Macro-ion Characterization - ACS Publications - American Chemical

Chapter 15

Role of Multivalent Cations in Condensation of DNA Victor A. Bloomfield, Chenglie Ma, and Patricia G. Arscott

Downloaded by UNIV QUEENSLAND on October 4, 2013 | http://pubs.acs.org Publication Date: December 13, 1993 | doi: 10.1021/bk-1994-0548.ch015

Department of Biochemistry, University of Minnesota, St. Paul, MN 55108

We critically survey the ways in which multivalent cations may induce the condensation of DNA, and describe results using alcohol-water mixtures which shed some light on the relative roles of electrostatics and solvent structure in the condensation process. DNA condensation arises from the interplay of several forces, including stiffness, electrostatic repulsion and counterion fluctuation attraction, hydration structure, crossbridging by condensing ligands, and interhelical binding between bases whose normal intra-duplex pairing and stacking have been disrupted by interactions with solvent or ligand. DNA is often studied by physical techniques in dilute solution, where it is a wormlike or random coil with a veiy low segment density (e.g. 4xl0" for T4 DNA). However, in biological systems such as cells and viruses, DNA is typically very tightly packaged, with a fractional occupancy of its domain that may approach 0.5. This enormous decrease in volume of the molecular coil is termed condensation. A fundamental problem in molecular biology, and in polymer physical chemistry, is to understand how DNA condensation is established and maintained. Model systems that can produce condensation in solution are of great interest for understanding DNA behavior inside cells and viruses. A general review of polymer condensation has been written by Frisch et al (i), and there are several recent overviews of DNA condensation mechanisms (2-5). Our purpose in this paper is to critically survey the ways in which multivalent cations may induce the condensation of DNA, and to describe our results using alcohol-water mixtures which shed some light on the relative roles of electrostatics and solvent structure in the condensation process. Many different types of substances cause condensation of DNA. In aqueous solutions, condensation is caused by multivalent cations: the naturally occurring polyamines spermidine*" and spermine"" (6-S), the inorganic cation hexaammine cobalt (ΙΠ), Co(NH3)6 (9,10\ which we shall henceforth abbreviate as CoHex(m), cationic polypeptides such as polylysine (11) and histone HI (12). Divalent metal cations do not provoke condensation in water at room temperatures, but they will do so at somewhat elevated temperatures (73) or in water-methanol mixtures (14,15). Ethanol at high concentrations is widely used as a precipitant in the purification of DNA, but under properly controlled conditions it can produce particles of defined morphology (16,17). Even neutral polymers, such as polyethylene glycol, at high concentrations and in the presence of adequate concentrations of salt, can provoke 5

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In Macro-ion Characterization; Schmitz, K.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


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DNA condensation (18). The resulting structure has been termed psi-DNA, or ΨDNA, the acronym for Polymer-and-Salt-/nduced which describes die condensation process. Ψ-DNA is also produced by anionic polymers, such as polyaspartate, polyglutamate, and the anionic peptides found in the capsid of bacteriophage T4 (19). In addition to the basic fact of condensation, the size and shape of the condensed particles is characteristic and presumably holds clues to underlying mechanisms. DNA condensation by multivalent cations leads to ordered particles. They are usually toroidal or rod-shaped in aqueous solutions, and rather uniform in size distribution regardless of DNA length (4), though closed circular DNA produces somewhat smaller particles (20). The size and shape of die condensates produced in nonaqueous solutions, or at high temperature, is more highly varied. Toroids and rods are produced as well as lateral aggregation of DNA into multistranded,fibrousstructures (17). Permethylated spermidine produces a higher proportion of rods (21). Even when extensively aggregated networks are formed after long times, as predicted by statistical thermodynamic equilibrium theory (5), the initially formed ordered particles maintain their compact, discrete morphologies. Comprehension of the fundamental mechanisms of DNA condensation is needed to understand packaging of DNA (and RNA) in viruses (22, 25), and the functional arrangement of DNA in eukaryotic nucleic and prokaryotic nucleoids (24,25). DNA functions in compact environments stabilized by multivalent cations. In vitro, such environments can be produced by multivalent cations. They are probably a major factor in stabilizing compaction in vivo, as are proteins, RNA crosslinks (24), and crowding/excluded volume effects (18). Viral DNA packaging usually, though not always, requires polyamines (26). DNA condensation is required for efficient catenation and recombination (27), and changes in intracellular DNA compaction produced by varying polyamine levels affect susceptibility to radiation damage (28). Excluded volume and higher local concentration in condensed DNA may lead to a speedup of several orders of magnitude in renaturation of single stranded to double helical DNA (29). In biotechnology, DNA packaging is important for genetic transfer using viral vectors and liposomes. The obvious importance of coulombic interactions in a polyion as highly charged as DNA might lead to the assumption that electrostatics is the dominant factor in regulating condensation. In the presence of divalent and higher valence cations, however, DNA behavior is not governed just by simple electrostatics. Most obviously, the very fact that condensation implies attractive forces means that other forces must overcome the coulombic repulsion between DNA phosphate charges. Hydration, counterion fluctuations, bridging by condensing ligands, and distortion of DNA secondary structure by binding interactions are also involved in condensation and other functionally significant changes of DNA behavior. Our purpose in this paper is to examine critically these various factors, to move closer to a balanced assessment of their relative importance. Interaction of multivalent cations with DNA Many of the fundamental features of counterion interaction with DNA appear to be well understood. Polyelectrolyte theories, rangingfromcounterion condensation to Poisson-Boltzmann and beyond (30, 31) agree pretty well on the thermodynamic consequences of the strong field of the DNA charges for the behavior of the counterions, and on the localization of a relatively constant number of counterions near the DNA regardless of bulk ionic concentration (territorial binding in counterion condensation terms). Equilibrium dialysis measurements of the salt dependence of polyamine binding (32) shows that the predictions of counterion condensation theory are very well obeyed for cations of charge +2, +3, and +4. A study of CoHex(m) binding (33) showed that over the range from 52 to 159 mM Na , the binding constants of CoHex(III) and spermidine* to double stranded DNA are essentially the +

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197 Multivalent Cations in Condensation of DNA

same and have the salt dependence predicted by polyelectrolyte theory. The 5-6 fold greater efficiency of CoHexÇHI) in producing DNA condensation (9) must therefore be due to some specific factor not reflected in binding constants. The predominantly coulombic interaction of polycations with DNA is buttressed by calorimetric measurements (74). For binding of spermine * to salmon sperm DNA, ΔΗ = 0. For poly (L-lysine) binding, ΔΗ = -300 cal/mol DNA-P; and for M g binding, ΔΗ = +350 cai/mol DNA-P. All of these enthalpies are very small, indicating that association of cations with DNA is entropy-driven. NMR studies of cation interaction with DNA have confirmed some of the basic ideas of territorial binding, but have also demonstrated some ion- and base sequencespecific tight binding. Wemmer et al (55) used NMR to study polyamine binding to a defined DNA sequence. They found no broadening of the spermine linewidth when most of it is bound to DNA, and weak: positive NOEs. (Negative NOEs would be expected if spermine tumbled with the DNA relaxation time.) Similar results were obtained for spermidine. Polyamine mobility thus appears to be independent of its binding to DNA. This is true even in spermine-induced aggregates of DNA. Pulsed field gradient NMR measurement (36) of the self-diffusion coefficient D of permethylated spermidine (PMS) fits best to a two-state model, in which the PMS is eitherfree,or bound to DNA (of a range of lengths) where it has a diffusion coeffient equal to that of the DNA. The extent of binding is computedfromthe Poisson-Boltzmann cell model. This implies a considerable diminution of D when bound («3-5% of Do) in contrast to the territorial binding model, which predicts abound « 1/3 Do. This could arise either because of high localization, or by trapping along thefinitelength of a DNA molecule. The latter appears more plausible in light of the results of Wemmer et al (55). Co and ^Na NMR measurement of competitive interactions of CoHex(III) and Na with helical B-DNA showed relatively minor changes in chemical shift and relaxation time, indicating that the octahedral coordination shell of CoHex(III) remains intact (57). More recent C o NMR measurements (38) of CoHex(UI) in the presence of DNA of varying GC content show evidence for at least three classes of bound CoHex(m): a small, transiently localized class; a larger, highly mobile class; and a very small,tightlybound class observed only with M. lysodeikticus DNA, which contains 72% G+C. Crytallography shows a different side of oligocation-nucleic acid interactions. Since only those ligands which are stably bound to reproducible sites appear in crystal structures, ions which are territorially bound will not be seen. Indeed, the results surveyed previously in this section would suggest that no ions should ever be seen bound to DNA. But this is not the case, and the contradiction is instructive. Early ideas on the interaction of polyamines with DNA camefromthe x-ray crystal structure of spermine-HCl (59). The polyamine was supposed to bind in the minor groove of the DNA, with its NH's making Η-bonds with the DNA phosphates. Thefirsttwo polyamine charges neutralize phosphates on one chain, and the other two bridge across the minor groove and neutralize phosphates on the opposing chain. Seemingly consistent with this picture, x-rayfiberphotographs of the s^nnine-DNA complex showed the B-form of DNA at high humidity (40). Calculation of intensities was consistent with the minor groove-binding model. However* the interhelix distance increased with hydration, and reached an upper limit. This implied interhelical crosslinking, which was also compatible with computedfiberpatterns. Actual crystal structures (as opposed tofiberdiffraction and model building) show a very different story. Spermine is a common component of crystallization buffers for nucleic acids. ThefirstB-DNA structure, of a dodecamer (41), showed that spermine was bound in the major groove, and was associated with a bend at the GC-rich end of the duplex. This behavior is also found by computer simulation (42). This association of bound spermine with loops or distortions of regular double helix structure wasfirstnoted in tRNA crystal structures (43). In a review (44) of 44


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crystallographic evidence about metal and spermine binding to tRNA, two types of electronegative binding sites for cations were identified: pockets and clefts. "Pockets are formed by high concentrations of negative charge at a point produced by a sharp bend or loop in the negatively charged phosphate backbone or by the juxtaposition of accessibel Ν and Ο atoms on adjacent bases. Clefts ... are formed when rows of backbone atoms approach each other closely." Metals prefer pockets, polyamines prefer clefts. Spermine is in the major (not minor) groove of the RNA double helix. It is in a slightly coiled conformation, rather than in the extended, all-trans form expectedfromthe crystal structure of spermine-HCl. There is a strong contrast of these crystallographic studies of spermine binding, which show only two binding sites to tRNA, with equilibrium dialysis (45) which shows that there are 13-15 strong binding sites of spermidine to various tRNAs, and that binding is governed predominantly by electrostatic forces. It is probably generally true that there is much delocalized binding in addition to a few localized sites for most multivalent cations. Returning to complexes of spermine with B-DNA, the highest resolution crystal structures are those of Williams et al (46). Spermine binds in the major groove of complexes of d(CGATCG) with the anthracycline drug 4'-epiadriamycin. A "continuous hydrophobic zone" is formed by the 5-methyl and C6 of thymidine, C5 and C6 of cytidine, and the chromophobe of the anthracycline. Spermine, through its methylene residues, makes extensive van der Waals contacts with this hydrophobic zone. Different drug complexes have different geometries, but the flexibility of the spermine allows hydrophobic contacts to be made in all cases. About 5-7 water molecules are displaced by a spermine, indicating strong hydrophobic stabilization. Spermine binds near the floor of the major groove in these complexes, where the calculated electrostatic potential is highly negative. Therefore, favorable coulombic interactions are important in stabilizing the complexes. The amino groups of spermine make Η-bonds to the DNA bases, phosphates, and sugars in some but not all cases; some of these are water-mediated. Thus, electrostatics, Η-bonding, and hydrophobic interactions arc all important in polyamine-DNA binding. When the base sequence of the DNA permits (alternating purine-pyrimidine), binding of polyamines or CoHex(HI) may provoke a transitionfromthe right-handed Β form to die left-handed Ζ form (47). In this initially solved structure, it was found that the spermine does not bind in the grooves, because they are very narrow. Instead, it binds between the molecules in the crystal. A very recently determined structure of the pure-spermine form of Z-DNA (magnesiumfree)at 1-Λ resolution showed some striking features (48). Crystals of the DNA hexamer d(CGCGCG)2 in the Zconformation, when crystallized with Spm as the only multivalent cation, have structural features quite differentfromthose of other Z-DNA crystals which had generally been crystallizedfromMg-spermine solutions. The relative orientations and positions of the DNA molecules in the crystal are changed, and the DNA structure itself is different: The helix axis is compressed, the base pairs are shifted into the major groove, and the minor groove is narrower. Most important for a potential understanding of the role of polyamines in DNA condensation, each spermine interacts with three DNAs (and each DNA oligomer with three spermines). Thus crosslinking could stabilize the condensed form of DNA. This interhelical bridging has been found to be generally true for Z-DNA, but not for B-DNA. The spermine is quite long (15.4 ÂfromNl to N14 in the pure-spermine form) but is not always completely stretched out. Its flexibility, as well as the dispersion of various sorts of interactions - electrostatic, Η-bonding, and hydrophobic - along its length, allows for a wide variety of possible interactions with different DNA molecules. When CoHex(m) and Mg are the crystallizing agents instead of spermine, crosslinks between molecules are not observed, but the structural basis for stabilization of the Z-form is clear (49). CoHex(m) stabilizes the Z-form of DNA at concentrations four orders of magnitude lower than does Mg . X-ray crystallography shows that the CoHex(III) ion forms five Η-bonds to groups on the surface of 2+

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Multivalent Cations in Condensation of DNA 199

d(CGCGCG)2, binding to 06 and N7 of G as well as to a phosphate group in the Zn conformation. In contrast, M g forms only three Η-bonds through its hydration shell, to N7 and 06 of G. The greater number of Η-bonds, specific steric fit, and higher charge all contribute to die greater stability of the CoHex complex. These x-ray results (and similar hintsfromNMR) show that, in addition to the delocalized binding predicted by polyelectrolyte theory, specific complexes of polyamines and CoHex(III) with DNA and RNA can form. These may have distorted geometries and crosslinking between helices. The relevance of these observations for the mechanism of DNA condensation by multivalent cations is obvious. 2+


How multivalent cations may cause DNA condensation There is strong evidence that electrostatic forces are important in condensation. The laws of physics require that something must be done to neutralize the strong repulsive interactions as negatively charged DNA segments approach closely; this is the most obvious reason that high salt is required in Ψ-condensation (18). Numerous studies (9, 10, 14, 50) make quantitative the requirement for reduction of coulombic interactions: approximately 90% of the DNA charge must be neutralized by counterion condensation before DNA collapse can occur. As reported later in this paper, this result holds for alcohol-water mixtures as well as for pure aqueous solvent. However, there are some subtleties in this conclusion. ΟοζΝΗβ^^ is a 5-6 fold more effective condensing agent than spermidine**" (9,50) despite equal binding of these isovalent compounds (33). Therefore, factors other than valence are important. Further, as Benbasat (50) snowed, equal percentage charge neutralization does not necessarily mean equal residual linear charge densities for all types of DNA. She studied wild-type and mutant forms of | W14 phage DNA, in which some of the thymidines are derivatized to give average charges per phosphate rangingfrom-0.76 to -1.24 (compared to -1 for normal DNA). In all cases except the lowest charge density, condensation with spermidine occurred when 89% of the DNA charge was neutralized. Thus the key factor seems not to be the effective linear charge density, but rather the neutralization of each fixed unit phosphate charge. A further subtlety is that the electrostatic repulsion determined by osmotic stress measurements cannot be explained simply by screened coulombic interactions between parallel rods (51). At large interhelical distances, greater than 30 A, the force-distance curve depends on ionic strength with a decay length about half that predicted by Debye-Hiickel theory. This is due to fluctuations of the flexible DNA helices, maximizing configurational entropy and bringing charged groups closer together than expected for an orderly array of parallel cylinders. Substantial reduction of the repulsive electrostatic interaction between DNA helices does not in itself account for the attractive interaction stabilizing the condensed form. Induced dipole interactions between the polarizable ion atmospheres surrounding the polyions can lead to a net attraction, as first proposed by Oosawa (52). As estimated by Marquet and Houssier (5), this attraction should be sufficient to stabilize condensed DNA with trivalent cations in water or divalent cations in watermethanol mixtures. Analytical calculations in our laboratory (Rouzina & Bloomfield, in preparation) show that similar results can be obtainedfroma model based on fluctuating interactions between closely apposed surface charge lattices. Rather elaborate grand canonical Monte Carlo calculations show a net attraction between hexagonally packed DNA cylinders in the presence of divalent counterions (53,54). These theoretical results seem to show that coulombic interactions have the strength to account for both the repulsive and attractive aspects of DNA condensation. But that does not mean that they are the whole story. Not only can hydration forces (see below) apparently give an equally good account of the phenomena. But our results with water-alcohol mixtures clearly show that while the solvent dielectric constant is a controlling variable, other factors are also at work. 3


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Despite the intuition that electrostatics should dominate, the reality is more complicated. Force-distance measurements of DNA parallel helices as a function of salt concentration and valence, with monovalent and divalent cations, show a surprising result: all ionic influences nearly disappear when the separation between DNA surfaces is 5-15 Â. The repulsive pressure decays exponentially, with a characteristic length of 2.5 - 3.5 A, behavior which has also observed for forces between phospholipid bilayers and musclefibers.This was interpreted by Rau et al (55) to demonstrate the overwhelming importance of hydration structure forces as the DNA surfaces approach. These forces are presumed to be due to water structure imposed not by hydrophobic groups, as in the familiar hydrophobic bonding, but rather by the water-soluble, polar and charged groups at the DNA surface. At interaxial distances of 26-30 A typical of DNA packing in bacteriophage, the packing energy per base is 0.1 - 0.4 kcal/mol, corresponding to a DNA pressure of 1.2 - 5.5 χ 10 dyne/cm . This is about one order of magnitude less than the electrostatic repulsion estimated by Riemer and Bloomfield (56), but greater than the conformational energies estimated in that work. These hydration structure forces can under suitable conditions be attractive as well as repulsive. Rau and coworkers (57,58) found that condensing agents such as CoHex(m), spermidine, spermine and chromium exert an attractive force that brings DNA molecules only to an 8 - 10 A separation. If the molecules are pushed further together by osmotic stress, an exponential repulsion with a 1.3 to 1.5 Â decay length half the normal value - is observed. This type of behavior is explained by a theory that postulates the rearrangement of surface-bound water by condensing ligands, to create regions of hydration attraction, or water bridging, between helices. Thermodynamic analysis shows that a significant amount of extra CoHex(III) is bound upon condensation. The collapse transition is largely entropy-driven (58). A chaotropic anion, perchlorate, favors the collapse of Mn-DNA because the accompanying displacement of bound water into the bulk has a more favorable AS. In contrast to CoHex(III), there is no net binding of M n when condensation occurs, implying that a subtle rearrangement of cations causes water restructuring and a switch to net attraction. In view of these observations, Rau and Parsegian (57) give reasons why they feel that forces between DNA helices cannot be characterized by balance of repulsive double-layer and attractive van der Waals interactions. (1) At distances of 10-15 A, the repulsive force decreases exponentially with a 3 A decay length which is independent of ionic strength or ion type. The coefficient of the force differs for different counterions, but magnitudes are similar. (2) At distances shorter than the collapse minima, the decay length is 1.3 to 1.5 A, independent of ionic strength and valence or chemical nature of the condensing ion. (3) Added NaCl should screen repulsions and enhance net attraction according to double-layer theory. Instead, it causes the condensate to dissociate. (4) The same decay lengths are seen in other systems: e.g. phospholipid bilayers and collagen. These arguments are cogent, but perhaps not definitive. For example, while meanfield Poisson-Boltzmann theory cannot explain the exponential decay and the independence on counterion concentration and valence, the situation is not so clear for a polyelectrolyte theory that takes fluctuations and correlations into account And objection (3) is explicable even in terms of counterion condensation theory, since addition of NaCl displaces multivalent counterions, thereby lowering the fractional neutralization of DNÂ backbone charge. Perhaps the most obvious cause of DNA condensation is crosslinking by the condensing ligand. There are several pieces of evidence favoring this idea. Suwalsky et al (40) used X-ray diffraction to measure the distance between DNAfibersin DNA-spermine complexes, as a function of relative humidity. The distance increased with hydration, but reached an upper limit consistent with spermine crosslinking. Allison et al (59) condensed DNA with homologs of spermidine, in which the butyl 7



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moiety was replaced by longer pentyl through octyl groups. Aggregation became a more prevalent occurrence relative to condensation as the length of the end chain increased, suggesting an important role for crosslinking. Schellman and Parthasarathy (60) used spermidine analogs of the structure NH3 -(CH2)3-NH2 (CH2)n-NH3+ with η = 3 , 4 , 5 , and 8 and aliphatic diamines NH3+-(CH2)n-NH3 with η = 2,3,4,and 6 to collapse DNA. They found that the interhelical spacing varied systematically with the length of the methylene bridge, and that the ionic strength of the solution had no effect on the spacing. This suggests that the arrangement of DNA in the complexes is determined by the structure of the polycation, not by long-range electrostatic repulsive and attractive forces. However, the crosslinking mechanism is challenged by results with M g in water-methanol, where the spacing is as much as 3 2 Â , much greater than the diameter of a hydrated Mg ion (57). It must also be emphasized that in B-DNA crystals where spermine or C O ( N H 3 ) O has been localized, it has never been found in a crossbridging location. Instead, as noted above, it is generally associated with some loop or distortion of a double helix. However, linking by spermine between molecules of a Z-DNA hexamer has been observed (48,61). There is increasing evidence that at least part of the glue that holds condensed DNA together comesfromdistorted secondary structure induced by the ligands. This idea is supported by numerous observations that condensing cations induce conformational changes in DNA. The chemical specificity of 2 ° and 3 ° structural changes contrasts with the nonspecificity predicted by polyelectrolyte theories based solely on cation size and radius. It is by now widely accepted that DNA is quite a plastic molecule, able to adopt a wide range of conformations both within the Β family and in other helical families (A,C,D,Z, etc) (62) depending on sequence, humidity, superhelical stress and other factors. Raman spectroscopy and crystallography of oligonucleotides show that a rather large number of sequences [not just the canonical (dCdG) ) or (dCdA) )] are either strongly or weakly susceptible to being in the Ζ conformation under high salt conditions (63, 64). Using chemical probes, Johnston (65) has shown that with increasing negative supercoiling, Z-DNA sequences can extendfroman initial 1 4 bp region that is mainly alternating purine-pyrimidine, to flanking regions that have less alternating character. Polyelectrolyte theories have been remarkably successful in explaining and predicting thermodynamic properties of DNA and its complexes. Perhaps because of this success, it is not widely appreciated that, in those cases where structures have been determined, multivalent cations seem to be generally associated with distortions of the double helix. An early demonstration of this was by Eichhorn and Shin (66), who showed that a wide variety of divalent cations, especially transition metals that bind to bases in preference to phosphates, cause apparent optical melting of DNA at temperatures well below those expected on the basis of ionic strength. The rapid reversibility of the hyperchromicity upon addition of EDTA suggests that bases were locally unpaired or unstacked, without extensive unwinding of the backbones. Under low ionic strength conditions, Mg provokes the B-Z transition in poly[d(G-me C)] (67). IR and CD studies of DNA in the presence of N#+ show helical distortion (68); the N i appears to form a strong inner-sphere complex with N7 of adenine (69). Extrusion of cruciforms by an S-type pathway depends on ionic size in a series of divalent cations, suggesting that specific ion-binding cavities are formed by the geometry of the four-way junction (70). The formation of extreme bends in kinetoplast minicircles is also induced by binding of Zn , Co , Ba , and M n (71). The distortions induced by spermine*" have been noted earlier. We have reviewed the evidence that DNA is structurally plastic, and that structural variations are induced by the same multivalent cations that cause condensation or aggregation. An intriguing possibility is thus suggested, that condensed DNA structures are stabilized to a significant extent by interaction +

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between helical segments that have been perturbed from the normal Β conformation. There is a range of circumstantial evidence supporting this hypothesis. Cytosine residues of Sa phage DNA which has been condensed by poly-lysine are reactive to HSO3, indicating perturbed 2° structure (72). Much data comesfromZ-forming polymers, particularly poly[d(G-C)] and poly[d(G-me C)]. Van de Sande and Jovin (75) showed that the left-handed form of poly[d(G-C)] (designated Z* DNA) produced by 0.4 mM MgCh and 20% ethanol, or 4 mM MgCh and 10% ethanol, sedimented readily out of solution at low speed, indicative of extensive aggregation. Kinetic turbidity studies indicate that Z* DNA is formed by a nucleated condensation mechanism (74). Detailed studiesfromour lab and elsewhere (75-78) have shown that collapse/aggregation and B-Z transition generally occur together for poly[d(GC)] and poly[d(G-meC)] in a wide variety of conditions. Chaires and Sturtevant (79) have shown by differential scanning calorimetry and spectroscopy that the B-Z transition in poly[d(G-C)] is followed by a transition with the characteristics of an aggregation reaction; both are reversible at low Mg and well below T . Electron microscopy often shows close parallel association of DNA strands under condensing conditions. Some helical modifications observed with poly[d(G-C)], such as fourstranded hairpins and loops built up by the sticking together of two segments of DNA, have also been seen with natural sequence plasmid DNAs (75). Further insight into the mechanism of aggregation comesfromuse of transition metal ions which prefer to bind to the bases, in contrast to Mg which preferentially binds to phosphate. An aggregating, left-handed form of poly[d(G-C)] can be produced in the absence of EtOH by Mn , N i and Co"*- (80). The ability of transition metals to aggregate natural sequence calf thymus DNA at elevated temperatures has been extensively studied in our lab (75). There is a strong correlation between the effectiveness of the divalent cations in producing aggregation, their effectiveness in producing apparent thermal melting of DNA (6P transition is notreversedon cooling or adding MeOH, but is instantaneously reversed when water is added, indicating no extensive strand separation in the P-form. Similar unstacked but rapidly renatured DNA is observed at lower MeOH in the presence of divalent ions (75). 5


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Effects of alcohol on DNA condensation If electrostatic interactions are important in condensation, then solutions with lower ε should need less, or lower valence, condensing agent because electrostatic interactions leading to counterion condensation are stronger (74). To test this, we varied εfrom80 to 50 by adding various amounts of alcohols (methanol, ethanol, and isopropanol) to water. We used laser light scattering and electron microscopy (EM) to study effects of dielectric constant on changes in aggregation number, hydrodynamic radius, and morphology of plasmid pUC 18 DNA condensed with CoCSH^)^* We determined therelationbetween ε and the critical Co(NH3)* concentration required to induce condensation. Thefractionof DNA phosphate charge neutralized by both Na (held constant at 1 mM) and Co(NH3)$ was calculated using a modification of 3+

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the two- variable theory of counterion condensation. EM allowed estimation of the number of DNA molecules per particle, and the detection of a trend in particle morphology from toroidal through rodlike to fibrous states as ε decreases. The closed circular plasmid pUC 18 DNA (2686 base pairs) was isolated and purified by standard procedures, and analyzed by agarosç gel electrophoresis and restriction endonuclease. The plasmid DNA was stored at 4 C as a concentrated stock solution, 50 μg/ml in 10 mM Tris Cl + 10 mM Na cacodylate, pH 7.0,1 mM EDTA. All static and dynamic light scattering studies were done with 10-fold dilutions of this stock solution (5 μg/ml DNA, 1 mM NaCl, 1 mM Na cacodylate) into alcohol-water mixtures of the desired composition. Highly purified Co(NH3)6Cl3 was purchased from Kodak and used without further purification. All buffers were filtered through 0.22 μπι GS Millipore filters before mixing. The weight percentages of alcohols used to obtain a specific ε are ε

80 MeOH 0.0 EtOH 0.0 i-PiOHO.O

75 11.7 8.2 7.4

70 22.0 17.0 14.2

65 32.1 25.9 21.1

60 42.6 34.7 28.0

55 50 53.1 63.0 43.6 52.4 34.8 41.7

To compute the extent of counterion condensation and DNA charge neutralization in a solution with two cation species 1 and 2 of different valences, we solved numerically the coupled equations (14) 1 + In ( T ^ ) =

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Ζ

θ

1 ^ * 1 1 • Ζ2θ2)β-*>

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9i is the number of associated counterions of species i per DNA phosphate charge, Ci and Zi are the molar concentration and valence of ion i, e is the base of natural logarithms, κ is the Debye-Huckel screening parameter, b is the linear charge spacing of the DNA in the absence of condensed counterions. ξ is the familiar counterion condensation parameter, q ^BTb. For DNA in aqueous solution, b = 1.7 Â and ξ = 4.2 at 25°C. Vpj is the volume per mole of phosphate in which the condensed counterions are territorially bound, 2

ν ΐ = 4πΝΑ(1+Ζ0(ξ-Ζι-1)1)3 ρ

(3)

where N A is Avogadro's number. Once B\ and Θ2 are known, the fractional charge neutralization of the DNA is given by Γ = Ζιθι + Ζ θ . 2

2

(4)

Dynamic light scattering autocorrelation functions were analyzed by second order cumulant analysis to obtain the z-average diffusion coefficient, from which the hydrodynamic radius RH was derived by Stokes* law. All scattering measurements were made at a scattering angle of 90° with an Ar+ laser operating at 488 nm. For electron microscopy, a drop of sample was deposited on a carbon coated 200 mesh copper specimen grid and allowed to stand for 1.5 minutes. The grids were previously glow-discharged for 1.5 minutes to render them hydrophilic and thereby facilitate staining. Each grid was stained by a drop of 1% uranyl acetate solution and blotted withfilterpaper after 30 seconds. The grid was then allowed to dry in air for at least 10 minutes. EMs were taken at a magnification of 50,000 with an Hitachi H6010 microscope operating at 75 kV. Negatives were optically projected at 6x


204


enlargement, and the images of individual particles were traced on paper. These traces were scanned and digitized. For each toroid, inner and outer radii were the average of four perpendicular measurements. The random coil conformation of DNA in buffered aqueous or low to moderate concentrations of alcoholic solution has little scattering power compared to condensed DNA. When DNA molecules condense to a smaller structure, or when the average number of DNA molecules in a scattering particle is increased by aggregation, the intensity of scattered light greatly increases. Figure 1 shows the scattering intensity two hours after addition of Co(NH3)6 , plotted against ε of EtOH-water mixtures. The intensity increases as ε decreases, reaching a maximum at ε = 70. It then decreases to a plateau as ε further decreases. The increase in scattering intensity as the concentration of Co(NH3)$ increases indicates that the DNA molecules get more compact and/or the aggregates get larger as larger amounts of condensing reagent are added. Similar behavior is seen with MeOH and PrOH. RH, measured two hours after addition of the condensing reagent, decreases moderately as ε decreases from 80 to 70, and then increases as ε is further lowered. At ε = 70 and above, RH is independent of the species of alcohol. These results indicate chain compaction (along with multimolecular association) down to ε = 70, then further growth of the particles at lower dielectric constant. We were unable to monitor reliably the dynamic scattering behavior of condensed DNA molecules at ε less than 65. This is due to the low scattering intensity, partial precipitation, and polydispersity of the remaining soluble DNA particles in the presence of multivalent cations and large amounts of alcohol. Under these circumstances, EM shows that the molecules are crosslinked and may produce a gel-like structure. When increasing amounts of the condensing cation are added, an abrupt increase in the light-scattering intensity-is noted at a critical concentration. The critical concentrations Ccrit of C o f N î ^ needed for collapse in water and alcohol solutions with ε ^ 70 are ε: 80 75 70 Qrit rcalc leak Ccrit calc Ccrit MeOH 30±2 .894 28±2 .900 25±2 .905 EtOH 30±2 .894 27±2 .899 23±2 .905 PrOH 30±2 .894 24±2 .898 18 ± 1 .902 3+


3+

r

As expected, Ccrit generally decreases with decreasing ε, but Ccrit is not the same for different alcohols at a given ε. The values of r at Ccrit* calculated according to equations 1-4, all confirm the empirical rule that DNA condensation occurs at about 89-90% DNA phosphate charge neutralization. light scattering kinetic measurements were made with the first point measured 15 seconds after addition of CoiNHi^ *. Decreasing ε by increasing the alcohol concentration speeds up DNA condensation and produces more strongly scattering particles. There are two phases: an initial veryrapidrise,followed by a slower increase over a couple of hours.The initial phase is greater in magnitude andrate,the longer the hydrocarbon side-chain of the alcohol. However, kinetic measurements at equivalent r values show that the concentration of condensing agent is an even more important factor than the dielectric constant or hydrophobicity of the alcohol. The EM morphology of condensed DNA particles was found to be dependent on ε, but not on die species of alcohol. In the range of alcohol concentrations used, secondary structural transitions should not occur in the absence of condensing reagent (75). The proportion of rods increases sharply from 4.9% to 85% as ε decreases from 80 to 65. At ε = 60 or below, multistrandfibersare dominant The observation of toroids in aqueous solution is commonplace by now, but the high fraction of rods has not previously been observed in plasmid DNAs condensed in low concentration of alcohol solutions. Rods had been observed in very high EtOH solutions (95% v/v) or in solutions containing poly-(lysine) or PMS as condensing agents (77,77,27). 3




15. BLOOMFIELD ET XL

50

55

60

65

70

75

80

ε 3+

Figure 1. Scattering intensity two hours after addition of Co(NH3)$ , plotted against ε of EtOH-water mixtures.


206


From the interhelical spacing of 27.5Â determined for Co(NH3)* - condensed DNA (60), an assumption of hexagonal packing, and the measured dimensions of toxoids, we can estimate the number of plasmid molecules per condensed particle. In aqueous solutions this number is fairly stable, remaining about 5.5 for up to a month. The molecularity in alcohol solutions with ε = 75 is less reproducible and changes more with time. The average, however, is 5.2 molecules/toroid, not significantly different from the value in aqueous solutions. Discussion


3+

DNA, in the presence of Co(NH3)$ , collapses into different types of compact structures when subjected to different degrees of alcohol dehydration. Electrostatic, hydration, and kinetic factors are probably all involved in determination of morphology (4,5). The mechanism of formation of condensed particles, and the factors leading to different relative amounts of toroid or rod even in aqueous solution, are still matters of debate. Marx and Ruben (83) have shown that DNA in toroidal condensates is circumferential wrapped; each DNA molecule winds severaltimesaround the toroid. Formation of toroids requires continuous bending of the DNA double helix with a positive free energy related to the persistence length and radius of curvature. Plum et al. (21) suggested that morphological differentiation of condensates is controlled by a kinetic process. They presented data to show that PMS induces condensation at a slower rate and produces many more rods than either Co(NH3>6 or unmodified spermidine. Formation of rods requires sharp bending or kinking of the DNA helix at the ends of the rod. These deformations require more energy locally than constant circumferential bending. Thus they might require more activation energy and therefore be slower than smooth wrapping of the DNA. However, bending occurs continuously and kinking occurs only at the ends of rods. They are therefore expected to have similar total energies. Matters become even more complicated when alcohol is added to the mix. When the concentration of alcohol is low, the water structure around the DNA helix remains unchanged, but the dielectric constant decreases. This leads to a higher degree of charge neutralization, causing both reduced electrostatic repulsion and also a lower persistence length or greaterflexibility.This should permit compact, strongly curved toroidal or rodlike particles to form more readily. Further increasing the alcohol concentration leads to progressive dehydration of the DNA. This has been shown to elicit extensive changes in the hydrodynamic properties of the DNA helix and can lead to the formation of compact structures. Multivalent cations could modulate this dehydration, particularly by augmenting the associated distortion of helix geometry. Alcohol at higher concentration also decreases DNA solubility due to the hydrophobicity of alcohol molecules and shielding the negatively charged backbones of interacting helical segments, thus facilitating the contact of DNA molecules. It is interesting that ethanol and water have maximal enthalpy of mixing in the concentration rangefrom35 to 60% (84). This is just the range where the transitionfromrods to multi-strand fibers occurs, suggesting that some thermodynamic or structural property of the mixed solvent system must be important in detennining morphology. Kinetic factors may also be important in these morphological differences. It seems likely that DNA in solutions containing multivalent cations at low dielectric constant may be subjected to such strong condensing forces that there is insufficient time for bending or kinking. Widom and Baldwin (9) showed that condensation by Co(NH3>0 in aqueous solutions to yield toroidal particles took about 1/2 hour. Thus DNA molecules may pack together more rapidly by aligning side by side. 3+

3+





Summary An overview of all these results indicates that DNA condensation arises from the interplay of several forces. Stiffness sets limits ontightcurvature. Electrostatic repulsions must be overcome by high salt concentrations or binding of multivalent cations. While mean field polyelectrolyte theories of the Poisson-Boltzmann type may not account for net attraction, counterion fluctuation mechanisms cannot be ruled out. Hydration must be adjusted to allow a cooperative accommodation of the water structure surrounding surface groups on the DNA helices as they approach. Unfortunately, hydration forces cannot be convincingly computed from first principles for comparison with experiment at the present time. Repulsive excluded volume interactions with other polymers in the solution can force the DNA segments closer together. Additional attractive free energy may be provided by bridging through condensing ligands (but there are at least some instances where necessary bridging distances are too large), and/or by interhelical binding between bases whose normal intra-duplex pairing and stacking have been disrupted by interactions with solvent or ligand. In cation-induced condensation of random sequence DNA, only a smallfractionof the base pairs can be disrupted since the CD spectrum is very similar to that of B-form DNA. However, the evidence in favor of the involvement of such disrupted helices is sufficiently great, and suggests such novel possibilities, that it deserves serious consideration. Acknowledgments This research was supported by NIH Grant GM28093. Literature Cited (1) Frisch, H. L.; Fesciyan, S. J. Polym. Sci., Polym. Lett. Ed. 1979, 17, 1-7.

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