DOPC + OTAB + DNA Complexes – Effect of Ionic Strength and Surface Charge Density on DNA Condensation

Synthetic neutral phospholipid DOPC was purchased from Avanti Polar Lipids (Alabaster, Alabama, USA) and used without further purification. Herring testes DNA (sodium salt), highly polymerized (average M_r of nucleotides = 308), was obtained from Sigma Aldrich (St. Louis, Missouri, USA). EtBr was purchased from Merck (Darmstadt, Germany). Cationic surfactant OTAB was from Sigma-Aldrich Chemie (Steinheim, Germany). NaCl of analytical purity was purchased from Lachema (Brno, Czech Republic). Chemical formulae of used chemicals are shown in Fig. 1.

Figure 1.

Structures of DOPC, OTAB and EtBr.

DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine, OTAB: octadecyltrimethylammonium bromide, EtBr: 3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide

EtBr is a cationic fluorescent probe that intercalates between the bases of uncondensed DNA. In solution, EtBr molecules transition from the excited state to the ground state mainly through nonradiative processes that involve the transfer of a proton from the amino group to the solvent molecules. However, when EtBr is intercalated, it is isolated from the solvent, blocking the nonradiative decay pathway. This leads to a dramatic increase in the EtBr fluorescence intensity upon intercalation (Izumrudov et al., 2002).

We performed a set of measurements at 15 different DNA/EtBr mole ratios to determine the optimal ratio. The effect of ionic strength on this ratio was verified for five hydration media in the range of 5–150 mmol l⁻¹ NaCl. A linear increase in emission fluorescence intensity was recorded approximately up to a DNA/EtBr mole ratio of 4–6 depending on the ionic strength (Fig. 2a). In the linear part, the binding sites of the DNA for EtBr are fully saturated. At higher mole ratios, the dependences are no longer linear due to excess DNA.

Figure 2.

(a) Dependences of the emission fluorescence intensity I of complex DNA + EtBr on DNA/EtBr mole ratio at different NaCl ionic strengths. Error bars are within the size of the symbols. Lines represent the best linear fits of respective experimental data. (b) Dependences of the fluorescence intensity I of DNA/EtBr = 5:1 mol mol⁻¹ complex (▪) on the ionic strength compared to pure EtBr (). Curves are a guide to the eyes only.

For further measurements, we chose a constant mole ratio DNA/EtBr = 5 for all studied ionic strengths. This finding is consistent with the results of other authors (Barreleiro & Lindman, 2003; Eastman et al., 1997; Lengyel et al., 2011).

The ionic strength significantly influences the fluorescence intensity of EtBr. We detected a nonlinear decrease in fluorescence intensity with increasing ionic strength (Fig. 2b). The DNA + EtBr complex had up to 15.3-fold higher fluorescence than the probe alone in 5 mM NaCl solution, but in 150 mM NaCl, only a 3.8-fold increase in fluorescence intensity was detected. This is mainly caused by the shielding of electrostatic interactions between DNA and EtBr by the solution at high ionic strength, resulting in reduced affinity of EtBr for DNA-binding sites (Vardevanyan et al., 2001, 2016). Ionic strength also influences the conformation of DNA, stabilizing the double helix structure and preventing unwinding. These changes are making intercalation energetically less favorable as it requires significant changes in the double-helix structure (Kas'yanenko, 2006; Manning, 2015; Zipper et al., 2004).

When DNA interacts with cationic liposomes, cationic polymers, or multivalent cations, it leads to the formation of complexes in which the opposite charges are canceled out. The driving force for the formation of the complexes is the high entropic gain from the release of small counterion during the mutual neutralization of the opposing charges (Flock et al., 1996; Rädler et al., 1997). Cancelation of more than 90% of the negative charge of the DNA phosphate groups leads to condensation of DNA, during which DNA changes its conformation from coil to tightly packed toroidal or globule particles (Bloomfield, 1991). Condensed DNA is no longer accessible to intercalating dyes such as EtBr, which will lead to the displacement of intercalated EtBr molecules and a subsequent drop in fluorescence intensity (Eastman et al., 1997; Izumrudov et al., 2002). This enables us to use fluorescence spectroscopy to evaluate the ability of DOPC + OTAB liposomes to form complexes with DNA.

In our experiment, we modulated the surface charge density of liposomes through the OTAB/DOPC mole ratio and the ionic strength of the solution by the addition of NaCl. At first, we determined the ability of DOPC + OTAB liposomes to condense DNA in 5 mM NaCl (Fig. 3a). For most of the OTAB/DOPC compositions, the increase in the concentration of cationic liposomes expressed as the charge ratio OTAB/DNA leads to a significant drop in the fluorescence intensity, reflecting the extent of DNA condensation. However, the liposomes prepared at OTAB/DOPC = 0.2 mol mol⁻¹ seem to be ineffective for DNA condensation. We detected only ~13% of condensed DNA at the highest studied OTAB/DNA charge ratio. The inability of liposomes with OTAB/DOPC = 0.2 mol mol⁻¹ to efficiently condense DNA even at a high OTAB/DNA charge ratio demonstrates that the surface charge density of cationic liposomes is an important factor for successful DNA condensation. With a small increase in the OTAB/DOPC ratio to 0.3 mol mol⁻¹, the ability of cationic liposomes is significantly enhanced, reaching almost 60% at a OTAB/DNA charge ratio of 10:1. However, only liposomes with OTAB/DOPC = 1 mol mol⁻¹ were able to exceed 90% of condensed DNA.

Figure 3.

(a) Dependence of normalized fluorescence intensity I_norm of DOPC + OTAB + DNA + EtBr complexes on the OTAB/DNA charge ratio at different OTAB/DOPC mole ratios in 5 mmol l⁻¹ NaCl. The points are connected to guide the eyes. Error bars are smaller than the symbol size. (b) Dependence of normalized fluorescence intensity I_norm of DOPC + OTAB + DNA + EtBr complexes on ionic strength at a charge ratio of OTAB/DNA = 10 and at different OTAB/DOPC mole ratios.

Our results agree well with those of the published literature. An increase in surface charge density generally leads to better efficiency in condensing the DNA up to a certain point at which the maximum efficiency is reached. In addition, at least the critical surface charge density of the cationic liposomes is required to condense DNA. This critical surface charge density can vary depending on the lipids used and the ionic strength of the solution (Ryhänen et al., 2003; Wang et al., 2001).

To evaluate the effect of ionic strength on DNA condensation by DOPC + OTAB liposomes, we chose the maximal tested concentration of liposomes with OTAB/DNA charge ratio = 10. The dependence of the normalized fluorescence intensity I_norm of DOPC + OTAB + DNA + EtBr complexes on the ionic strength at the studied OTAB/DOPC mole ratios is shown in Fig. 3b. The I_norm increases with increasing ionic strength for all studied OTAB/DOPC mole ratios. The minimal values of fluorescence intensity were recorded for the highest mole ratio, OTAB/DOPC = 1 mol mol⁻¹, in all studied NaCl solutions. At low ionic strength (5 mM NaCl), DNA condensation reached 90%, while only ~55% of DNA was condensed in 150 mM NaCl (I_norm = 45.6%). For the least efficient OTAB/DOPC mole ratio of 0.2, we even recorded I_norm over 100% in 150 mM NaCl, indicating that the addition of liposomes with low surface charge density in physiological ionic strength caused a conformational change in DNA that increased its accessibility for EtBr.

The general trend of the ionic strength reducing the ability of cationic liposomes or polymers to condense DNA was observed through the exclusion of intercalated fluorescent dyes and is well documented in the literature (Coelho et al., 2021; Eastman et al., 1997; Even-Chen et al., 2012; Hirsch-Lerner et al., 2005; Hubčík et al., 2014; Jing et al., 2004). It is attributed to the screening of electrostatic interactions between DNA and cationic liposomes by the small ions in the solution.

A high concentration of small ions in the solution also reduces the entropy gain from the counterion release during the formation of the complexes (Harries et al., 2013), which reduces the condensation efficiency.

Other techniques such as titration calorimetry also show suppression of DNA condensation by cationic liposomes in physiological ionic strength compared to 5 mM NaCl. In the work of Pullmannová et al. (2012), titration calorimetry was used to assess the endpoint of the condensation process with cationic liposomes containing DOPC and the cationic surfactant ethane-1,2-diylbis(dodecyldimethylammonium bromide) (C2GS12) in 5 and 150 mM NaCl. While at low ionic strength, the condensation endpoint was reached at the charge ratio of 2:1, at physiological ionic strength, it was reached at the charge ratio of 9:1. The molar enthalpy per mole of DNA base pairs for DNA condensation was reduced from 15.1 kJ mol⁻¹ in 5 mM NaCl to 4.7 kJ mol⁻¹ in 150 mM NaCl. This indicates that even with the higher required ratio of cationic lipid to DNA, the condensation was still more than three times less efficient in 150 mM NaCl. This aligns with our systems with the mole ratio OTAB/DOPC ≤0.5. Liposomes with a higher surface charge density were less affected by the increase in ionic strength.

At the investigated temperatures, fully hydrated DOPC forms a well-defined liquid-crystalline lamellar L_a phase (Nagle & Tristram-Nagle, 2000; Uhríková et al., 2005). In the SAXS pattern (Fig. 4), we observed two reflections corresponding to regularly packed bilayers. The repeat distance of the lipid bilayer stacking, including the thickness of the bilayer and the water layer, was determined from the position of the first lamellar peak L(1) according to Eq. 2.

Figure 4.

The SAXS patterns of DOPC in 5 and 150 mM of NaCl at 20 °C (a) and 50 °C (b). Intensity is in a logarithmic scale.

We found the repeat distance d = 6.28 ± 0.01 nm in 5 mM NaCl and d = 6.40 ± 0.01 nm in 150 mM NaCl at 20 °C (Fig. 4a), which is in agreement with the literature (Nagle & Tristram-Nagle, 2000; Pullmannová et al., 2012; Uhríková et al., 2005; Želinská et al., 2020). After heating to 50 °C (Fig. 4b), an increase in repeat distance was observed, which reached d = 6.34 ± 0.01 nm in 5 mM NaCl and d = 6.49 ± 0.01 nm in 150 mM NaCl. The water layer thickness increases more rapidly with increasing temperature, compared to the decrease of bilayer thickness, which ultimately manifests itself in increased repeat distance (Nagle & Tristram-Nagle, 2000).

It was reported that the OTAB forms complexes with DNA exhibiting a hexagonal structure (Kawashima et al., 2006). The use of DOPC as a helper lipid in DOPC + OTAB + DNA complexes preserve the lamellar phase at both tested OTAB/DOPC mole ratios (Fig. 5). However, the peaks in the SAXS region are broad, which indicates a significant decrease in the positional ordering of the stacked bilayers. The lamellar repeat distance d for the condensed lamellar phase of complexes was found in the range 6.21–6.51 nm for OTAB/DOPC = 1 mol mol⁻¹ and 6.48–6.83 nm for OTAB/DOPC = 0.3 mol mol⁻¹ (Fig. 6). The peaks that are typical for regularly ordered condensed DNA in between the lipid bilayers are visible only at low ionic strengths. At the mole ratio OTAB/DOPC = 1, the peaks indicating evidence of DNA–DNA organization were detected at q = 1.40 nm⁻¹ (5 mM NaCl) and q = 1.37 nm⁻¹ (15 mM NaCl). The resulting interhelical DNA–DNA distance was d_DNA = 4.5 ± 0.1 nm at 5 mM NaCl and 4.6 ± 0.1 nm at 15 mM NaCl. At the mole ratio OTAB/DOPC = 0.3, we determined the DNA–DNA distance d_DNA = 5.8 ± 0.1 nm. The peak of DNA is not distinguishable at higher ionic strengths, which means that DNA is weakly ordered between the lipid lamellae. The weak ordering of DNA at high ionic strength is in agreement with the condensation experiments which showed a large decrease in the fraction of condensed DNA with increased NaCl concentration.

Figure 5.

The SAXS patterns of DOPC + OTAB + DNA complexes at different ionic strengths and mole ratios OTAB/DOPC = 1 (a) and 0.3 (b) at 20 °C. For illustration, the data points are simulated with the best Lorentzian fit curves. Intensity is in a logarithmic scale.

Figure 6.

Dependence of the repeat distance d of the lipid bilayers stacking in the DOPC + OTAB + DNA system on ionic strength at the mole ratios OTAB/DOPC = 0.3 (dots) and 1 (circles) at 20 °C (blue line) and 50 °C (red line).

Another alternative is that a DNA peak could be overlapped with an L(1) peak. In cases where the two signals are close in q-space, their individual features could merge into a single, broadened peak that might be misinterpreted or simply be less distinguishable. High salt concentrations can lead to a more heterogeneous distribution of DNA within the lipid complexes. This heterogeneity could cause the DNA correlation peak to broaden significantly, reducing its intensity below the detection limit, even though DNA is still associated with the complexes.

The absence of DNA reflection in the SAXS region of diffractograms was also reported in studies of the structure of DOPC–DNA aggregates formed in the presence of calcium and magnesium ions (Francescangeli et al., 2003; Uhríková et al., 2005). The weak ordering of the condensed fluid lamellar phase L_α^c suggests that the interaction between DNA and cationic surfactant is weak. This could be an advantage during the release of DNA, after endocytosis, into the cell medium (Mel'nikova & Lindman, 2000).

The effect of the mole ratio OTAB/DOPC and the ionic strength on the repeat distance d in the lipid bilayer stacking of the DOPC + OTAB + DNA system at 20 °C and 50 °C is shown in Fig. 6. At the higher surface charge of the bilayer (OTAB/DOPC = 1 mol mol⁻¹), the repeat distance is systematically lower by ~0.25 nm, compared to OTAB/DOPC = 0.3 mol mol⁻¹, as a consequence of stronger electrostatic interaction between DNA and OTAB. With increasing ionic strength after a small drop at 15 mM NaCl, we observed a nonlinear increase of d for both OTAB/DOPC mole ratios and both temperatures, which suggests that the higher ionic strength screens the interaction between DNA and OTAB. As a result of the screening, the attractive force between DNA and positively charged lipid membranes that pulls the lipid lamellae closer together is weakened and the distance between the membranes is increased.

At 50 °C, we observed similar dependences; however, the d-values were systematically lower. This indicates that the temperature-induced decrease of the thickness of the lipid bilayer prevails against the diffusion of water molecules from the bulk water phase in between the lipid bilayers. The same effect was also observed in the lipoplexes with DOPC + Ca²⁺ and dilauroylphosphatidylcholine + cationic gemini surfactant (Uhríková et al., 2002, 2005).

The amount of bound DNA was verified by UV/Vis spectrometry in the supernatant after centrifugation of samples used for SAXS measurements. Surprisingly, free DNA was not detected in the supernatant (data not shown). This means that almost all DNA is bound in the lipoplexes, despite the peaks for DNA not being visible on the diffractograms and the condensation experiments showing only 55% of condensed DNA in 150 mM NaCl for the mole ratio OTAB/DOPC = 1 and less than 20% of condensed DNA for mole ratio OTAB/DOPC = 0.3. Thus, even in complexes with the majority of uncondensed DNA, almost all DNA is associated with the lipoplexes.

The apparent discrepancy between fluorescence spectrometry and UV/Vis determination of unbound DNA in the supernatant can be explained by the fact that DNA does not need to be fully condensed to be effectively removed from the supernatant. As the condensation of DNA requires 90% of its negative charge to be canceled out by the cationic lipid, a large fraction of the DNA can be associated in lipoplexes but is still partially accessible for EtBr. The EtBr assay is reliable for assessing condensation, but must be interpreted carefully alongside other data, as it does not directly measure binding efficiency.

A similar observation was also reported in the work of Even-Chen et al. (2012). The authors determined condensation of DNA below 50% in lipoplexes prepared in 150 mM NaCl, however, less than 5% of free DNA in the solution detected by UV/Vis spectroscopy in the supernatant and also by agarose gel electrophoresis.