyribose conformation of the DNA backbone, was visible indicating the presence of DNA at the interface at surface pressures above 5 mN‚m-1. Additionally, the intensity of the bands of the symmetric and asymmetric stretching of the phosphate group increased after adsorption of DNA to the monolayer (Figure 2, left). This increase is caused by the phosphate moieties of the DNA and possibly additionally by a change of the DMPE headgroup orientation. The distinction between these two events is difficult. Therefore, the presence of DNA can be proved only qualitatively. In the presence of DNA and NaCl in the subphase
Gromelski and Brezesinski
(no divalent cations), the DNA marker band at 970 cm-1 does not appear (Figure 2, left). Additionally, the intensities of the phosphate bands do not change significantly, compared to these of DMPE on water. In conclusion, DNA does not adsorb to the zwitterionic DMPE monolayer in the absence of divalent cations. Even though isotherm measurements show penetration of DNA into the lipid monolayer below 5 mN‚m-1, no DNA signals were detected at the interface below that pressure during the IRRAS measurements. Obviously, the amount of penetrated DNA is too small to be detected by IRRAS. The presence of the used divalent cations without DNA in the subphase did not have a significant influence on the DMPE monolayer behavior compared to that on water. In contrast to our results, Binder and Zscho¨rnig29 reported that the earth metal ions Mg2+ and Ca2+ shift the νas(PO2-) band of the zwitterionic POPC (1-palmitoyl-2-oleoyl-phosphatidylcholine) in the gel phase to higher wavenumbers compared to POPC on pure water, indicating a partial dehydration of the phosphate groups. They also observed that the ions Be2+, Cu2+, and Zn2+ shift the mean position of the νas(PO2-) band to smaller wavenumbers. They concluded that a relatively strong interaction of the nonesterified oxygens appears with these ions. The present results show almost no shift of the νas(PO2-) band of DMPE on 5 mM Mg2+ (Ca2+) + 1 mM Na+. Possibly the Na+ ions compensate for the dehydration effect of the divalent cations. Only at higher concentrations of divalent cations (50 mM) was a shift to higher wavenumbers observed, indicating a dehydration of the phosphate group due to interaction of the divalent cations with the phosphate moiety, which correlates with the IRRAS results of Flach et al.30 using DPPC on 5 mM CaCl2. IRRA spectra of DMPE on 0.1 mM calf thymus DNA in 10 mM citric buffer at pH 4 (no divalent cations) also showed adsorption of DNA to the interface, indicated by the presence of the characteristic band at 970 cm-1. The apparent pK of phosphatidylethanolamine is supposed to be e3.5.31 At pH 4, the DMPE headgroup is therefore positively charged, which enables an electrostatic interaction between DNA and DMPE even in the absence of divalent cations. The symmetric and antisymmetric CH2-valence vibrations give qualitative information about the conformation of the hydrocarbon chains. Figure 3 shows the change in band position of the symmetric CH2 stretching along the compression isotherm of DMPE on water and on DNA in the presence of the respective divalent cation. On water at low surface pressures (liquidexpanded phase), the DMPE acyl chains exhibit a high amount of gauche conformers (2855 cm-1). Compression to 40 mN‚m-1 shifts the band by ca. 5 cm-1 to smaller wavenumbers, indicating an all-trans conformation in the condensed state of the lipid monolayer. The shape of the resulting graph is similar to that of the π/A isotherm. In the phase transition region, all-trans and gauche conformations of the lipid chains are coexisting. At lower surface pressures, the presence of DNA and magnesium or calcium in the subphase causes a shift to smaller wavenumbers, indicating a higher fraction of all-trans conformation in the lipid chains. No remarkable difference is observed comparing DNA + magnesium and DNA + calcium in the subphase. Above 30 mN‚m-1, all examined systems show an all-trans conformation. 3.3. X-ray Reflectivity. Figure 4 shows X-ray reflectivity data of DMPE monolayers on different subphases at 20 mN‚m-1 and the corresponding fits using a two- or three-box model. On subphases containing DNA and divalent cations, the reflectivity (29) Binder, H.; Zscho¨rnig, O. Chem. Phys. Lipids 2002, 115, 39. (30) Flach, C. R.; Brauner, J. W.; Mendelsohn, R. Biophys. J. 1993, 65, 1994. (31) Papahadj, D. Biochim. Biophys. Acta 1968, 163, 240.
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Figure 2. (left) IRRA spectra of DMPE on water (A), on 1 mM NaCl + 5 mM MgCl2 (B), on 1 mM NaCl + 0.1 mM calf thymus DNA (C), and on 1 mM NaCl + 5 mM MgCl2 + 0.1 mM calf thymus DNA (D), for clarity curve D was shifted. (right) Subtracted IRRA spectra of DMPE on 1 mM NaCl + 5 mM MgCl2 (subtracted: DMPE on 1 mM NaCl) (A) and on 1 mM NaCl + 5 mM MgCl2 + 0.1 mM calf thymus DNA (subtracted: DMPE on 1 mM NaCl + 5 mM MgCl2) (B) and on 1 mM NaCl + 5 mM CaCl2 + 0.1 mM calf thymus DNA (subtracted: DMPE on 1 mM NaCl + 5 mM CaCl2) (C).
Figure 3. Symmetric CH2 valence vibrations at different surface pressures of DMPE on water (∆), on 1 mM NaCl + 5 mM MgCl2 + 0.1 mM calf thymus DNA (9), and on 1 mM NaCl + 5 mM CaCl2 + 0.1 mM calf thymus DNA (O) and π/A isotherm of DMPE on water (s).
Figure 4. Reflectivity R‚Qz4 as a function of Qz, the component of the momentum transfer perpendicular to the surface, of DMPE on 1 mM NaCl + 5 mM CaCl2 + 0.1 mM calf thymus DNA (A), on 1 mM NaCl + 5 mM MgCl2 + 0.1 mM calf thymus DNA (B), and on 1 mM NaCl + 0.1 mM calf thymus DNA (C) at 20 mN‚m-1. The curves are shifted for clarity.
data indicate an increased film thickness, compared to DMPE on water. Figure 5 shows the electron density profiles along the surface normal of the DMPE monolayers on the different subphases obtained with the Parratt program. Graph A represents a typical electron density profile of a lipid monolayer. It can be
Figure 5. Electron density F(z) obtained from the fitting parameters versus the thickness z of DMPE on 0.1 mM calf thymus DNA (A), on 5 mM MgCl2 + 0.1 mM calf thymus DNA (B), and on 5 mM CaCl2 + 0.1 mM calf thymus DNA (C) at 20 mN‚m-1.
divided into two slabs originating from the hydrocarbon chain region (17.8 Å) and the hydrophilic headgroup region (6.1 Å), which exhibits a higher electron density than the hydrophobic region. The values are in good agreement with data of DMPE on water.32 Therefore, the presence of DNA in the subphase does not change the electron density profile indicating again that DNA does not adsorb to the DMPE monolayer on water. The addition of divalent cations (magnesium or calcium) leads to clear changes in the reflectivity curves. An additional region of higher electron density, fitted by a third slab, appears. This slab indicates the presence of an adsorption layer close to the lipid headgroups with a thickness of ∼19 Å in the presence of Mg2+ and ∼18 Å in the presence of Ca2+. Combining the results of the IRRAS measurements with the performed reflectivity scans, it becomes clear that DNA adsorbs to the neutral DMPE monolayer only in the presence of divalent cations. 3.4. GIXD. X-ray diffraction measurements enable more quantitative and refined conclusions on ordered lipid structures and ordered adsorbed DNA layers. The experiments were performed at 20 °C. In Table 2, the structure data derived from the X-ray diffraction measurements of DMPE on different subphases are presented. Figure 6 shows selected contour plots of the corrected X-ray intensities as a function of the in-plane and out-of-plane scattering vector components Qxy and Qz of DMPE on subphases containing (32) Mo¨hwald, H. Annu. ReV. Phys. Chem. 1990, 41, 441.
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Table 2. Unit Cell Parameters a, b, and γ, Tilt Angle t, Projected Area Per Chain Axy, and Cross Sectional Area A0 of DMPE on Different Subphases π (mN m-l)
a (A)
12 21 29
4,92 4,89 4,89
10 20 25 30 40
4,92 4,89 4,87 4,86 4,84
10 20 30 40
DMPE on Calf Thymus DNA 4,91 5,02 117,8 22 4,87 4,93 118,7 15 4,90 4,88 120,1 8 4,84 4,84 120,0 0
10 20 25 30 40
Axy (Å)
A0 (Å2)
DMPE on Water 5,02 117,9 19 4,95 118,8 14 4,87 120,2 9
21,8 21,2 20,6
20,6 20,6 20,5
DMPE on CaCI2 5,04 117,7 20 4,96 118,8 15 4,91 119,4 11 4,86 120,0 0 4,84 120,0 0
22,0 21,2 20,8 20,4 20,3
20,7 20,4 20,5 20,4 20,3
21,8 21,0 20,6 20,4
20,4 20,5 20,5 20,4
DMPE on Calf Thymus DNA + CaCI2 4,91 5,02 117,9 20 21,8 4,87 4,93 119,2 12 20,9 4,90 4,88 120,2 8 20,6 4,85 4,85 120,0 0 20,4 4,84 4.84 120,0 0 20,3
20,7 20,5 20,6 20,4 20,3
b (A)
γ (°)
t (°)
calcium or calcium plus DNA at 10 and 40 mN‚m-1. On all subphases, the DMPE monolayers exhibit the same phase sequence, which is in correspondence with the results observed with magnesium, which were published recently.33 In the LC phase (10 mN‚m-1), three Bragg peaks are observed indicating an oblique lipid chain lattice. Upon further compression, the tilt angle decreases and the monolayer structure changes to orthorhombic packing of chains tilted in the nearest neighbor (NN) direction characterized by two Bragg peaks. The nondegenerated peak is located at zero Qz and the degenerated peak at Qz > 0. Such a phase is named L2.34 This phase transition is not seen in the isotherm. Further increase of the surface pressure leads to a kink in the isotherm, marking a second-order transition into a nontilted state. At surface pressures above the kink, only one Bragg peak at zero Qz is observed. The lipid chains are untilted and the monolayer possesses a hexagonal packing which is typical for the LS phase. For the second-order phase transition, one can assume that there is a linear dependence between the molecular area and the lateral pressure π.35 According to cos(t) ) A0/Axy, where A0 is the chain cross-sectional area and Axy the in-plane area of a chain, 1/cos(t) must be also linearly dependent on π. The extrapolation of 1/cos(t) vs π gives the transition pressure above which the chains are upright (t ) 0). For DMPE on water, on the saline subphases, and on DNA, the extrapolated pressure is ca. 32 mN‚m-1. The presence of calcium or magnesium ions and DNA shifts the value to 26.2 or 27.5 mN‚m-1, respectively. These calculated pressures agree well with the position of the kink in the isotherms. Figure 7 shows the tilt angles as a function of the surface pressure π. The surface pressures at t ) 0 are the extrapolated values. The presence of DNA and divalent cations (magnesium or calcium) in the subphase has a condensing effect on the DMPE monolayer in terms of decreased tilt angles compared to those of DMPE on saline subphases, only DNA or water. Comparing the effect of DNA in the presence of calcium or magnesium on the tilt angles, no significant differences were (33) Gromelski, S.; Brezesinski, G. Phys. Chem. Chem. Phys. 2004, 6, 5551. (34) Kaganer, V. M.; Mo¨hwald, H.; Dutta, P. ReV. Mod. Phys. 1999, 71, 779. (35) Brezesinski, G.; Kaganer, V. M.; Mo¨hwald, H.; Howes, P. B. J. Chem. Phys. 1998, 109, 2006.
Figure 6. Contour plots of the corrected X-ray intensities as function of the in-plane scattering vector component Qxy and the out-of-plane scattering vector component Qz for calf thymus DNA adsorbed to a DMPE monolayer in the presence of 5 mM CaCl2 (A and B) and 0.1 mM DNA + 5 mM CaCl2 (C and D) at 10 mN‚m-1 (left) and 40 mN‚m-1 (right).
Figure 7. Tilt angles of DMPE as a function of the lateral surface pressure on water (0), on 0.1 mM calf thymus DNA (9), on 5 mM MgCl2 (O), on 5 mM CaCl2 (b), on 5 mM MgCl2 + 0.1 mM calf thymus DNA (4) and on 5 mM CaCl2 + 0.1 mM calf thymus DNA (2). The lines are only to guide the eye.
observed. The tilting transition on DNA plus Ca2+ is shifted to slightly lower values compared with DMPE on DNA plus Mg2+. GIXD measurements at small 2θxy values show the appearance of an additional Bragg peak indicating an ordered adsorption of DNA to the DMPE monolayer. This additional Bragg peak at small Qxy values can be clearly seen in the contour plots presented in Figure 8. The interaxial distance between parallel DNA strands can be calculated by dDNA ) 2π/Qxy. Symietz et al.8 observed a decrease of dDNA by 22% upon compression of the positively charged TODAB monolayer on DNA solution from 10 to 40 mN‚m-1. Recent GIXD measurements of DMPE on DNA plus magnesium33 showed that compression from 10 to 40 mN‚m-1 leads to a decrease of dDNA from 46.2 to 42.7 Å (∼ 8%). The reproducibility of these values is rather bad. In the new experiments, dDNA in the presence of magnesium amounts to 38.6 Å on average, whereas no pressure dependence can be seen. It is now clear that dDNA strongly depends on the preparation of the DNA solution used in an experiment. Using Raman spectroscopy, Duguid et al.36 have demonstrated that the structural (36) Duguid, J.; Bloomfield, V. A.; Benevides, J.; Thomas, G. J. Biophys. J. 1993, 65, 1916.
DNA Condensation and Interaction
Figure 8. Contour plots (corrected X-ray intensities as function of the in-plane and out-of-plane scattering vector components Qxy and Qz) at small Qxy values of DMPE on 5 mM CaCl2 + 0.1 mM calf thymus DNA at 10 (A) and 40 mN‚m-1 (B).
perturbations induced by divalent cations are much greater for >23 kb genomic calf-thymus DNA than for 160 bp mononucleosomal DNA fragments. In the present experiments, calf thymus DNA solution was stirred overnight in a refrigerator. Gel electrophoresis shows that stirring results in fragments of >21 kb. This means that also very large DNA strands can be present in the solution. Obviously, the ratio of large DNA strands to small fragments differs for each DNA preparation, probably due to different geometries of the magnet stirrer or different stirring speeds. As large DNA strands are more strongly influenced by divalent cations, the experimentally observed dDNA values also differ for each DNA solution. Measurements of different zwitterionic lipids and mixtures on the same DNA solution plus Mg2+ show the same d-spacings (not shown here). Compression causes almost no change in the interaxial DNA spacing. The same behavior was observed for DMPE on DNA plus Ca2+: Compression from 10 to 40 mN‚m-1 (Table 3) does not change the average value of ca. 31.5 Å (Figure 9). These results show clearly that the observed dDNA value depends both on the stirring conditions and the kind of divalent cation used, rather than on the monolayer charge density or the kind of zwitterionic lipid headgroup. Comparing dDNA for the two divalent cations used in this work, one observes that the values are ca. 7 Å smaller in the presence of calcium. Possibly the different radii of the hydrated ions are responsible for this effect. Magnesium has the smaller ion radius (Mg2+, 0. 78 Å; Ca2+, 1.06 Å) but the bigger hydrodynamic radius (Mg2+, 4.31 Å; Ca2+, 4.12 Å). The effect of ion size on dDNA is reproducible and indicates that there is not just a simple bridging of DNA strands by divalent cations but a more complex mechanism. Divalent cations are known to reduce the effective charge density of DNA in solution.37 Using single molecule AFM, Cai and co-workers38 observed that the Mg2+ ions are likely to bridge the DNA intrastrand interaction. It was also found that divalent earth metal cations interact only weakly with the bases but more strongly with the phosphates in the following order: Ca2+ > Mg2+.36 These results indicate that divalent cations have a condensing effect on the DNA. This effect is larger in the presence of calcium due to its smaller hydrodynamic radius, which leads finally to smaller interaxial DNA spacings compared with magnesium. No Bragg peak arising from ordered adsorption of DNA was observed for DMPE on 0.1 mM calf thymus DNA + 10 mM citric buffer at pH 4 (no divalent cations), even though IRRAS (37) Teeters, M. A.; Root, T. W.; Lightfoot, E. N. J. Chromatogr. A 2004, 1036, 73. (38) Cai, X. E.; Yang, J. Biophys. J. 2002, 82, 357.
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showed an adsorption of DNA to the interface. This result confirms that divalent cations enable a side-by-side association of DNA fragments, causing an ordered adsorption structure. 3.5. BAM. BAM was used to identify the shape of condensed phase domains appearing in the first-order phase transition region between LE and LC. In Figure 10, BAM images of DMPE (7 mN‚m-1) on water (A), on DNA (B), on DNA plus magnesium (C), and on DNA plus calcium (D) solutions are presented. The comparison of images (A) and (B) shows that, in the presence of DNA, the domains are slightly smaller and the boundary is rough. One should note that the presence of only NaCl in the subphase has a very similar effect - the domains are slightly smaller compared to those on pure water. Additional experiments (not shown) exposed that MgCl2 leads to the appearance of a larger number of smaller domains, which are more round. These observations were even enhanced for DMPE on CaCl2 and might be explained by a stronger influence of the line tension and reduced dipolar repulsion. The presence of divalent cations leads to a lowering of the nucleation energy necessary for the formation of a critical nucleus. As a consequence, more domains are formed. If divalent cations are present together with NaCl, the domain shape resembled again that of DMPE on the NaCl solution. In contrast, the presence of divalent cations plus DNA and NaCl caused an obvious change in the domain shape: The domains are smaller, fuzzy, more frayed, and dendrite-like. Penetrated DNA can be seen as an impurity in the DMPE monolayer. Such impurities can impede the growth and are removed fast for a rough boundary which therefore is favored. This way they can cause a diffusion-limited aggregation process which may lead to fractal structures,39 observed in Figure 10, panels C and D. The presence of DNA and magnesium (calcium) causes a change not only in domain shape but also in size. Bright aggregates were additionally visible at all pressures investigated. Representative examples are presented in Figure 11. These aggregates exhibit mainly (curved) fiber-like structures, shown in picture (B), but also accumulations of more broad patches which were mainly oriented in the same direction (A) have been observed. Obviously these patches and fibres were present just below the monolayer, as the appearance and growth of DMPE domains was observed on top of these structures. Looking at the results from IRRAS, X-ray reflectivity, and GIXD, one can conclude that these bright parts consist of DNA, which is adsorbed to the monolayer in the presence of the two examined divalent cations. No pronounced differences could be found comparing the DNACa2+ and the DNA-Mg2+ systems, on one hand due to the diversity of the adsorbed patches and fibres and on the other hand due to the irregular branched and fuzzy shapes of the domains.
4. Discussion and Conclusions The in-plane molecular area Axy for DMPE on DNA + Ca2+ observed by GIXD is similar to the molecular area given by the isotherm of DMPE on water (Figure 12) but is smaller than that measured by the compression isotherm of DMPE on DNA + Ca2+. This can be easily explained by the fact that GIXD detects only ordered lipid structures. The disagreement between in-plane lipid molecular areas determined by GIXD and isotherm measurements shows that the monolayer is heterogeneous at lower surface pressures due to penetrated DNA into the headgroup region of the DMPE monolayer. In conclusion, the shift in the isotherm at lower pressures to larger areas per lipid molecule is the result of DNA penetration into the DMPE monolayer. The kink at ∼28 mN‚m-1 is due to a second-order tilting transition. (39) Miller, A.; Mo¨hwald, H. J. Chem. Phys. 1987, 86, 4258.
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Gromelski and Brezesinski
Table 3. In-Plane Scattering Vector Component Qxy, Full Width at Half Maximum fwhm, Interaxial Spacing dDNA, and Positional Correlation Length ξ at Different Surface Pressures DMPE on DNA + magnesium
DMPE on DNA + calcium
π (mN m-l)
Qxy (Å)
fwhm (Å-1)
dDNA (Å)
ζ (Å)
Qxy (Å)
fwhm (Å-1)
dDNA (Å)
ζ (Å)
10 20 25 30 40
0.166 0.161 0.161 0.162 0.163
0.041 0.032 0.040 0.045 0.039
37.7 39.0 38.9 38.7 38.6
140 181 140 126 145
0.198 0.199 0.198 0.201 0.201
0.022 0.023 0.030 0.022 0.022
31.7 31.5 31.7 31.2 31.2
276 266 195 277 271
Figure 11. Two examples of visualization of DNA aggregates by BAM: DMPE on 5 mM MgCl2 + 0.1 mM calf thymus DNA at 5 mN‚m-1 (A) and on 5 mM CaCl2 + 0.1 mM calf thymus DNA at 30 mN‚m-1 (B). Figure 9. Interaxial d spacings of ordered adsorbed DNA strands as a function of the lateral surface pressure π of DMPE on 5 mM MgCl2 + 0.1 mM calf thymus DNA (A) and on 5 mM CaCl2 + 0.1 mM calf thymus DNA (B).
Figure 12. π/A isotherms of DMPE on water (- - -) and on 5 mM CaCl2 + 0.1 mM calf thymus DNA (s) and the corresponding molecular in-plane areas Axy of DMPE on 5 mM CaCl2 + 0.1 mM calf thymus DNA (b) determined by GIXD.
Figure 10. BAM images of DMPE on water (A), on 0.1 mM calf thymus DNA (B), on 5 mM MgCl2 + 0.1 mM calf thymus DNA (C), and on 5 mM CaCl2 + 0.1 mM calf thymus DNA (D) at 7 mN‚m-1 and 20 °C.
From the IRRA spectra, one can conclude that the divalent ions link the negative phosphate moieties of the lipid headgroups and turn the zwitterionic monolayer cationic. The binding of Mg2+ and Ca2+ to the phosphate moieties of the DNA decreases the negative charge of the sugar-phosphate backbone40 and condenses the DNA.36,41 It seems reasonable that the DNA adsorption is mainly driven by electrostatic interactions between the remaining negatively charged regions in the DNA and the effectively positively charged phospholipid headgroup region. IRRAS and GIXD confirm the following model: The presence of DNA without divalent cations in the subphase does not cause any adsorption of DNA to the zwitterionic DMPE monolayer. (40) Sun, X. G.; Cao, E. H.; Zhang, X. Y.; Liu, D. G.; Bai, C. L. Inorg. Chem. Commun. 2002, 5, 181. (41) Bloomfield, V. A. Biopolymers 1997, 44, 269.
DNA adsorption appears only when divalent cations and DNA are together present in the subphase. The IRRA spectra of DMPE on water, on DNA, and on saline subphases are overall the same. In contrast, the presence of DNA and divalent cations in the subphase leads to the appearance of the band at 970 cm-1, which is an indicator for the presence of sugar moieties of the DNA backbone at the interface. X-ray reflectivity measurements proved the presence of an additional (DNA) adsorption layer with a thickness of approximately 19 Å at the interface. Referring to the diameter of a DNA strand in the B-form (20 Å), the X-ray reflectivity measurements demonstrate that the adsorption layer consists of one DNA layer. The thickness of this layer is slightly dependent on the size of the divalent ion, which condenses the DNA. The distance between ordered DNA strands in the adsorption layer is much more effected by the size of the divalent ions. The size difference between hydrated Mg2+ and Ca2+ ions influences strongly the interactions between DNA strands. A small change of the ion size just in the range of the typical values of the hydrated ion diameters may cause a transition from a purely repulsive interaction to an attractive one with a spontaneous assembly of DNA into an ordered phase42 leading to a drastic decrease in the interaxial DNA spacing dDNA. GIXD and IRRAS measurements of DMPE on calf thymus DNA at pH 4 (no divalent cations) showed that the DNA is electrostatically attracted by the mainly positively charged DMPE monolayer, but the DNA adsorption layer does not show any ordering. This result confirms (42) Lyubartsev, A. P.; Nordenskiold, L. J. Phys. Chem. 1995, 99, 10373.
DNA Condensation and Interaction
Figure 13. Schematic representation of the DNA-lipid interaction.
that divalent cations enable a side-by-side association of DNA fragments, causing an ordered structure. It has been proposed that a DNA molecule in solution spontaneously forms a loop with two sequence-separated sections in close contact. A condensing agent (like multivalent ions) binds to this contact and stabilizes the loop.41 In this way, DNA can form ordered structures observed by GIXD (Figure 8). Brewster angle microscopy images show significant differences between the examined subphases. DMPE domains on a subphase containing DNA and NaCl are similar to those observed on water but slightly smaller. They have clear but irregular flower shapes. Although there was no significant change in the DMPE monolayer structure, MgCl2 causes smaller domains which were more round. This effect is even bigger in the presence of CaCl2. If NaCl was added to the solution of divalent ions, the domain shape resembled again that on the NaCl solution, but the domains were slightly smaller. Surprisingly, it seems that monovalent cations can compete with divalent cations for interaction with lipid headgroups and have on this way some influence on the domain size. The coexistence of divalent cations and DNA in the subphase strongly changed the DMPE domain shape and size: The domains are smaller, more branched, and fuzzy. This indicates that dipolar interactions between lipid molecules and the line tension of the domains are changed. The line tension depends on the type of coexisting phases. Penetrated DNA can act as an impurity leading to fractal-like structures as observed in Figure 10, panels A and B. Figure 13 presents a schematic model of the DNA-lipid interaction. Divalent cations bridge the negative parts of the zwitterionic phospholipid headgroup. Therefore, the monolayer becomes effectively positively charged. If the cation is located between the lipid headgroups, one should expect a change in the area per lipid molecule and therefore a change in the tilt angle compared to DMPE on water. This effect is not observed. Possibly the bridging of DMPE molecules by the divalent cations is compensated by a change in the headgroup orientation. Therefore, the lipid headgroups occupy effectively the same area as on
Langmuir, Vol. 22, No. 14, 2006 6301
water indicated by the same tilt angle of the aliphatic chains observed by GIXD. Divalent cations also interact with the negative charges of the DNA phosphate moiety. In this way, they condense the DNA and lead to an ordered alignment of the DNA strands. The interaxial distance dDNA between the DNA strands depends on the kind of divalent cation used. Calcium decreases dDNA more than magnesium, due to its smaller hydrodynamic radius. Nevertheless, not all charges are screened by the divalent cations. Such condensed DNA aggregates remain partially negatively charged. They can now interact either via divalent cations with the lipid phosphate group or directly with the positively charged ethanolamine group of DMPE when the lipid phosphate groups are bridged by divalent cations. Since dDNA is not influenced by compression, we believe that there is no direct coupling between oppositely charged groups as in the case of a cationic monolayer. In cationic lipid-DNA complexes, a strong dependence of dDNA on the lateral pressure was observed.6 Using single molecule AFM, Cai et al.38 observed that the Mg2+ ions are likely to bridge the DNA intrastrand interaction. Therefore, it renders a long DNA molecule more likely to adopt a conformation facilitating interactions with an attractive surface, rather than directly bridging the DNA to the substrate. The DNA is electrostatically attracted by the positively charged monolayer. This leads to a change of the DMPE headgroup orientation. This change reduces the effective headgroup area which leads to a decrease of the tilt angle of the aliphatic chains. Since the compression of the lipid layer has no influence on the dDNA values, we assume that adsorbed DNA forms a kind of independent sublayer underneath the lipid monolayer. These observations confirm the results obtained earlier that concluded that calcium and the zwitterionic lipid DPPC form a bridging unit with “cationic” properties with which the negatively charged DNA interacts.43-45 Acknowledgment. We thank Prof. Dr. Ewa Rogalska (Universite´ Henri Poincare´ Nancy 1) for the opportunity to utilize the KSV Optrel BAM 300. The help of Kristian Kjaer with setting up the X-ray experiment is gratefully acknowledged. We thank HASYLAB at DESY, Hamburg, Germany, for beam time and providing excellent facilities and support. This work was supported by the VolkswagenStiftung within the framework of the “Complex materials” program and the DAAD (PROCOPE). LA0531796 (43) Kharakoz, D. P.; Khusainova, R. S.; Gorelova, A. V.; Dawson, K. A. FEBS Lett. 1999, 446, 27. (44) McManus, J. J.; Ra¨dler, J. O.; Dawson, K. A. J. Phys. Chem. B 2003, 107, 9869. (45) McManus, J. J.; Ra¨dler, J. O.; Dawson, K. A. Langmuir 2003, 19, 9630.
