Nucleic Acids Research

INTRODUCTION

The condensation of DNAs into compact structures is a common process for native genomes. The controlled bending and kinking of DNAs are demonstrated to be possible with enzymes (1), ions (2,3) or polyamines (4,5). For example, it was found that the Cro protein can induce DNA bending, and the bend angles of DNA were different for specific and non-specific sites (1). It is known from extensive studies that many factors could influence the packing processes. There is evidence which suggests the naturally-occurring polyamines in living cells such as spermine, spermidine and putrescine can exert a substantial effect on the packaging process of DNAs in bacteria (6) and viruses (7). However, the role of polyamines in the higher order condensation of chromatin as well as their effect on compacted DNA expression is yet to be fully understood.

Several studies (4,5) have demonstrated that in vitro polycations (including polyamines) can condense DNAs into three main types of structures depending upon the concentration and length of the DNA molecules: (i) in extremely dilute solutions (~1 ng/µl or below), long DNA molecules undergo a monomolecular collapse; (ii) in very dilute solutions (~10 ng/µl or below), microaggregates form with short or long molecules and remain in suspension. Experimental data show certain condensed structures such as toroids and rods (8-11) can be induced by polyamines in DNA solutions ~1-10 ng/µl; and (iii) in dilute solutions (~1 mg/ml), large aggregates are formed that sediment readily.

In the case of toroidal structures, as well as the important role of polyamines in vivo, understanding the organization of DNA within toroidal condensates and the dynamics of their formation became the focus of further studies. Toroidal structures have been extensively investigated by using electron microscopy (12), light scattering (13), circular dichroism (CD) and hydrodynamic measurements (8,14). Experimental data reveal that while the toroid sizes from independent preparations vary considerably (15-17), average toroid size is surprisingly unaffected by the length of DNAs being condensed. DNA molecules ranging from 400 bp to genomic length can experience monomolecular or multimolecular aggregation to produce toroids similar in size (4). Quantitative analysis has suggested that free energies including electrostatic energy, bending energy, hydration energy and mixing energy may be the main driving forces underlying DNA condensation. Among those factors, electrostatic is the dominant repulsive term, and condensation cannot occur unless ~90% or more of the charge along the DNA backbone is neutralized (13,20). In-depth and detailed discussions can be found in many studies (18,19).

Based on the biological implication of toroidal condensation, it was postulated that the DNAs in the toroid should be arranged with certain characteristics, as explained by a spool-like model (21,22), which postulates that DNAs are wound in an orderly way to form a toroid, and the constant radius of curvature model (23), which postulates that the DNAs are circumferentially wound with a constant diameter to form a toroid. Noguchi et al. (24) suggested that the formation of toroids is a coil-globule transition of polymers, with the folding process of toroid accompanied by the partly-coiled DNA chain. Recently, Dunlap et al. (25) used atomic force microscopy (AFM) to examine DNA in incomplete condensates induced by polyethylenimine and found clearly, for the first time, the individual DNA strands in the condensed state. In this experiment, DNAs were observed as being clearly arranged in parallel.

Moreover, some experimental data have indicated that local ordering exists in DNA-spermidine condensates. Very dilute solutions of high molecular weight DNA in the presence of the tetravalent cation spermine show CD spectra characteristic of a cholesteric helical supramolecular ordering of DNA, also called [psi]-type CD spectra (26). X-ray diffraction studies of the structure of the aggregates formed by spermidine or by trivalent cation spermidine with high molecular weight DNA molecules (27) resulted in a strong equatorial reflection, which corresponds to a 25.5 Å Bragg spacing. These results led to the assumption of a hexagonal lattice (crystalline or liquid crystalline). It was also discovered that over a large range of spermidine and DNA concentrations, short DNA molecules can form liquid crystalline phases (5,28). This result raised the questions of whether both the toroids and rods have similar local ordering, how the ordering structures are arranged and if these structures may be microdomains of a liquid crystalline or crystalline phase.

The aim of this study is to investigate the fine characteristics of the toroidal structures and other types of the DNA-spermidine condensates using mainly AFM. We believe the results could be beneficial to the understanding of the condensation process on a microscopic scale.

MATERIALS AND METHODS

[lambda]-DNA (0.49 mg/ml, 48 kb) under storage conditions of 10 mM Tris-HCl, pH 7.8, 10 mM NaCl, 1 mM/l EDTA buffer solution was obtained from Promega (USA). Spermidine trihydrochloride (purchased from Boehringer Ingelheim Bioproducts Partnership, research grade) was diluted with de-ionized distilled water to the desired concentrations.

The preparation of monomolecular toroids began with the DNA solution diluted with de-ionized distilled water to ~1-2 ng/µl before use. Condensates were prepared by adding 4 µl spermidine solution (200 µ[Mgr]) to 4 µl DNA solution. After 10 min reaction (at room temperature), ~2 µl of this mixed solution was distributed on freshly cleaved mica and immediately dried under an infra-red lamp prior to the AFM observations.

For the preparation of multimolecular toroids, condensates were formed by increasing the DNA final concentration to ~2.5-4.0 ng/µl and the reaction time from 10 to 35 min. The AFM sample was made by a similar process as described above.

Microscopic analysis was carried out by using a commercial atomic force microscope (Digital Instruments, Santa Barbara, CA) and polarizing microscope (Nikon Optipnot microscope). All AFM images were obtained in air at room temperature by using contact mode with a spring constant of 0.12 nN/nm. The cantilever was 200 µm in length.

Figure 1. AFM image of monomolecular toroidal structures of spermidine-[lambda]-DNA condensates. (a) Observations reveal the coexistence of complete toroids, incomplete toroids with gaps, and U-shaped rods. The scan size is 850 × 850 nm. (b) Close-in AFM image of a single toroidal condensate. The scan size is 200 × 200 nm. (c) Cross sectional profile of the toroid in (b) along the marked line. (d) A non-standard toroid. The scan size is 140 × 140 nm.

RESULTS

The observation of monomolecular toroidal structures (type I)

The toroid structures of [lambda]-DNA in extremely dilute solution were prepared as described previously. The corresponding ratio of spermidine to DNA base pairs was typically ~65. AFM observations, as shown in Figure 1a, reveal that the prepared condensed DNAs take the form of toroids as well as other structures, such as rod-like and U-shaped structures. The frequency of the occurrence of the toroid-like structure in this study is estimated at ~2/3.

Figure 1b is the AFM image of a single toroidal condensate normally seen in such preparation conditions. Close scrutiny reveals that there is an apparent gap in the circle whose appearance could be seen as an insufficiently bent rod. In addition, it appears that there are fine knots embedded in the toroids. Figure 1c is the cross-sectional analysis of this toroid, where the center of the toroid is at the same level with the mica, suggesting it is a real cave rather than a depression formed by subsiding the center of the DNA globe. Figure 1d is the AFM image of a non-standard toroid, showing similar fine structures.

Following the reported analyzing procedure, we only selected toroidal structure for dimensional statistics. It is worth mentioning here that the volume of toroids and irregular structures are in the same range. The average outer diameter of 32 toroids (including toroids with gaps) was determined to be 120 ± 15 nm (Fig. 2a). The average toroid diameter [as specified in (29)] from the center of one edge across the center of the hole to the center of the other edge is measured to be 56 ± 8 nm (Fig. 2b). The average thickness of the toroids was 17 ± 2 nm (Fig. 2c).

Figure 2. The histograms of the AFM measurements of the dimensions of monomolecular toroids seen in Figure 1. (a) The average outer diameter; (b) the average toroid diameter; (c) the average thickness of the toroids.

The approximate number of molecules per toroid (n) was estimated by using the equation proposed by Arscott et al. (17): n = 0.906 Vt/Vm, where 0.906 is the packing fraction for hexagonal close packing of parallel cylinders, Vt is the total volume per toroid and Vm is the volume of a single molecule, taken to be hydrated B-form DNA [L = 0.34 nm/bp, r = 1.47 nm (27)]. This equation is based on the model for circumferentially wound DNAs [also named as the spool-like model which means that DNAs are wound in an orderly way to form toroids (21,22)] and is considered suitable for a rough estimate of the compact degree of toroids. The above measurements of the average diameter and the thickness of the toroids suggest it consists of a volume roughly equivalent to a complete single [lambda]-DNA molecule. In comparison with previous electron microscopy data for monomolecular condensed [lambda]-DNA [outer diameter was ~86-106 nm (30,31)], the dimensions of toroids of our AFM measurements are reasonably consistent. The discrepancy may be related to the specific cation used for the condensation, whose binding and cross-linking properties could change the flexibility of DNA strands and the packing fraction with which they pack (32). Other factors such as local concentration of condensing agent, temperature and pH values may also play important roles for the development of toroids.

It is worth mentioning here that the AFM measurements of the heights and widths of many biological samples are subject to changes under loads, humidity, etc., and in some cases are lower than the expected values. It is also acknowledged by a number of groups that by repeated measurements under controlled conditions, the measured height could still provide characteristic information about samples such as DNAs (35). The numerical data obtained in this work were recorded at the same force setpoint (load) and comparable humidity. Therefore, we consider that the data should be consistent, and we would rather use the above measured values as a relative measure of the observed structures.

In our AFM observations, it was found that the toroids have two prevalent forms, namely the toroid with a gap and the standard toroid or completely self-connected toroids (type I). Though the electron microscopy observations frequently found the standard toroids, it is reasonable to believe that some reported standard toroids were actually the toroids with gaps, considering that the sample preparation process in electron microscopy could hamper the observation of the fine characteristics of toroids (e.g. the uneven knots on the toroids in our AFM images). Previous reports also show that toroids sometimes co-exist with other structures such as rods and U-shaped rods, yet the frequency of toroids is much higher. By using the arguments in (17), toroids were formed by winding DNAs circumferentially, while the rods were formed by abrupt bending or kinking of DNAs (which would need more energy than toroids) and the toroids with gaps were formed by mainly bending rods (17).

By examining the DNAs in incomplete condensates induced by polyethylenimine, Dunlap et al. (25) suggested that condensates were formed through folding rather than winding the DNA. The co-existence of irregular structures with toroids leads to the belief that these structures are thermodynamically equivalent. The variations in the final structures could be affected by factors such as reaction time and mixing sequence. The apparent gaps observed in some toroids may be direct proof that toroids were formed by bending rods (Fig. 1b). Toroids with tail-like structures also indicate the further development of a curved rod (Fig. 1d). We believe that more investigations are necessary in order to clarify various stages of the toroidal formation process.

The observation of multimolecular toroidal structures(type II)

As mentioned in the Introduction, though multimolecular toroidal condensates of short DNA molecules have been reported, few data are available on multimolecular toroidal condensates for high molecular weight DNA molecules such as [lambda]-DNA or calf thymus DNA. Here we report the AFM image of multimolecular toroidal condensates formed by increasing the DNA final concentration to >2 ng/µl and the reaction time from 10 to 35 min. The corresponding ratio of spermidine to DNA base pairs is ~20. Figure 3a is the AFM image of these multimolecular condensates showing their appearance. AFM observations reveal that the appearance of toroids is variable (from circular to elliptical) and the dimensions of these structures are much greater than monomolecular condensates of [lambda]-DNA (>600 nm in diameter in multimolecular toroids), as discussed in the previous section. Careful observations show that some of the toroids have more than one layer with spherical particles as subunits. Figure 3b is an AFM image of an elliptical structure, whose particle-like subunits can be seen clearly. Figures 3c and d are the AFM images of circular and elliptical toroids, respectively. It seems that these toroids were formed as a spiral structure with a string of aggregated subunits. Cross-sectional analysis (Fig. 3e) shows that the center of the toroid in Figure 3d is at the same level as the mica, suggesting that it is a real cave rather than a hole formed by subsiding the center of the DNA globe.

Figure 3. AFM image of multimolecular toroidal structures of spermidime-[lambda]-DNA condensates. (a) Multimolecular toroidal structures (also named as type II structure in this study) observed by AFM . The scan size is 7 × 7 µm. (b) An elliptical structure with well resolved particle-like subunits. The scan size is 1.7 × 1.7 µm. (c) AFM image of an apparent spiral-up elliptical structure. The scan size is 1.5 × 1.5 µm. (d) An apparent spiral-up round toroidal structure. The scan size is 900 × 900 nm. (e) Cross-sectional analysis of a single toroidal condensate in (d).

Statistics of the AFM measurements are shown in Figure 4. The average short axis of 30 toroids was determined to be 680 ± 80 nm (Fig. 4a). The average long axis was determined to be 1100 ± 260 nm (Fig. 4b). The thickness of the toroids varies in different places. We measured the four highest points of each toroid where the long axis and the short axis passed. Figure 4c shows the height distribution of these points, which reveals the height distribution is discontinuous, namely 11 ± 4, 20 ± 5, 30 ± 6 and 40 ± 3 nm in multiple Gaussian simulation. This result indicates that the multimolecular toroids were probably formed by layers ~10 nm in height. We also measured the diameters of the subunits in the multimolecular toroids. The measured average diameter of these particles was 100 ± 10 nm (Fig. 4d).

Figure 4. Statistics of the AFM measurements of the dimensions of the mutimolecular toroids seen in Figure 3. (a) The histogram of the distribution of short axis of the observed toroids (round and elliptical structures). (b) The histogram of the distribution of long axis of the observed toroids, giving an averaged long axis of ~1100 $ 260 nm. (c) Height distribution of the points along the long axis and the short axis (four points marked `a', `b', `c' and `d' in each toroid). A discontinuity in the height distribution is discernable. (d) The average diameter of the subunits within the multimolecular toroids.

This structure (type II) is significantly different from the type I structure described in the previous section. The multimolecular toroids we observed here are interesting not only because of their size but also because of the fine structures displayed. This cannot be accounted for by simple aggregation of type I condensates. We have found that the concentration of DNAs and the reaction time can appreciably influence the toroidal structures, in the sense of changing the sizes (outer diameter from ~100 to 600 nm and above). These results suggest that it is important to have fine control of the environment in the process of condensation.

Moreover, AFM images, together with the dimensional measurements, reveal that multimolecular toroids are formed by particles which are arranged nearly ordered. These subunits (fine particles) appeared as anisotropic. As to the driving force for this self-organization process, a comparison could be drawn to the measurement of Rau et al. (33) of the intermolecular forces between counterion-condensed DNA double helices. The analysis suggests that polyvalent ligands bound to DNA double helices appear to act by reconfiguring the water molecules between macromolecular surfaces to create attractive long range hydration forces. Therefore, the toroid formation process by using anisotropic subunits is a somewhat orderly process similar to a nucleation and growth process, namely the subunits have a tendency to self-assemble into a string of particles, which has a certain long range interaction and has the tendency to wind by itself. As this nucleation and growth process continues, the spiral-up toroidal structures will be formed (Figs 3c and d).

On comparison with the measured results of monomolecular toroids, we found the size of the subunits in multimolecular toroids (average diameter was 100 nm and the thickness ~11 nm) is very close to that of single monomolecular toroids (average outer diameter was 110 nm and the average thickness was 16 nm). This implies that the subunits in multimolecular toroids may originate from the monomolecular toroids, which have the ability to self-assemble into multimolecular structures as discussed in the above paragraph.

The observation of other ordered structures

DNA-spermidine complexes in AFM observation taking the form of the multimolecular toroids were further studied by using polarizing microscopy. Approximately 50 µl of this solution which was similarly prepared as in Figure 3 were deposited on an area ~1cm² in glass and evaporated into ~5 µl solution. The preparation was observed between linear crossed polarity in a Nikon Optipnot microscope.

By enriching the DNA-spermidine complexes in very diluted solution, branch-like structures constructed by similar subunits were observed by using AFM. Figure 5 is the AFM image of typical branch-like structures. Figure 6 is the polarizing microscopy image of the aggregates prepared from the enriched DNA-spermidine condensates. The image indicates that the condensed DNA is in an ordered state. This observation is consistent with Pelta's observation of short DNA molecules induced by polyamine in dilute solutions (5,28). The drying process will indeed cause changes in concentration. Numerical estimation tells us that drying the solution from 50 to 5 µl would increase the concentration from 5 to 50 ng/µl. This later value is still close to the range for `very dilute' solutions and is far below the typical range for `dilute' solution (1 mg/ml or 1 µg/µl). In addition, we believe that since the drying process is a rather fast one the particles should more or less remain at their equilibrium states. Therefore, we consider the result of the polarizing microscopy could still be helpful for studying the possible intrinsic ordering of the condensates.

Figure 5. The AFM image of the branch-like structures in the enriched condensate solution. The scan size is 10 × 10 µm.

Figure 6. Polarizing microscopy image of the solution prepared from the enriched DNA-spermidine condensates. A typical pattern for crystal or liquid-crystal-like sample is present. Magnification ×450.

DISCUSSION

There have been conjectures for the toroidal formation. Noguchi et al. (24) suggested that condensation is a coil-globule transition. In this model, a ring-like structure is generated along the chain at first, then the remaining coiled parts gradually wind around the ring, and finally a stable toroid is formed. Another explanation by Bloomfield et al. (4) considers that the toroid formation may be a self-assembly process, in which toroids can be formed by wound DNA, circumferentially primarily or by bending rods. Dunlap et al. (25) first directly observed that the subunits in toroids are parallel DNA chains. The condensates appeared as being formed through folding rather than winding the DNA. Most toroids appear to result from the annularization of rods.

Our results present the likelihood of different types of toroids. Type I is the small toroid which had been observed by a number of groups. The considerably high frequency of variant structures (rods of various shapes) co-existent with toroids and non-standard toroids naturally gives the implication that these toroids and rods are basically thermodynamically equivalent. Furthermore, a new kind of multimolecular toroids was observed with much larger sizes (type II). Their formation is possibly a self-assembling process of the anisotropic particles. It is difficult for us to determine if the small toroid (type I) in our observations has a similar formation process to the type II. Nevertheless, deeper investigations about the intermediates of these structures should be carried out.

Porschke (34) suggests that condensation in large DNAs occurs in three phases: (i) an induction period when ligand binds to the DNA; (ii) a rapid intramolecular condensation as the bound ligand reaches a critical concentration; and (iii) a slow intermolecular condensation. This could account for the observed variations of the dimension of the condensates with reaction time.

Our results show that the average dimension of the subunits in type II toroids closely resembles that of the type I toroid. Perhaps this similarity, as well as the formation results with time elapsed, means that the condensation is a multi-staged self-assembly process, where by introducing polyamine, monomolecular toroids formed and collapsed into further condensed particles, which were subsequently organized to form type II structures.

In addition, we have also experimented with the sample prepared by vacuum evaporation, similar to SEM sample preparation except without metal coatings. The resulting sample's characteristics are nearly identical to those of the sample prepared by the drying process described in this report. This makes us believe that the drying process we used in this study is comparable with the standard procedure for SEM practices, though both techniques involved inevitable dehydration of samples to a certain degree. We would suggest that since either vacuum evaporation or accelerated heat drying is a relatively fast process, the condensates are likely to remain not very far from their equilibrium state during such treatments. The drying process may indeed cause some variations at the interface between sample and substrate. We consider this effect should not substantially affect the observed multilayered nature of the condensates and the fine characteristics of the top-most sample surface. It is considered that a very helpful test could be provided from the fluorescence microscopy or light scattering experiments which could measure the dimensions of the condensates in solutions. Unfortunately, from our knowledge we do not know of the results obtained by these techniques which are accurate enough to allow quantitative comparisons.

CONCLUSIONS

By using atomic force microscopy and polarizing microscopy, we found monomolecular (type I) and multimolecular (type II) toroids could be formed in different spermidine-DNA concentrations. Measurements show that the average diameter of the subunits in type II toroids is similar to the outer diameter of the monomolecular toroids, and the thickness of the multimolecular toroids had a multi-layer structure with increments of 11 nm, indicating that the multimolecular toroidal structures may be constructed by monomolecular toroids. Polarizing microscopy images show that the highly concentrated result of the DNA-spermidine complexes in very diluted solution for type II may be crystalline or liquid crystalline, suggesting there is an ordered self-assembly process in DNA-spermidine complexes.

ACKNOWLEDGEMENTS

This work is supported by the Foundation of Chinese Academy of Sciences and National Natural Science Foundation of China.

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*To whom correspondence should be addressed. Tel: +86 10 6256 8158; Fax: +86 10 6255 7908; Email: clbai@infoc3.icas.ac.cn

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