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Department of Physics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan
Correspondence: Address reprint request to Kenichi Yoshikawa, Tel.: 81-75-753-3812; Fax: 81-75-753-3779; E-mail: yoshikaw{at}scphys.kyoto-u.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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r from 80 to 170. Single-chain observations of changes in the conformation of giant DNA were performed by transmission electron microscopy and fluorescence microscopy, with tetravalent spermine (SPM4+) as a condensing agent. At a fixed dielectric constant, single DNA chains fold into a compact state upon the addition of spermine, whereas at a constant spermine concentration single DNA chains unfold with an increase in r. In both cases, the transition is largely discrete at the level of single chains. We found that the critical concentration of spermine necessary to induce the single-chain folding transition increases exponentially as the dielectric constant increases, corresponding to 87–88% of the DNA charge neutralized at the onset of the transition. We also observed that the toroidal morphology of compact DNA partially unfolds when r is increased. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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), such as polyamines (Gosule and Schellman, 1976), multivalent metal cations (Widom and Baldwin, 1983), cationic surfactants (Mel'nikov et al., 1995), or neutral polymers to a DNA solution (Laemmli, 1975). In fact, the folding transition between the elongated coil state and the compact state is largely discrete at the level of single chains and can be described as a first-order phase transition (Yoshikawa et al., 1996). The attractive force between negatively charged monomers is an example of a more general phenomenon called like-charged attraction, which is responsible for various types of macromolecular organization in charged systems, such as bundling and cross-linking of F-actin (Tang and Janmey, 1996; Wong et al., 2003), precipitation of flexible polyelectrolytes (Olvera de la Cruz et al., 1995) and clustering in colloidal suspensions (Grier, 2003). Since the pioneering interpretation by Oosawa (1971), like-charged attraction has been the subject of many theoretical works (Borukhov et al., 2001; Ha and Liu, 1997; Rouzina and Bloomfield, 1996; Shklovskii, 1999; Deserno et al., 2003) and numerical simulations (Grønbech-Jensen et al., 1997; Lyubartsev et al., 1998; Stevens, 1999), which all show, despite fundamental discrepancies, the crucial role of counterions, especially those that are localized in the vicinity of the charged chain and usually referred to as "condensed" (in fact, counterions are continuously distributed around the chain, as shown recently by anomalous x-ray scattering; Das et al., 2003). However, the like-charged attraction phenomenon has not been unambiguously clarified yet, probably due to the paucity of precise experiments on model systems with a variable number of condensed counterions. Furthermore, many previous studies on charged polyelectrolytes have not distinguished between the single-chain phenomenon (e.g., DNA compaction) and multiple-chain aggregation. To obtain fundamental knowledge on like-charged attraction in a single polyelectrolyte chain induced by counterion condensation, we studied the folding transition of single DNA chains under various dielectric fields.
In the so-called Manning-Oosawa condensation theory (Manning, 1969; Oosawa, 1971), a fraction 
of counterions "condense" on the chain when the average spacing b between charges along the chain becomes smaller than the Bjerrum length lB, the distance at which the electrostatic energy equals kBT:
 | (1) |
0 is the dielectric constant of vacuum, r is the relative dielectric constant, kB is the Boltzmann constant, and T is the temperature. Counterions condense so that the effective distance between charges equals lB. Thus, for counterions of valency Z, we have
 | (2) |
For DNA in pure water at 20°C, b = 0.17 nm, lB = 0.71 nm, and = 0.76 in the case of monovalent counterions. Therefore, can be decreased by decreasing lB or increasing r at constant temperature. For instance, the Bjerrum length of a solvent such as N-methylformamide can be decreased with temperature, from 0.45 nm at 70°C to 0.30 nm at 10°C (Bass et al., 1964; Sehgal and Seery, 1998). In this study, the dielectric constant of water at room temperature was enhanced by the dissolution of zwitterionic species, with r ranging from 80 to 170, lB from 0.71 to 0.33 at 20°C, and from 76% to 48% (in the case of sole monovalent counterions). The condensation of DNA in the presence of zwitterionic species to achieve a high dielectric constant has been studied in detail by Houssier et al. (Flock et al., 1995, 1996; Flock and Houssier, 1997). However, in these pioneering experiments, DNA concentration was quite large; i.e., the phenomenon was observed in the ensemble of a large number of chains where the single-chain compaction can not be studied due to the effect of multichain precipitation. To our knowledge, we report here the first experimental study on the folding transition of single double-stranded DNA molecules as a function of an increasing dielectric constant. Direct single-chain observation in bulk was performed by fluorescence microscopy, and transmission electron microscopy was used to study the detailed morphology of the compact states. The results have enabled us to establish the phase diagram of single-chain conformation as a function of an increasing dielectric constant, i.e., with a decrease in the extent of counterion condensation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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r ranging from 80 to 170. As a condensing agent, we used spermine tetrahydrochloride (Nacalai Tesque), a tetravalent cationic polyammonium salt, abbreviated SPM4+.
Fluorescence microscopy
Very dilute DNA solutions were prepared at 0.1 µM (in nucleotides) with 0.1 µM of the fluorescent dye 4'6-diamidino-2-phenylindole, abbreviated DAPI (Wako Pure Chemical Industries, Osaka, Japan). Fluorescent microscopic observations were performed using an Axiovert 135 TV (Carl Zeiss, Aalen, Germany) microscope equipped with a 100x oil-immersed lens. Images were recorded using an EB-CCD camera and an image processor Argus 10 (Hamamatsu Photonics, Hamamatsu, Japan). Samples were placed in custom-built microscope cells (made of glass previously cleaned by baking at 500°C for 1 h), illuminated at 365 nm, and observed at 
420 nm. Due to the blurring effect of fluorescence light, the apparent sizes of fluorescent images are 
0.3 µm larger than the actual sizes (Yoshikawa and Matsuzawa, 1995). Under these experimental conditions, we can directly observe the bulk conformation of a large number of individual DNA chains. The coil and compact states of individual chains are clearly distinguishable: compared to folded compact DNA, which appears as a bright fast-diffusing spot, DNA in the coil state has a much larger apparent long-axis length (longest distance in the outline of the DNA image), has a much lower translational diffusion coefficient, and exhibits characteristic intra-chain thermal fluctuations.
Transmission electronic microscopy
Transmission electronic microscopic (TEM) observations were performed at room temperature using a JEM-1200EX microscope (JEOL, Tokyo, Japan) at an acceleration voltage of 100 kV and with an H-7000 microscope (Hitachi, Tokyo, Japan) at 75 kV. We used carbon-coated grids with a mesh size of 300 and uranyl acetate (1% in water) as staining agent.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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r on the general properties of individual DNA chains in the absence of spermine. Using fluorescent microscopy, we observed that, for r ranging from 80 to 158, all individual chains are in the characteristic elongated coil state. Image analysis has allowed us to measure the long-axis length L of individual DNA molecules. Fig. 1 shows L, averaged for 150 individual molecules, plotted as a function of r. Within the limits of our experimental accuracy, L is independent of r and equals 4.4 µm. This value suggests that the persistence length lp remains essentially constant (50 nm; Yoshinaga et al., 2002) regardless of changes in r. In the Odijk or Skolnick and Fixman theories (OSF) for rigid polyelectrolytes in the presence of monovalent salts, lp is the sum of the bare persistence length l0 and the electrostatic persistence length lOSF 
1/(4
2lB) (Odijk, 1977; Skolnick and Fixman, 1977). –1 is the Debye length and 2 = 8
lBCs where Cs is the total salt concentration. In the conditions of our experiments where 10 mM of monovalent salts come from the buffer solution, we can estimate 
Therefore, in the range of r investigated here, the variation of lp is <25%. The corresponding variation in the long-axis length is 10%, which cannot be detected within the experimental accuracy of our experiments.
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r on the secondary structure of DNA, circular dichroism (CD) measurements were performed as well. These experiments were performed at a DNA concentration of 15 µM (in nucleotides), which under our conditions is the lowest DNA concentration that allows for CD measurement with a reasonable signal to noise ratio. On the other hand, it has been shown that above 10 µM, T4 DNA chains start to interact (Iwataki et al., 2004). Therefore, strictly speaking, a 15 µM DNA solution cannot be considered a dilute solution. However, under our experimental conditions, neither precipitation nor aggregation was observed in the prepared specimen, which indicates that DNA chains interact in a very weak manner. Thus we can assume that the CD spectra obtained under these conditions reflect the CD properties of individual chains. Fig. 2 shows CD spectra for 15 µM DNA solutions at various dielectric constants r. Within the limits of our experimental accuracy, all curves have a similar shape and present two characteristic bands: negative at = 245–250 nm and positive at = 275–285 nm. The low DNA concentration results in somewhat larger experimental error compared to usual conditions. Thus, we will not discuss the minor changes observed in the CD spectra. The presence of the two characteristic bands shows that individual DNA chains are in the B-form throughout the entire range of r investigated here. The secondary structure, or the spatial arrangement of bases, is thus unaffected by the dielectric constant, which essentially controls the electrostatic interactions. Therefore, in the absence of condensing agents, regardless of r within the range 80–158, all chains are in an elongated coil state, have a similar characteristic size, and are composed of B-DNA.
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r = 80). As long as the spermine concentration [SPM4+] is <4.5 µM, all chains are in the coil state (a) and their long-axis length is independent of [SPM4+]. In contrast, for spermine concentrations above 5.2 µM, all individual chains are in the compact state (c). The integration of fluorescence signals confirms that each compact object is composed of one single chain. For intermediate spermine concentrations, 4.5 µM < [SPM4+] < 5.2 µM, chains in the elongated coil and compact states coexist (b), and the fraction of chains in the compact state increases with an increase in [SPM4+]. The largely discrete nature of the conformational change and the coexistence region at intermediate spermine concentrations are the signature of the first-order nature of the single-chain folding transition of giant DNA (Yoshikawa and Matsuzawa, 1995; Yoshikawa et al., 1996).
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rr on the DNA single-chain conformation in the presence of spermine. For very low spermine concentrations, [SPM4+] < 4.5 µM, i.e., DNA in the coil state at r = 80, there was no change in conformation with an increase in r. Conversely, at a constant spermine concentration >5.2 µM, i.e., DNA in the compact state at r = 80, we systematically observed that DNA chains unfold with an increase in r in a largely discrete manner at the level of single chains, as illustrated by Fig. 4. In this figure, the spermine concentration is fixed at 20 µM. In water at r = 80, which corresponds to panel c in Figs. 3 and 4, all chains are in the compact state. When r is increased, all chains remain in the compact state as long as r < 98. With a further increase in r, chains individually unfold in a largely discrete manner. At r > 101.5, all chains are in the elongated coil state (Fig. 4, panel e), with a typical long-axis length of 4.4 µm (see Fig. 1). For intermediate dielectric constants, 98 < r < 101.5, the compact and elongated coil states coexist (Fig. 4, panel d), and the proportion of chains in coil state increases with an increase in r. These features are typical of a first-order phase transition at the level of single chains. It is important to note that this first-order unfolding transition was observed over a wide range of spermine concentrations: from 8 µM to 7 mM. Regardless of [SPM4+], the unfolding transition is always largely discrete at the level of single chains, and the critical dielectric constant at which the transition occurs increases with an increase in [SPM4+]. To our knowledge, these results constitute the first evidence of the largely discrete unfolding transition of individual DNA chains with an increase in r.
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rr on the folding transition of DNA induced by spermine. For this purpose, again using fluorescence microscopy, we characterized the conformation of single chains upon the addition of spermine at various dielectric constants. Regardless of r, single chains undergo a first-order folding transition between the elongated coil state and the compact state in response to the addition of a sufficient amount of spermine. This transition shows the same features as that observed in pure water (see Fig. 3). The only difference is the critical spermine concentration [SPM4+]crit at which the transition occurs. The phase diagram in Fig. 5 summarizes the experimental observations. In this graph, the critical spermine concentration at which the compact state first appears is represented by blue points, and red points show when the last coils disappear. Hence, the phase diagram is divided into three zones. The coiled state is observed below the border formed by blue points and the region above the red points corresponds to the compact state. In the intervening region the two phases, i.e., coil and compact states, coexist. Fig. 5 shows that the critical spermine concentration at which the folding transition occurs is a strong increasing function of r. Moreover, this function is independent of the chemical nature of the zwitterion used for r enhancement. This means that the retardation of the DNA folding transition observed in these experiments is entirely due to the increase in r and is not related to any specific chemical interaction between a particular zwitterion and the DNA chain. As shown in Fig. 5, this strong dependence of [SPM4+]crit was noted over a range of spermine concentrations that spanned four orders of magnitude; within the limits of our experimental accuracy, [SPM4+]crit increases exponentially as a function of r according to the following relationship (first appearance of the compact state; blue points in Fig. 5):
 | (3) |
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r. Conversely, the appearance or disappearance of the coil state, which is related to monomer-monomer "like-charged" attraction, is very sensitive to r: i), at a fixed spermine concentration, single DNA chains unfold in a first-order manner upon an increase in r; and ii), at a fixed dielectric constant, DNA chains undergo a first-order folding transition upon the addition of spermine, and the critical concentration of spermine necessary to induce this transition is a strong increasing function of r. This demonstrates the crucial importance of counterion condensation in the like-charged attraction process of the DNA single-chain folding transition. Therefore, it seems essential that we more precisely characterize the effect of r on the like-charged state, i.e., the compact state of individual DNA chains. For this purpose, we used TEM to characterize the compact state of DNA as a function of increasing r.
TEM of compact DNA as a function of r
These experiments were performed at a DNA concentration of 1 µM, which facilitates single-molecule detection, whereas fluorescence microscopy was performed using 0.1 µM solutions. Moreover, as mentioned in the section Coil state, the single-chain properties of T4 DNA are independent of concentration for a DNA concentration lower than 10 µM (Iwataki et al., 2004). Therefore, concentration effects can be neglected and the DNA solution considered dilute (no interaction between individual chains). In particular, the single-chain phase diagram shown in Fig. 5 is still valid under the experimental conditions for these TEM observations. Fig. 6 shows a series of typical TEM pictures of single DNA chains in the compact state for various dielectric constants. In pure water at r = 80 and [SPM4+] = 20 µM, all chains are compacted into a highly ordered toroidal morphology (picture c) with an average outer diameter of 90 ± 10 nm, in agreement with classical observations (Gosule and Schellman, 1976; Hud and Downing, 2001). Pictures f–h show typical morphologies observed for higher r: 103, 115, and 131, respectively. For each dielectric constant, the spermine concentration was chosen so that the corresponding compact state is in the same relative position in the phase diagram with respect to the transition point (see black points labeled c, f, g, and h in Fig. 5). Indeed, it is known that at a fixed dielectric constant, a large increase in the concentration of the condensing agent can induce a swelling of the compact toroidal state before complete unfolding (Yoshikawa et al., 1999). By comparing points in the same relative position, we can focus solely on the effect of r, regardless of the concentration of spermine. First, we observed that, for all dielectric constants, chains are compacted in a toroidal conformation (pictures f–h), as observed in pure water (picture c). Furthermore, within the limits of our experimental accuracy, we found that the average outer diameter was independent of r and equaled 90 ± 10 nm. However, it is interesting to note that the highly ordered toroidal structure is progressively destabilized with an increase in r. At r = 103, the toroidal morphology is very similar to that observed in water; at r = 115, the edge of the toroid becomes fuzzy, and at r = 131, small parts of the chain that are locally in the coil state form loops surrounding the toroid (arrow in picture h). At a much higher dielectric constant, only disordered and loosely packed structures were observed. An example of such a structure is shown in Fig. 7, for r = 157 and [SPM4+] = 10 mM (point i in Fig. 5). In this case, a very loose toroid-like structure (indicated by arrow 1) coexists with bundles (arrow 2) and parts of the chain that are locally in the coil state (arrow 3). In summary, Figs. 6 and 7 show that the compact state becomes less ordered with an increase in the dielectric constant.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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r = 80–180) while keeping constant the solvent quality. Although the dielectric constant seems to be the main parameter that controls the DNA compaction properties, one should discuss the eventual effects of the specific interaction between zwitterions and DNA. Recently, Hong et al. (2004) have shown that glycine betaine is strongly excluded from the DNA surface and that the DNA chain is preferentially hydrated. The local dielectric constant around the chain is thus expected to be different from that in the bulk solution. In the case of alcohols, Hultgren and Rau (2004) have also shown that such exclusion mechanisms can affect the DNA precipitation by multivalent counterions such as spermidine. Similarly to glycine betaine, the distribution of the zwitterions used in this study around DNA is probably not homogeneous, and exclusion from the DNA chain may be expected. Thus, different amplitudes in the exclusion mechanism as a function of zwitterion size and concentration could qualitatively explain the increase in [SPM4+]crit shown in Fig. 5. However, by the use of various solvent mixtures with dielectric constants smaller than that of water, Mel'nikov et al. (1999) have shown that the dielectric constant of the solvent is the key factor that determines the conformational behavior of single DNA molecules in solution. Moreover, the effect of the dielectric constant shown in their study is in quantitatively good agreement with the results of this work (Figs. 3–5
). Therefore, even if preferential hydration may be an important factor in the conformational properties of DNA, we will focus in the discussion below on the effects of the dielectric constant.
First of all, it has become clear that DNA in the compact state unfolds in a largely discrete manner at the level of single chains with an increase in r (Fig. 4). This directly illustrates the role of counterion condensation in the DNA folding transition and, more generally, in like-charged attraction phenomena. Indeed, with all other parameters constant, an increase in r corresponds to a decrease in counterion condensation (electrostatic energy weakens, whereas the translational entropy of counterions is constant). The unfolded coil state (like-charged repulsion) is preferred at low counterion condensation (high r) and the compact state (like-charged attraction) is only observed at sufficiently high counterion condensation (low r). We also noted that at fixed dielectric constant, individual DNA chains fold in a first-order manner with an increase in the spermine concentration (Fig. 3), but the critical spermine concentration [SPM4+]crit at which the transition occurs is a strong increasing function of r (Fig. 5). To correlate this increase with counterion condensation, we estimated the fraction of the DNA charges neutralized by the counterions of valencies Z1 = 1 (mainly coming from the buffer solution) and Z2 = 4 (tetravalent spermine), respectively. In the two-state model of Manning-Oosawa, a fraction of these counterions, 1 and 2 respectively, are "bound" to the chain to decrease the linear charge density of the chain, whereas the other fraction remains free in the solution. Minimization of the free energy with respect to 1 and 2 leads to the following system of equations (Manning, 1978; Wilson and Bloomfield, 1979):
 | (4) |
 | (5) |
is the adimensional Manning parameter (lB/b), and –1 is the Debye length. The system was solved numerically by taking for v1 and v2 their limiting values (infinite dilution) and the total neutralization was calculated as
 | (6) |
The total neutralization was calculated at the onset of the folding transition (blue points in Fig. 5) and is plotted as a function of r in Fig. 8. It shows that the total neutralization is remarkably constant at the onset of the transition and equals 0.875 for all dielectric constants in the range 80–170, independent of the zwitterion species used for r enhancement. This confirms the original observation of Wilson and Bloomfield (1979) that the onset of the folding transition depends only on the total charge neutralization of the DNA chain, which is mainly governed by counterion condensation. However, it is important to note that the Manning-Oosawa theory is only applicable when all DNA chains are in the coil state. This means that the above calculation is only valid for the coil state and, in the extreme limit, at the onset of the folding transition. Although the Manning-Oosawa theory describes remarkably well when the transition is initiated, it is thus inappropriate to explain the mechanism of the folding transition. This is clearly shown by the fact that chains in the coil state can coexist with chains in the compact state, where full neutralization is almost reached (e.g., panel b in Fig. 3). Hence, previous studies have shown that the mechanism of the folding transition is mostly driven by ion exchange between condensed monovalent and free multivalent counterions (Murayama and Yoshikawa, 1999). Thus, the difference in free energy between the coil (GCoil) and folded (GFolded) states depends on the chemical potential of free multivalent counterions, here spermine, abbreviated 
and can be expressed as follows:
 | (7) |
r is increased. To estimate the dependence of 
GF/C on r, we measured, in our fluorescence microscopic observations, the ratio of the relative population of compact (PF) and coil (PC) states (PF/PC) in the region of coexistence as a function of r. Fig. 9 shows ln(PF/PC) as a function of r for a constant spermine concentration of 20 µM (i.e., conditions in Fig. 4). This graph confirms that the compact state is preferred at low dielectric constant and shows that, within experimental error, ln(PF/PC) depends linearly on r. We can thus estimate GF/C as follows:
 | (8) |
GF/C:
 | (9) |
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 | (10) |
 | (11) |
Finally, the change in morphology with a change in the dielectric constant, as shown in Figs. 6 and 7, may also be of scientific value. The somewhat swollen morphology at higher dielectric constant suggests a decrease in the energy barrier between the coil and compact states. In a first-order phase transition, fluctuations are generally enhanced with a decrease in the free energy barrier. From this point of view, the highly disordered conformation presented in Fig. 7 illustrates the enhancement of fluctuations near "criticality".
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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r had no remarkable effect on the coil state (secondary structure and characteristic size) but strongly influenced the appearance and characteristics of the compact state (like-charged attraction state). We found that at a constant spermine concentration, single chains unfold in a first-order manner with an increase in r, which shows that the compact state is preferred at high counterion condensation. At constant r, DNA chains fold individually (first-order phase transition) when the spermine concentration increases, but the critical concentration [SPM4+]crit at which the transition occurs is an exponentially increasing function of r. A rough estimation of the total DNA charge neutralization, using the Manning-Oosawa condensation theory, shows that the onset of the folding transition always occur when 87%–88% of the charges are neutralized by condensed counterions. The compact state of individual chains consists of a toroidal structure with a diameter of 90 nm, which becomes loose and less ordered with an increase in r. These features directly illustrate the crucial role of counterion condensation in the single-chain folding transition of giant DNA, which is a typical example of a like-charged attraction phenomenon. |
ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGEMENTSREFERENCES |
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This work was supported by the Japanese Society for Promotion of Science and by a Grant-in-Aid for the 21st Century Center of Excellence "Center for Diversity and Universality in Physics" from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Submitted on January 12, 2005; accepted for publication February 22, 2005.
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), 103 (*), 131 (
), and 158 (
). DNA solutions are in pure water, glycine 1 M, 4-aminopropanoic acid 1M, and 6-aminohexanoic acid 1M, respectively.
), glycine (
), 4-aminopropanoic acid (•), and 6-aminohexanoic acid (
). Black points labeled from a to i correspond to the experimental conditions for the microscopic pictures presented in 
