DNA packing in stable lipid complexes designed for gene transfer imitates DNA compaction in bacteriophage

Abstract

The structure of complexes made from DNA and suitable lipids (lipoplex, Lx) was examined by cryo-electron microscopy (cryoEM). We observed a distinct concentric ring-like pattern with striated shells when using plasmid DNA. These spherical multilamellar particles have a mean diameter of 254 nm with repetitive spacing of 7.5 nm with striation of 5.3 nm width. Small angle x-ray scattering revealed repetitive ordering of 6.9 nm, suggesting a lamellar structure containing at least 12 layers. This concentric and lamellar structure with different packing regimes also was observed by cryoEM when using linear double-stranded DNA, single-stranded DNA, and oligodeoxynucleotides. DNA chains could be visualized in DNA/lipid complexes. Such specific supramolecular organization is the result of thermodynamic forces, which cause compaction to occur through concentric winding of DNA in a liquid crystalline phase. CryoEM examination of T4 phage DNA packed either in T4 capsides or in lipidic particles showed similar patterns. Small angle x-ray scattering suggested an hexagonal phase in Lx-T4 DNA. Our results indicate that both lamellar and hexagonal phases may coexist in the same Lx preparation or particle and that transition between both phases may depend on equilibrium influenced by type and length of the DNA used.

Synthetic gene delivery systems containing cationic lipids are attractive tools for the application of gene therapy. The design of plasmid DNA-cationic lipid complexes or lipoplexes (Lxs) (1) must overcome several obstacles, which exist from Lx administration to the production of a therapeutic molecule in targeted cells (2, 3). Because plasmid DNA (pDNA) is a large and highly biodegradable molecule, it must be protected and condensed in the delivery particles while outside of targeted cells, and then it must be decondensed inside cells in a functional form. The reversible DNA condensation process, a critical feature for Lx efficacy, generally is considered as a fundamental feature of the replicative cycles of organisms ranging from viruses to higher eukaryotes: the specificities of this intriguing process are still to be established (4–6). In vitro DNA condensation with the use of inorganic and organic compound materials under various conditions has been described (5–10). These studies have revealed that the structure of Lx appears to be crucial for their efficacy, and the investigation on DNA organization in Lx would improve such technology.

The experimental conditions by which Lx complexes are formulated needs to be precise to obtain a high efficacy of gene transfer, which is especially true for i.v. gene delivery, where Lx structure is critical (3, 11–15). We have demonstrated that the stability of Lx is greatly influenced by its structure (16), and the efficacy of gene transfer may be maintained up to 5 months or more after Lx preparation (data not shown). Definitive conclusions regarding Lx structures cannot be drawn since DNA has never, thus far, been observed in Lx particles. In this report, the use of cryo-electron microscopy (cryoEM) has enabled us to visualize DNA macromolecules in the Lx structure. This approach combined with other analytical methods has provided critical insight into the phenomena of DNA packaging in Lx.

Materials and Methods

Nucleic Acids.

pDNA consisted of the pBKd1RSV-luc construct (10,400 bp) previously designed and used as an episomal/reporter expression vector containing the luciferase gene (11). Linear single-stranded DNA (M13mp8, 7,229 bases) was derived from M13 bacteriophage. Linear double-stranded DNA (M13mp8; 7,229 bp) is the intracellular replicative form of the M13mp8 phage DNA produced by chronic infection in Escherichia coli. M13mp8-derived DNA contained a multiple cloning site within a portion of the Lac-z gene (Sigma). Oligodeoxynucleotides (ODN) used were 27 bases long (XbaI, Promega). T4 phage DNA (169,372 bp) was purchased from Sigma.

Lx Preparation.

Small unilamellar vesicles (SUV) were formed by mixing 1 mg of dioctadecylamidoglycylspermidine (Biosepra, Paris), 1 mg dioleoyl phosphatidylethanolamine, and 0.5 mg cardiolipin (Sigma), in ethanol (20 μl/mg lipid) and subsequently by adding excess pure water (6.25 mg lipid/ml, final). Formation of Lx was carried out by mixing SUV suspension with DNA at a 7.8 lipid/DNA weight ratio (0.16 mg/ml DNA, final concentration in water). The zeta potential of Lx was −32.3 mV as measured with a Zetasizer 3000 (Malvern Instruments, Paris), indicating a slight anionic global surface charge. No salt was added to Lx preparation, and the osmolarity was 420 mosm. Lx were stored at 4°C and used within 3 months after preparation.

CryoEM.

For cryoEM experiments, the various formulations were diluted to approximately 1 mg/ml concentration. A drop is applied onto a holey carbon film, which was rendered hydrophilic by a glow discharge. The excess of liquid was blotted by a piece of filter paper and the samples were plunged into liquid ethane cooled with liquid nitrogen (17). The grid then was transferred in a Gatan 626 cryoholder and observed under low-dose conditions in a Philips CM12 microscope equipped with an additional anticontamination device. SO163 films were developed in D19 full strength in 12 min.

Small Angle X-Ray Scattering (SAXS).

X-ray patterns were recorded at station D43 at the Laboratoire pour l’Utilisation du Rayonnement Electromagnetique (Orsay, France) by using the synchrotron source. The radially averaged scattering intensity I(Q) was plotted as a function of the scattering wave vector Q = 4π/λ sin θ where λ is the wavelength of the incident x-ray beam (λ = 1.445 Å) and 2 θ the scattering angle.

Dynamic Light Scattering.

Particle size was measured by dynamic light scattering (Zetasizer 3000, Malvern Instruments). Measurements were carried out in automatic mode with the following parameters: scattering angle, 90°; medium viscosity, 0.890 cP; temperature, 25°C, and refracting index, 1.33. Width (width at half peak height) is indicative of the homogeneity of size distribution.

Results and Discussion

Lx complexes are formed by adding DNA to cationic SUV. SUV are carefully prepared to obtain a highly homogeneous population in regard to size (mean size: 198 nm, and width: 54 nm) as measured by dynamic light scattering size analysis and structure (>95% unilamellar vesicles) as observed by cryoEM (data not shown). The bilayer measured 5.0 nm. The addition of pDNA (pBKd1 RSV-luc; 10.4 kbp) to highly homogenous SUV, under precise experimental conditions, resulted in the formation of stable lipid-pDNA complexes. As shown in Fig. 1A, cryoEM examination revealed spherical particles exhibiting two kinds of structures: a multilamellar organization of concentric patterns (>90% of total particles) and a more rare punctate pattern (<10%). Distinguishing the former concentric ring motif and the number of concentric layers depended on the spherical condensate observed. Typically 5–10 concentric shells were observed to surround a central area of a punctate pattern. No free SUV could be observed on the squares of the grids. All the complexes were highly sensitive toward the electron beam, indicating high material density, and consequently, indicating the presence of DNA in Lx. CryoEM (Fig. 1A) revealed that the concentric ring pattern is made of alternative layers appearing transversally striated and layers with low density.

Figure 1.

(A) CryoEM images of Lx-pDNA forming multilamellar particles. Striations are detected in the outer and inner high density layers (full arrows, Upper). The lamellar structure sometimes can be observed up to the center of the particle. Punctate array are occasionally observed (empty arrows, Lower). Faint striations are detectable on the edge of the latter Lx particles. (B) Scheme of cryoEM observation of a portion of Lx-pDNA particle and average values (n = 15). (C) Detection of the same pDNA molecule outside (arrow A) and complexed inside (arrow B) a Lx-pDNA particle. This Lx-pDNA was prepared as described in Materials and Methods, but dioleoyl phosphatidylethanolamine was omitted in the composition, resulting in incomplete formation of Lx particles and enabling the visualization of DNA. (Bars = 50 nm.)

Based on the cryoEM images, the thickness of the striated layers had a value of 5.3 nm and perpendicular striation of 2 nm. Furthermore, we observed a periodicity of 7.5 nm because of the stacked bilayers. This periodicity was confirmed by optical diffraction (data not shown). Perpendicular to these layers, faint striations could be seen mainly on the edge but also inside the particles. These striations were 2.0 nm thick with a periodicity of 3.4 nm (Fig. 1 A and B). The particles with punctate array (Fig. 1A) are confined by an outer shell. This outer shell occasionally contained the same thin striations as those found on multilamellar structures. In addition, the punctate structures occasionally were associated with these multilamellar structures. Empty SUV (data not shown) do not show any internal structure, thus confirming that the striation and the punctate pattern observed in the other particles is caused by the presence of DNA. When dioleoyl phosphatidylethanolamine was omitted from the formulation, incomplete formation of Lx particles occurred whereby the DNA molecules appeared to converge at the striated layer at the periphery of incompletely formed particles (Fig. 1C). Previous optical microscopy (18) or cryoEM (19, 20) studies observed fingerprint-like patterns, but details of the ordered layers were not revealed and DNA chains intercalated between membranes could not be detected. Freeze-fracture EM study (21) revealed a tubule-like structure formed of lipid bilayer-covered DNA.

The lamellar symmetry in Lx structures has been previously demonstrated by means of SAXS (18, 20). Rädler et al. (18) and Lasic et al. (20) showed the multilamellar nature of Lx and a periodicity of 6.5 nm. Analysis by SAXS of our Lx sample (Fig. 1B) revealed three diffraction peaks: one indicating a repetitive spacing of 2π/0.094 = 6.9 +/− 1 nm and the second and fourth order indicating the multilayer structure (2π/0.18). The width of x-ray diffraction peaks suggested highly ordered particles made of at least 12 concentric layers. Thus SAXS clearly confirmed the multilamellar structure of Lx and the periodicity observed with cryoEM. We could not reliably detect a peak corresponding to the DNA sandwiched between lipid bilayers, as previously observed (18, 20). SAXS was performed on centrifuge-pelleted Lx to maintain the dispersity of Lx particles. By contrast, detection of the DNA peak by SAXS as previously made by Radler et al. (18) and Lasic et al. (20) was possible by preparing Lx in the capillaries in which aggregates formed, allowing high local density. This discrepancy could be the result of the beam intensity that did not allow for the observation of the weak DNA-DNA correlation peak, or it also could suggest that the DNA chains were not arranged in a parallel array between lipidic layers as assumed by those authors.

Based on our results, it is feasible that each lamella represents a DNA-lipid complex i.e., packed rows of DNA duplexes on the surface of a bilayer. Concerning the molecular arrangement, we have two hypotheses: (i), the spacing between two striations (3.4 nm) observed on the particle multilayers could correspond to the second periodicity (≈3.7 nm) previously detected (18, 20) and thus to the DNA in an interhelical packing regime, sandwiched between lipid bilayers; and (ii), the striations observed in Lx exhibit the same width as the B-DNA form (2.0 nm). Consequently, we hypothesize that this transverse striation pattern could correspond to the transmission of a solenoid made of the superhelical conformation of DNA. We propose a three-dimensional model in which DNA exhibits a superhelix with a pitch of ≈3.4 nm that is absorbed on to lipid bilayers with a width of 5.3 nm. This model is based on the hypothesis that DNA will compact in Lx in a superhelical conformation with such molecular constraints.

Examination of Lx by dynamic light scattering indicated a mean size of 254 nm (width: 108 nm) and a monomodal population with a polydispersity of 0.193 (Fig. 2B). Size analysis and cryoEM illustrated in Fig. 1 were carried out 3 weeks and 2 months, respectively after complex formation, pointing out the high stability of the Lx. In contrast, other recently described Lx preparations (15, 19, 20, 22) must be used the same day or the day after preparation. The Lx formulation used in this study is the result of meticulous optimization enabling the production of efficient gene delivery particles whose preparation is reproducible and stable. They are composed of a poly-cationic lipid (dioctadecylamidoglycylspermidine) (23), a neutral lipid (dioleoyl phosphatidylethanolamine), and an anionic phospholipid (cardiolipin) that interact with DNA, a poly-anionic macromolecule. Such mixture of heterogeneously charged compounds are a priori not prone to form stable complexes at concentrations of between 1 and 6 and 0.2–2.0 mg/ml of lipids and DNA, respectively, because binding reactions involving multivalent surfaces are difficult to control and inclined to promote aggregation (24). Except in the case of rare Lx formulations (13, 16) heterogeneous aggregates typically occur when mixing DNA with cationic lipids in salt-containing buffer and DNA concentrations >0.2 mg/ml. Multilamellar Lx as formulated here are of great interest as they exhibit near neutrality or a negative net charge (upon lipid-to-DNA ratio), high homogeneity, low mean size, and stability. Hence, this optimized formulation makes the Lx preparation well suited for systemic administration. Thus, up to 50 ng/ml human factor IX was detected in mouse plasma after a single i.v. injection of Lx formulated with pDNA containing the gene coding for this blood coagulation factor (unpublished data). Thus, this formulation appears appropriate to facilitate the intricate interactions leading to the formation of a stable supramolecular organization.

Figure 2.

(A) SAXS scan of Lx-pDNA reveals a multilamellar structure. 1, 2, and 4 indicate the first, second, and fourth diffraction orders, respectively. (B) Population size analysis by dynamic size analysis that shows the monomodal distribution of Lx-pDNA particles with a mean size of 254 nm (width, 108 nm) and a 0.192 polydispersity.

Lx is a multicomponent system governed by a combination of interactions caused by charge neutralization. In the Lx formed here neutralization of DNA charges is caused by the presence of cationic spermidine-containing lipids (23). The side-by-side alignment of DNA in a liquid crystalline phase may be further promoted by spermine (7). Tight supercoiling structures can be obtained in vivo by polycationic molecules or proteins such as spermine, spermidine, histones, or histone-like proteins (25, 26). At critical concentrations and charge densities, liposome-induced DNA collapse and DNA-dependent liposome fusion are initiated (27). We suggest that this phenomenon induces thermodynamic forces toward compaction of the DNA macromolecule in the complex through a concentric winding where DNA is adsorbed onto the cationic head groups of the lipid bilayers. This winding results in a membrane-like complex leading to a concentric structural motif, as observed in Fig. 1B. Tight control of the parameters for Lx preparation, however, enables us to obtain thermodynamically stable and more completely formed Lx particles. In this respect, previous workers have demonstrated that careful design of the formulation method was crucial: when the same reagents and same concentrations, but different formulation procedures are used, the resulting thermodynamic and biological stability of Lx differ greatly (13, 16). It is noteworthy that such a concentric pattern already was hypothesized for intermediates that may be involved in the generation of Lx formed from detergent/cationic lipid micelles mixed with DNA in detergent (22). Moreover, our data and the data from recent studies (18, 20) on the structure of Lx formed by different formulation procedures and lipid compositions seem to indicate that similar mechanisms are responsible for the formation of ordered multilamellar structures in a liquid crystalline phase.

We also have formulated Lx, using the same method and same carrier lipids, but with different types of nucleic acids such as linear single-stranded DNA from the M13mp8 phage and ODNs. Their respective structures were visualized by cryoEM (Fig. 3), and we observed a multilamellar structure with a concentric pattern, similar to that observed for Lx/pDNA. However, there were some discrepancies in the details of these structures. Lx particles with M13mp8 virus DNA had a round shape with an outer layer (arrow A) of 2.0-nm thickness, then a small number of inner lamellae of 4.5-nm thickness with a periodicity of circa 6.8 nm (arrow B) and an inner core presenting a punctate structure (arrow C). Thus the structure of the particles observed for a single-stranded DNA was roughly the same as the one observed for the pDNA. The layers were not as dense as for double-stranded DNA, reflecting the lower mass present in them. Particle mean size of this preparation was found to be 212 nm (width: 234 nm) (data not shown). Lx particles formed with the intracellular replicative form of M13mp8 DNA (double-stranded DNA) presented the same concentrically layered and inner punctate structures as for pDNA and single-stranded M13mp8 DNA (data not shown). These particles had an outer layer of 2.0-nm thickness, then a small number of layers of 5.5 nm with a periodicity of 7.8 nm. Dynamic light scattering revealed a mean size of 209 nm (width: 114 nm). Lx-ODN particles exhibited the similar multilamellar ordering of Lx-pDNA (ordering, 7.7 nm; width of the striated layer, 5.7 nm), but they were far more heterogeneous in shape as noted by the presence of condensates with spherical shape, as well as smaller particles with angular even planar morphologies (Fig. 3B). No punctate pattern was observed in Lx-ODN. The presence of smaller particles was confirmed by dynamic light scattering (mean size of 175 nm; width: 93 nm) (data not shown).

Figure 3.

CryoEM images of Lx formed with the linear single-stranded DNA of M13 mp8 virus phage (A) and ODN (B). Concentric-ring like patterns are clearly visible in the two types of Lx. Outer layer (arrow A), inner layer (arrow B), and inner punctate structure (arrow C). (Bar = 50 nm.)

Our results demonstrate that we can obtain a similar structural morphology in Lx formulated with all the nucleic acids tested despite highly different structures and sizes as with pDNA (circular supercoiled DNA of 10.4 kbp, PM ≈6,870,600) and ODN (linear single-stranded DNA of 30 bases, PM = 9,900). Furthermore, Ghirlando et al. (9) indicated that DNA segments as short as 20 bp were able to condense in micellar aggregates and, in general, condensation of DNA by multivalent cations seems caused by mechanisms independent from the length of the individual DNA molecules (5, 6). Our results confirm that DNA condensation in multilamellar complexes was insensitive to DNA size up to at least 10.4 kbp when using SUV, as previously found when using micelles (9). In this study, Lx was formulated with the same lipid/DNA ratio, thus suggesting the existence of an intermolecular process for DNA accompanying charge neutralization.

We have packaged T4 phage DNA in Lx by using this same formulation. CryoEM showed particles of spherical shape exhibiting a punctate array surrounded by a thin layer and particles formed by lamelae in a network (Fig. 4A). The latter structure might correspond to an intermediate stage of DNA condensation with lipid bilayers. A smaller number of particles contained the multilamellar structure as previously described for Lx composed of other DNA types tested here. As observed in cryoEM, the size distribution of these punctate T4 DNA-filled particles have a mean diameter ranging from 30 to 130 nm (n = 38). At times, some elements of striation are barely detectable in Lx-T4 and T4 phage particles (Fig. 4 C and D). SAXS patterns are completely different from those obtained for Lx-pDNA as shown in Fig. 4B. Three reflexions are observed with spacing ratios of 1:√3:√4 in unit Q. Two very weak reflexions with spacing ratios of 1:√7:√9 also are sometimes detected (not visible on Fig. 4B). These data could indicate an hexagonal structure between parallel DNA chains surrounded by lipids. The parameters a_H (spacing between two DNA molecules) of this hexagonal lattice seems to depend on specimens. Values ranging from 7 to 8 nm were measured. Conversely, concentric motifs are the majority species in Lx-pDNA cryoEM images corroborating with the lamellarity detected by SAXS. As punctate and multilamellar structure were associated in some Lx particles, it might be possible that both liquid crystalline and hexagonal phases coexist in the same particle. As T4 DNA is more than 16 times longer than the pDNA used in this study, there may exist constraints of length and type of DNA that influence phase transition in the particles.

Figure 4.

CryoEM images of Lx-T4 DNA (A and C) and a T4 tail deletion mutant phage particle (D) (courtesy of J. Lepault, Centre National de la Recherche Scientifique) showing morphological similitary. Arrows indicate end-on views of T4 particles. (B) SAXS of Lx-T4 DNA (solid line) in comparison with that of Lx-pDNA (dotted line). [Bar = (C and D) 50 nm and (A) 125 nm.]

The cryoEM images of punctate Lx particles formed with T4 DNA that we observed (Fig. 4C) are strikingly similar to the images of phage lambda (28), complete tail-deletion mutant of T4 (28) (Fig. 4D), and T7 (29) phages. T4 phage mutants produce a normal mature head but no tail, which enables a better observation of DNA organization (28, 29). These T4-related phage particles showed a spherical shape and an average diameter of 80 nm (28). Recent investigations (28, 29) performed by using tailless mutants indicated the presence of DNA packing domains in viral particles. It is noteworthy that T7 tail-deletion mutants yield cryoEM images with both a concentric and punctate ring motifs as observed for Lx-pDNA. Cerritelli et al. (29) indicated that the state of DNA compaction in these viral particles should correspond to the three-dimensional hexagonal crystalline phase of DNA predicting the viability of such a model for other phage heads.

Despite these similarities, there are numerous discrepancies for DNA packaging between Lx and bacteriophage particles. Among them are: (i) the packing density of DNA in bacteriophage heads is much higher than in Lxs; and (ii) in bacteriophage, DNA is packaged into preformed procapsids by a terminase/translocate enzyme at the expense of ATP hydrolysis (30). Whatever the sequence of events in Lx formation, it must be quite different from the bacteriophage prototype.

In vitro complexing of DNA with viral DNA-condensing agents such as polyamines resulted in liquid crystalline condensed phases (31). The polyamines spermine or spermidine are ubiquitous compounds that stabilize the DNA double helix in vivo, and which, consequently, were intensively studied as a valuable model system for studying DNA organization in biological structures (25, 32). Lx used in this report were partly composed of spermine-containing lipids and DNA and could constitute another model. Organization of such nucleotidic supramolecular assemblies is relevant for prebiotic chemistry (33). As postulated by Lipowsky (34), cellular life might have begun with a membrane vesicle containing just the right mixture of polymers. In light of the numerous observations made on DNA packaging in nature or by various organic or inorganic condensing agents, our data corroborate the notion that a parallel between natural and synthetic DNA compaction can be drawn. We have demonstrated some of the structural similarities between a synthetic supramolecular organization and viruses.

Abbreviations

Lx

lipoplex

cryoEM

cryo-electron microscopy

SAXS

small angle x-ray scattering

SUV

small unilamellar vesicles

pDNA

plasmid DNA

ODN

oligodeoxynucleotides