The in vitro nuclear aggregates of polyamines Aldo Di Luccia 1,2, *, Gianluca Picariello 1, *, Giuseppe Iacomino 1, *, Annarita Formisano 1 , Luigi Paduano 3 and Luciano D’Agostino 1,4 1 Institute of Food Sciences, National Research Council (CNR), Avellino, Italy 2 Department PROGESA, University of Bari, Italy 3 Department of Chemistry, University of Naples ‘Federico II’, Italy 4 Department of Clinical and Experimental Medicine, University of Naples ‘Federico II’, Italy Self-assembly of polyamines – putrescine (Put), spermi- dine (Spd), and spermine (Spm) – with phosphate ions was previously described by our group [1]: the interca- lation of a phosphate anion between the N-terminal ends of two polyamines determines, by electrostatic interaction, the formation of basic cyclical structures that further aggregate into supramolecular complexes [2] by means of hydrogen bonds, thus producing three different structural classes of molecular aggregates that interact with the genomic DNA [1,3,4]. These com- pounds were named nuclear aggregates of polyamines (NAPs). Interestingly, other authors have described the phosphate-induced self-assembly of polyamines in a different biological setting [5]. Polyamine and phosphate self-aggregation is reputed to be an important phenomenon in directing DNA orga- nization and functions [1]. In our earlier studies, Caco-2 cells were used to assess the biological properties of NAPs, but investigations concerning NAPs extracted from nuclei of many different cell types have also been described [1,3]. However, only preliminary observations concerning the in vitro production of these compounds have been reported [1,3,4]. In addition, the mecha- nism(s) regulating the supramolecular self-aggregation of polyamines and phosphates and the cooperative action of NAP–DNA aggregates have yet to be defined. For this reason, we determined the conditions neces- sary for the aggregation of polyamines in a simplified Keywords DNA interactions; nanostructures; polyamines; self-assembly; supramolecular chemistry Correspondence L. D’Agostino, Department of Clinical and Experimental Medicine, University of Naples ‘Federico II’ Ed. 6, Via S. Pansini, 5, 80131 Naples, Italy Fax: +39 081 7462707 Tel: +39 081 7462707 E-mail: luciano@unina.it *These authors contributed equally to this work (Received 16 September 2008, revised 9 February 2009, accepted 11 February 2009) doi:10.1111/j.1742-4658.2009.06960.x Natural polyamines (putrescine, spermidine, and spermine) self-assemble in a simulated physiological environment (50 mm sodium phosphate buffer, pH 7.2), generating in vitro nuclear aggregates of polyamines (ivNAPs). These supramolecular compounds are similar in structure and molecular mass to naturally occurring cellular nuclear aggregates of polyamines, and they share the ability of NAPs to interact with and protect the genomic DNA against nuclease degradation. Three main ivNAP compounds were separated by gel permeation chromatography. Their elution was carried out with 50 mm sodium phosphate buffer supplemented with 150 mm NaCl. Freezing and thawing of selected chromatographic fractions obtained by GPC runs in which the mobile phase was sodium phosphate buffer not supplemented with NaCl yielded three different microcrystallites, specifically corresponding to the ivNAPs, all of which were able to bind DNA. In this study, we demonstrated that in vitro self-assembly of polyam- ines and phosphates is a spontaneous, reproducible and inexpensive event, and provided the indications for the production of the ivNAPs as a new tool for manipulating the genomic DNA machinery. Abbreviations DLS, dynamic light scattering; EtBr, ethidium bromide; GPC, gel permeation chromatography; ivNAP, in vitro nuclear aggregate of polyamines; NAP, nuclear aggregate of polyamines; Put, putrescine; Spd, spermidine; Spm, spermine. 2324 FEBS Journal 276 (2009) 2324–2335 ª 2009 The Authors Journal compilation ª 2009 FEBS in vitro model in order to investigate some of the fea- tures of the polyamine–phosphate interactions. Specifi- cally, we examined the role played by each polyamine in the self-assembly of in vitro NAPs (ivNAPs) and their ability to interact with genomic DNA. Further aims of the present study were to investigate the mech- anisms that regulate the interactions among polyam- ines and phosphate ions that induce the assembly of these supramolecular structures, and to gather addi- tional conceptual elements for molecular modeling and determination of NAP functions. In this article, we report findings indicating struc- tural and functional analogies among extractive and synthetic NAPs: therefore, according to their mole- cular masses, and in keeping with the terminology of natural NAPs [3], we named the synthetic compounds l-ivNAP, m-ivNAP, and s-ivNAP (in vitro large, medium and small), respectively. Furthermore, for the first time, we show images of crystallized aggregates of polyamines and phosphates interacting with genomic DNA. Results and Discussion Gel permeation chromatography (GPC) analysis of ivNAPs In vitro aggregation of polyamines and phosphate ions generated supramolecular compounds, the ivNAPs, characterized by an extended electronic delocalization detectable by a distinctive absorbance peak at k = 280 nm in the UV spectrum, which is completely absent for unassembled polyamines (data not shown). Representative GPC profiles of ivNAPs are shown in Fig. 1, where it is also possible to analyze GPC chromatogram modifications by varying the concentra- tion of one of the three polyamines at a time (range 5–48 mm), while keeping the concentrations of the other two constant (24 mm). Three main peaks with different intensities and esti- mated molecular masses of  8000, 5000 and 1000 Da, according to increasing elution time and corresponding to l-ivNAP, m-ivNAP and s-ivNAP, respectively, were detected. Although polyamine concentrations of 24 lm were able to produce detectable GPC peaks [1], we noted that the peak variations were more appreciable when a 24 mm polyamine solution was used. The GPC profiles and the estimated molecular masses of the ivNAPs were similar to those of naturally occurring NAPs, particularly those found in the nuclei of the cells at the top of their replication phase [1]. Attempts to assemble ivNAPs in phosphate- free buffers failed. In fact, no GPC peaks were detected at k = 280 nm when polyamines were dissolved in 100 mm Tris ⁄ HCl pH 7.2 buffer (data not shown). Fig. 1. Self-assembly of polyamines assayed by GPC with detec- tion at k = 280 nm. Chromatograms were obtained by progres- sively increasing (in the range 5–48 m M) the concentration of (A) Spm, (B) Spd and (C) Put, keeping the concentration of the remain- ing two polyamines constant at 24 m M. A. Di Luccia et al. ivNAPs FEBS Journal 276 (2009) 2324–2335 ª 2009 The Authors Journal compilation ª 2009 FEBS 2325 The ivNAP chromatographic peak areas as a func- tion of the stepwise change of polyamine concentra- tions are reported in Table 1. In all three sets of experiments, the peak area corresponding to m-ivNAP remained the most prominent. The peak area of m-ivNAP – 50.3 min retention time – was only slightly affected by the polyamine concentration. The increase in concentration of the three polyamines caused a pro- gressive decrement in l- ivNAP areas (retention time: 54.3 min), whereas only minor fluctuations were observed for the s-ivNAP areas (retention time: 44.6 min). Another interesting feature of this kind of polyamine assembly was the complete fusion of the l-ivNAP peak with that of m-ivNAP (Fig. 1A), recorded at 48 mm Spm. Self-assembly is a process by which molecular subunits spatially organize in well-defined supra- molecular structures through noncovalent interactions. The structures generated in molecular self-assembly are usually in equilibrium states (or at least in metasta- ble states). Self-assembled molecular compounds have been recognized in biological systems [1,3–6], and designed for the generation of advanced materials [7] by means of the aggregation of nanoparticles. At the moment, self-assembly is the most general strategy uti- lized for generating nanostructures [7]. Self-assembly of polyamines and phosphates is, in our case, substantiated by the detection at 280 nm of a discrete set of aggregates with estimated molecular masses ranging from 1000 to 8000 Da, arising from low molecular mass species, and by the absence of covalent interactions in the aggregates. Furthermore, the appearance of the absorbance band around 280 nm, missing in single polyamine solutions (data not shown), is the demonstration that the aggregation of the single components determines an impressive change in their electronic properties. The absorbance band at 280 nm is due to the establishment of an electron delocalization favored by the electrophilic properties of the polyamines and the cyclic structure of the unimers. Surprisingly, whatever the polyamine concentrations – assayed in the range 24 lm to 48 mm – used, the for- mation of three ivNAP compounds was observed, and these compounds had estimated molecular masses very close to those of the ‘biological’ aggregates. This spe- cial chemical–physical behavior indicates that some sort of molecular mass set point regulates polyamine– phosphate ion self-assembly. Thus, the formation of these complexes can be attributed to an existing chemi- cal and thermodynamic equilibrium between reagents (polyamines and phosphates) and products (ivNAPs) [8]. Furthermore, our data suggest that self-structuring of polyamines and phosphate ions occurs within well- defined ratios, as predicted [1,3,4], indicating that this kind of aggregation is a finely self-regulated chemical– physical event. One of the principles of self-organization is the tran- sition from a disordered to an ordered state by sponta- neous symmetry breaking. The transition from a disordered into an ordered phase takes place through changes in thermodynamic or physical field strengths. Such changes may be of temperature and chemical potential (concentration, pH value, salt addition), of mechanical fields (pressure, shear, extension, ultrason- ics), or of electric and magnetic fields. In our case, it seems that the increase in polyamine concentration, the sole variable, functioned as an ‘actuator’ and ‘sta- bilizer’ of symmetry, producing an ordered state. This last condition is characterized by the facts that individ- ual molecules are located at restricted three-dimen- sional regions, and that a localization is always accompanied by a decrease in the number of realizable states and, hence, a loss of entropy. Furthermore, in phosphate-buffered solution or in a phosphate ion-rich environment (in vivo), enthalpy Table 1. Percentage distribution of ivNAPs. Relative amounts of ivNAPs were estimated by integrating the peak area of the GPC chromatograms (Fig. 1) obtained from the separation of polyamine solutions prepared by changing the concentration of a single poly- amine and keeping the concentrations of the other two constant (24 m M). In the case of variation of Spm concentration, the mean ± standard deviation (SD) values were calculated from three observations (at 5, 10 or 24 m M), as the m-ivNAP and l-ivNAP areas fused at 48 m M. ND, not detected. Polyamine concentration l-ivNAP (% relative) m-ivNAP (% relative) s-ivNAP (% relative) Put (m M) 5 34.9 50.4 14.7 10 13.2 63.7 23.l 24 12.9 64.7 22.4 48 11.4 62.1 26.5 Mean ± SD 18.1 ± 11.2 60.2 ± 6.6 21.7 ± 5.0 Spd (m M) 5 18.2 59.6 22.2 10 16.6 64.6 18.8 24 13.5 66.7 19.8 48 5.1 68.7 26.3 Mean ± SD 13.3 ± 5.8 64.9 ± 3.9 21.8 ± 3.3 Spm (m M) 5 30.8 49.5 19.7 10 23 58.6 18.4 24 12 64.4 23.4 48 ND 82.8 17.2 Mean ± SD 21.9 ± 9.4 57.5 ± 7.5 20.5 ± 2.6 ivNAPs A. Di Luccia et al. 2326 FEBS Journal 276 (2009) 2324–2335 ª 2009 The Authors Journal compilation ª 2009 FEBS changes are due to cooperating short-range attractive and long-range repulsive forces established by charged polyamines [9]. All of these principles can be evoked to give a possible explanation for the exclusive aggre- gation of the polyamines and phosphates into three molecular complexes. Another intriguing point is the relationship existing between the three-dimensional arrangement of these structures and the regular production of only three main compounds, whatever the solute (polyamine) concentration was. We are persuaded that the number of hydrogen bonds is crucial in defining both the three-dimensional outlines and the molecular masses. In our previous papers [3,4], we proposed a hierar- chical process of supramolecular polymerization based on the assembly of polyamines and phosphates (the extractive NAPs). The initial step is the self-arrange- ment of polyamines in disk-like unimers by means of their terminal interactions with the phosphate groups. The formation of ring-like unimers can be attributed to the low equilibrium constant for isodesmic polymer- ization [10], which characterizes the system, whereas the successive formation of the medium and large assemblies is an expression of a ring stabilization pro- cess. A clear example of this multistep process of supramolecular assembly is m-NAP, which in solution – unbound to the DNA – was depicted as structured in a two-dimensional planar (not columnar) disk-like arrangement resulting from the oligomeric aggregation of five s-NAP unimers [3] (Fig. 2). Our modeling should be considered in line with an isodesmic supra- molecular polymerization [10] for the further reason that, since this theory predicts the production of only oligomers and a preferential disposition of the unimers in a linear chain, rather than their columnar stacking, if the hydrogen bonds are single and arranged in a chain [3]. The data reported here concerning the ivNAPs support this belief, as a linear chain-type assembly fits better with the constant and reproducible detection of low molecular mass aggregates (oligomers) than with a columnar stacking of disks (polymers) that, by means of the serial aggregation of available disk-like monomers, should ultimately generate com- pounds with greater molecular masses. However, it is interesting to note that, in the case of their interaction with the DNA, the assembly of these supramolecular structures can be imagined, without contradiction, to be in a columnar form. In fact, the establishment of two or more hydrogen bonds among adjacent disk-like unimers can ultimately lead to the formation of supramolecular nanotubes enveloping the entire DNA [4]. The process of interaction and colum- nar disposition of the unimers along the DNA grooves is probably driven by the phosphates of the DNA, which can in part replace (two for each ring) the phos- phates terminally linking the polyamines [4] (Fig. 2). A similar mechanism, based on the recognition of specific helically distributed chemical groups, has been already described in biological systems, e.g. for the assembly of the protein capsid of tobacco mosaic virus along the polynucleotide chain. Namely, it is well established that in the helical columnar assembly of the tobacco mosaic virus protein coat, the viral RNA acts as a template and provides additional stability to the columnar aggregate after formation. However, infor- mation governing the hierarchical self-assembly process is, for the most part, encoded within the protein com- ponents, as, under certain pH conditions, the capsid subunits are able to self-assemble in the absence of the RNA strand. In this biologically occurring example of strict self-assembly, as well as in our case, the com- ponents spontaneously aggregate without external guidance into ordered structures [11]. A B Fig. 2. Proposed model for polyamine and phosphate group assem- bly. (A) A multistep process of supramolecular assembly occurs in solution. The electrostatic interactions between the amine termini of polyamines and the phosphate groups generate cyclic ivNAP uni- mers, which further aggregate to form disk-like supramolecular compounds. (B) The interaction of these compounds with the DNA and ⁄ or their in loco aggregation produces the DNA shielding, and promotes and assists the DNA conformational changes. The ulti- mate result of the hierarchical self-assembly is the formation of organized polyamine–phosphate nanotubes that wrap but do not constrict the double helix. A. Di Luccia et al. ivNAPs FEBS Journal 276 (2009) 2324–2335 ª 2009 The Authors Journal compilation ª 2009 FEBS 2327 Composition of GPC peaks To determine the relative ratios among the individual polyamines forming ivNAPs, collected GPC fractions were derivatized with dansyl chloride and analyzed by RP-HPLC (Fig. 3). Table 2 shows the concentration of the polyamines constituting the ivNAPs. Spm was the major com- ponent in both l-ivNAP and m-ivNAP, Spd was pre- dominant in s-ivNAP, and Put was completely absent in l-ivNAP. Total recovery values, also reported in Table 2, were 87.7% for Spm, 68.3% for Spd, and 16.5% for Put. Recovery was not quantitative, indicating that a frac- tion of polyamines escaped detection at k = 280 nm, probably because they did not aggregate in cyclic structures. The recovery values for Put were generally lower than those for Spd and Spm, and the highest percent- ages of Put were found in s-ivNAP. Recovery of Spm, the major constituent of l-ivNAP, progressively increased with the ivNAP size. In contrast, recovery of Put and Spd followed an inverse trend. Put recovery was significantly lower than that of the other polyamines. The differences in recoveries reported in Table 2 could be indicative of a thermo- dynamic equilibrium among the free polyamines and the supramolecular aggregates, which depends not only on the different concentrations of the solutes but also on the electrostatic interactions in the solution. Molecular masses estimated by GPC (Table 2) are quite similar to those reported for NAPs extracted from cell nuclei [1,3]. Our data, however, do not per- mit the definition of simplest formulas, as self-assem- bled compounds present in broad GPC peaks have to be considered as resulting from a Gaussian distribution Fig. 3. Quantitative determination of polyamine in ivNAPs by RP-HPLC analysis of dansyl chloride derivatives. Chromatograms of the deriva- tized polyamines from (A) l-ivNAP, (B) m-ivNAP, and (C) s-ivNAP. Table 2. Relative concentrations and recoveries of polyamines in ivNAPs. Polyamines were quantified by RP-HPLC after derivatiza- tion with dansyl chloride. In vitro NAPs were in this case obtained by pooling 48 m M polyamines in 50 mM phosphate buffer solutions (pH 7.2). A typical GPC chromatogram is shown. Concentrations of polyamines in the ivNAPs are expressed as m M. ND, not detected. Putrescine (% recovery) Spermidine (% recovery) Spermine (% recovery) Estimated molecular mass (Da) l-ivNAP ND 0.75 (4.1) 10 (55.0) 8000 m-ivNAP 0.23 (2.3) 8.3 (18.6) 10.4 (23.5) 5000 s-ivNAP 1.9 (14.2) 6.1 (45.6) 1.5 (9.3) 1000 Total recovery 16.5 68.3 87.8 ivNAPs A. Di Luccia et al. 2328 FEBS Journal 276 (2009) 2324–2335 ª 2009 The Authors Journal compilation ª 2009 FEBS of the molecular masses of several coeluting compounds. On the other hand, attempts to confirm the proposed molecular formulas by means of ‘soft’ MS techniques (MALDI-TOF and ESI-MS, in appropriate conditions for detecting noncovalent interactions) were unsuccessful, most likely because ivNAPs ⁄ NAPs were destructured in the ionization because of the weakness of the interactions involved. Influence of NaCl on ivNAP stability In vitro NAPs were separated by GPC in the presence or absence of 150 mm NaCl in 50 mm phosphate buf- fer (pH 7.2) as mobile phase. Even though the yield of ivNAPs was significantly increased in the presence of NaCl, chromatographic patterns were only slightly affected by ionic strength. However, extraphysiological modifications of salt concentration and ⁄ or pH destabi- lize the supramolecular assembly, making the com- pounds undetectable by GPC analysis. In vitro NAPs isolated in NaCl-enriched sodium phosphate buffer were freeze–thaw stable (Fig. 4A). Conversely, ivNAPs isolated in sodium phosphate buf- fer not supplemented with NaCl contained macro- scopic precipitates (Fig. 4A). Figure 4B–D clearly illustrates that the precipitates were due to the forma- tion of crystallites. The crystallite shapes from s-ivNAP and m-ivNAP solutions were similar, and showed mainly tetragonal forms, whereas l-ivNAP crystallites had a more complex dendritic–broad-branched appear- ance (Fig. 4). Interestingly, isolated polyamines did not give rise to precipitates if frozen and thawed in sodium phosphate buffer not supplemented with NaCl. In order to determine the presence of polyamines in the crystallites, we resolubilized them and repeated the RP-HPLC analysis, obtaining chromatograms of the derivatized polyamines similar to those reported in Fig. 3 (data not shown). These analyses showed the presence of distinct polyamine patterns in the crystallites. We have taken into account the possibility of cocrystallization in the genesis of the crystallites. Cocrystallization of polyamines and phosphates seems to be less probable than crystallization of ivNAPs, on the basis of the following experimental observations: (a) precipitation of the sole phosphates was easily excluded, as polyamines were recovered in the crystallites – fur- thermore, previously reported data [12] showed that NaH 2 PO 4 did not precipitate at all under freeze–thaw conditions, even at high concentrations (0.5–1 m); (b) formation of crystallites is a property of the NAPs only, as it was not observed at all for single polyamines dis- solved in phosphate buffer (with or without NaCl), even after several freeze–thaw cycles; and (c) crystallites, in microscopy analysis, assume distinct shapes for each one of the three ivNAPs. For all of these reasons, we are inclined to believe that each ivNAP crystallizes with conservation of its supramolecular assembly. However, we think that a definite answer to this question will be given by X-ray diffraction studies. Defrosted ivNAPs I-ivNAP m-ivNAP s-ivNAP AB CD Fig. 4. In vitro NAP crystallization. (A) The defrosted ivNAPs solution obtained by GPC in which the mobile phase was sodium phosphate buffer not supplemented with NaCl exhibits turbidity if compared to the unfrozen control. (B–D) Crystallites of the ivNAPs were clearly distinguishable in these defrosted GPC fractions (l-ivNAP, m-ivNAP, or s-ivNAP). Images were acquired by phase contrast microscopy at · 400 magnification. The scale bars correspond to 20 lm. A. Di Luccia et al. ivNAPs FEBS Journal 276 (2009) 2324–2335 ª 2009 The Authors Journal compilation ª 2009 FEBS 2329 The role of NaCl as a phase separator factor in our experimental conditions is supported by studies con- cerning silica precipitation [5,13]. These studies describe: (a) the mechanisms by which long-chain poly- amines, consisting of 15–21 repeating units of N-meth- ylpropyleneimine attached to Put, undergo phase separation and form microemulsions in the presence of either phosphate or other polyanions; and (b) the abil- ity of polyamines (with molecular masses ranging from 1000 to 1250 Da) to promptly precipitate silica nano- spheres from a silicic acid solution. This occurrence is strictly dependent on the presence of phosphate ions and on ionic strength. In our case, the phase separa- tion observed after freezing of soluble and natural (small-sized) polyamines, in the presence of phosphate ions and in an environment lacking NaCl, is a surpris- ing phenomenon that signifies the reassembly of small structures (ivNAPs) into larger and insoluble supra- molecules. The role played by NaCl can be also be satisfacto- rily explained by referring to the theory of polyampho- lytes [14]: in the absence of salt, the attraction of the fixed charges leads to molecular collapse in globular forms and to consequent insolubility; with low salt, as in our system, the charge shielding of the molecules by mobile ions prevents their globularization, thus leading to solubility and increasing molecular network swell- ing; with high salt, salting-out effects lead again to insolubility or association. Similar effects occur even under nonisoelectric conditions. Furthermore, when saline solutions are cooled to subzero temperatures, H 2 O freezes as pure ice, and ions are ejected into the unfrozen part of the system. This event occurs only when the solution temperature over- comes the eutectic point of a given salt [15,16] (in our system, )21.1 °C for NaCl and )9.9 °C for NaH 2 PO 4 ⁄ Na 2 HPO 4 buffer). As the freezing process progresses, a salt concentration gradient, as well as a temperature gradient (due to latent heat release), establishes across the freezing front. This leads to the occurrence of mac- roscopic instabilities due to the formation of pockets of unfrozen salt-concentrated brine [17,18]. Therefore, considering that the saline bonds are at the basis of NAP ⁄ ivNAP formation, it can be inferred that, in NaCl-free solutions, polyamine–phosphate salt precipi- tation occurs more easily in a crystalline form than in an amorphous one [16]. In our case, in these pockets of unfrozen salt-concentrated brine, greater suprastruc- tures assembled and finally precipitated, forming crystallites as a consequence of the increased concentra- tions of polyamines and phosphate salts [16,19]. We are persuaded that the influence of NaCl in determining the size and shape of the aggregates is quite delicate, and needs to be investigated in detail. Dynamic light scattering (DLS) measurements can be useful for clarifying this matter. Preliminary DLS data indicate that, in the absence of NaCl, ivNAP solutions have a natural tendency to form large aggregates. At room temperature, the process is time-dependent: a sample left for several hours on the bench becomes opalescent. Low temperatures or freeze–thaw processes speed up the superaggregation of ‘NaCl-free’ ivNAPs. Every way, the aggregation produces micrometer-sized particles that, for their dimension, are outside the DLS detection range. In contrast, 150 mm NaCl l-ivNAP, m-ivNAP or s-ivNAP solutions remained clear in all of the above-mentioned conditions. DLS measurements performed on these solutions after a freeze–thaw cycle gave reproducible and fitting results about the hydro- dynamic size of the superaggregates, the radii of which ranged between 200 and 500 nm. These dimensions could be ascribed to both large hydration shells and shape effects of the compounds. However, to obtain information on these aggregates at the mesostructural and microstructural scales, a specific study based on DLS and small-angle neutron scattering measurements would be required. In any case, the analysis of both the correlation function and the corresponding distri- bution function of the hydrodynamic radii revealed a quite small polydispersity in size of the complexes (Fig. 5). These data indicate that ivNAPs can remain struc- turally stable in appropriate saline conditions. It is likely that the presence of ions in the hydration sphere of ivNAPs induces an orientation of the electric water dipoles and ⁄ or repulsion among the charges that stabi- lizes the aggregates and restrains their further growth into macrocomplexes. Further studies are also needed to provide an understanding of these underlying chem- ical and physical mechanisms. However, it is clear that, in our systems, fusion phenomena are drastically depressed by the presence of NaCl in the solutions. The role played by NaCl in conferring stability on these supramolecular aggregates is a rough indication of the degree of difference in complexity between the in vitro and in vivo nuclear settings. For instance, it is easy to suppose that both the presence of many other ions in the cell and the complicated system of regula- tion of polyamine metabolism [20] modulate their formation and functions. ivNAP–DNA interaction The interaction of ivNAPs with genomic DNA was studied using ivNAPs obtained from equimolar 48 mm polyamines in 50 mm sodium phosphate (pH 7.2) ivNAPs A. Di Luccia et al. 2330 FEBS Journal 276 (2009) 2324–2335 ª 2009 The Authors Journal compilation ª 2009 FEBS solutions and separated by GPC with NaCl-free 50 mm sodium phosphate buffer, in order to prevent the influence of NaCl on DNase I activity [21,22]. As reported in Fig. 6A, the three ivNAPs protected geno- mic DNA from DNase I degradation more efficiently than did single polyamines (Fig. 6B), which were coas- sayed as controls at the highest concentrations found in the chromatographic fractions of ivNAPs (Table 2). This suggests that the interaction of ivNAPs with the genomic DNA leads to shielding of the phosphodiester bonds, so protecting the DNA against hydrolytic attack. The three ivNAPs exhibited comparable protec- tive abilities in preventing DNA degradation, as shown by absorbance analysis (Fig. 6). Furthermore, the detection of ivNAP crystallites in phoshate buffer not supplemented with NaCl prompted us to verify their 2.4 A B C 10 0 0.9 I-ivNAP m-ivNAP s-ivNAP 100% R = 443 nm 100% R = 265 nm 100% R = 447 nm 0.7 0.5 0.3 0.1 10 1 10 2 10 3 nm 10 0 10 1 10 2 10 3 nm 10 0 10 1 10 2 10 3 nm 0.9 0.7 0.5 0.3 0.1 0.9 0.7 0.5 0.3 0.1 2.2 2.0 1.8 1.6 1.4 1.2 1.0 1E5 1E4 1E3 0.01 0.1 t(ms) g 2 (t) –1 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 2.0 1.8 1.6 1.4 1.2 1.0 g 2 (t) –1 g 2 (t) –1 1 1E5 1E4 1E3 0.01 0.1 t(ms) 1 1E5 1E4 1E3 0.01 0.1 t(ms) 1 Fig. 5. DLS features of ivNAPs in 150 mM NaCl phosphate buffer solution. The correlation function and the corresponding distribution function of the hydrodynamic radius (insets) for l-ivNAP, m-ivNAP or s-ivNAP are shown. The narrow hydrodynamic radius distribution functions indicate low polydispersity of the systems. Average hydrodynamic radius measured values are also reported. A B Fig. 6. In vitro NAPs protect genomic DNA against DNase I degra- dation and influence the DNA conformation. The electrophoretic migration at 37 °C of genomic DNA preincubated with ivNAPs and exposed to DNase I. Whole genomic DNA and DNA fully digested by DNase I were used as controls. (A) Lane 1: DNA + DNase I + l-ivNAP (11 lL). Lane 2: DNA + DNase I + m-ivNAP (11 lL). Lane 3: DNA + DNase I + s-ivNAP (11 lL). Lane 4: DNA + DNase I + sodium phosphate buffer (11 lL). Lane 5: DNA + sodium phosphate buffer (11 lL). Lane 6: DNA + DNase I + H 2 O (11 lL). (B) Incubation of genomic DNA with DNase I in the presence of single polyamines. Lane 7: DNA + DNase I + Spm (10 m M). Lane 8: DNA + DNase I + Spd (6.1 m M). Lane 9: DNA + DNase I + Put (2 m M). Lane 10: DNA + DNase I + sodium phosphate buffer (11 lL). Lane 11: DNA + sodium phosphate buffer (11 lL). Lane 12: DNA + DNase I + H 2 O (11 lL). A. Di Luccia et al. ivNAPs FEBS Journal 276 (2009) 2324–2335 ª 2009 The Authors Journal compilation ª 2009 FEBS 2331 potential ability to interact with the genomic DNA. DNA localization was determined by ethidium bro- mide (EtBr) staining and microscopy analysis, carried out on the same field of view both with fluorescence and with bright field light. The images (Fig. 7A,B) clearly show that fluorescent DNA labeling perfectly corresponds to l-ivNAP, m-ivNAP or s-ivNAP crystallites observed in bright field light (Fig. 7A). No fluorescence was detectable when the acquisition of images was performed in the absence of DNA (Fig. 7B). It is noteworthy that, despite their morphological diversities, the three kinds of crystallites are all able to interact with genomic DNA. In Fig. 7, we show, for the first time, microscopic images of genomic DNA wrapping the polyamine–phosphate superaggregates. As revealed by the EtBr staining in comparison with bright field light microscopy, fluorescence localized precisely, and exclusively, on crystallite structures, thus confirming the ability of ivNAPs to interact with geno- mic DNA. Therefore, our data indicate that: (a) the latter is a typical attribute of both NAPs and their in vitro equivalents; and (b) the ivNAPs, similarly to the cellular analogs, are able to protect genomic DNA from DNase I digestion. Finally, the images illustrat- ing the genomic DNA–ivNAP crystallite interaction suggest that other important aspects of DNA physiol- ogy, such as conformation and packaging, can be exploited by these supramolecular aggregates, as already proposed [3,4]. Structural and functional features All NAP functions were proposed by us to be per- formed by tunnel-like supramolecular structures, entirely enveloping the genomic DNA [3,4], of the helical face-to-face rosette nanotube type [23]. The basic modules, formed by the intercalation of a phos- phate anion between the N-terminal ends of two polyamines and arranged in macro(poly)cyclic struc- tures, were further assembled by the hydrogen bonds into a polymeric supramolecular system [24]. Such a molecular organization, which has structural properties that are considered to be favorable for maximizing and optimizing the functional DNA machinery [2], recently found support in a crystallographic study by Ohishi et al., showing a water–polyamine nanowire compound that was able to bind DNA minor grooves [25]. Even though in vitro and ‘natural’ NAPs share a series of structural characteristics, in the present article we are describing the in vitro assembly of polyamines and phosphates in conditions that are different from those present in the biological setting. Explicitly, in this work we demonstrate that the self-assembly hap- pens under conditions of thermodynamic equilibrium and independently of the presence of the DNA template. However, our data clearly indicate that it is possible, by mimicking in vitro the physiological con- text (pH and ionic strength), to obtain supramolecular compounds similar to the extractive ones. 200x Fluorescence A NAP + DNA + EtBr Brightfield NAP + EtBr Fluorescence B Bright field Fig. 7. DNA interaction with crystals of ivNAPs demonstrated by EtBr staining and fluorescence microscopy analysis (· 200 magnification). (A) Fluorescence detection of DNA–EtBr complex after incubation with ivNAP crystallites. The images can be matched with those acquired by bright field light microscopy. Fluorescent DNA exactly corresponds to the ivNAP crystallite shapes. (B) No fluorescence was detectable when ivNAPs were incubated with EtBr in the absence of DNA. ivNAPs A. Di Luccia et al. 2332 FEBS Journal 276 (2009) 2324–2335 ª 2009 The Authors Journal compilation ª 2009 FEBS Altogether, our data concerning the ivNAPs do not contradict the NAP model, but indicate that the stabil- ity and formation of the ‘natural’ supramolecular structures has to be ascribed to more complex mecha- nisms. For instance, the concentrations that we used in order to obtain comparable protective effects on geno- mic DNA were in the millimolar range (about 1000 times higher than the physiological concentration). Thus, it is possible to infer from our results that NAPs are more efficient, as well as more stable, than the ivNAPs, and that polyamine introduction into the complexes could be, at least for Put, which is a nones- sential component of l-ivNAP, actively regulated in the cell nuclear environment. Hence, although we believe that the thermodynamic forces involved in the assem- bly of ivNAPs are basically the same as those involved in the production of the biological analogs, additional regulatory processes should be investigated in the cell setting. This kind of molecular aggregation seems to be more effective than other types of polyamine aggrega- tion; in fact, polyamine dendrimers, which also interact with dsDNA, barely protect it from DNase I [26]. Nevertheless, all the known types of polyamine aggre- gate are more effective than single polyamines in the carrying out of the crucial functions of the dsDNA protection and conformation, thus indicating that polyamine aggregation is a prerequisite for their inter- action with the DNA. It is not surprising, then, that the functions of one supramolecular structure, DNA, are regulated by others, the NAPs–ivNAPs, as the hierarchical organization of supramolecules is consid- ered to be fundamental for the integrated function of biochemical structures [27]. Conclusions Our data indicate that ivNAPs can be produced by means of an easy, fast, reproducible and inexpensive synthetic method. The products are stable if the GPC separation is performed in the presence of NaCl, are able to interact with the genomic DNA and, conse- quently, are potentially utilizable in many fields of research in which polyamines are involved [4]. Further- more, starting from individual polyamine–phosphate aggregates, we produced definite crystallized forms that were able to imprint the genomic DNA. It is our conviction that the ivNAPs, which mimic naturally occurring NAPs, are components of a new class of biologically relevant supramolecular com- pounds and that they represent an excellent example of the fundamental working strategy of nature: to achieve great results with the simplest and cheapest tools. Experimental procedures Polyamines (Put, Spd, and Spm) and reagents were pur- chased from Sigma-Aldrich (Milan, Italy). All chemicals and reagents used in the study were of analytical grade. HPLC-grade acetonitrile was obtained from Baker (J. T. Baker, Deventer, the Netherlands). Milli-Q water, obtained through a Millipore filter system (Millipore Co., Bedford, MA, USA) with conductivity < 18.2 lSÆcm )1 , was used throughout to prepare aqueous buffers. Human genomic DNA was isolated from peripheral blood leuko- cytes. DNA was extracted and purified using a standard phenol ⁄ chloroform procedure, and then resuspended in Tris ⁄ EDTA buffer. The in vitro self-assembly was performed at room tem- perature by incubating polyamines (Put, Spd, and Spm) in 50 mm sodium phosphate buffer (pH 7.2) for 10–15 min. The concentration of each polyamine was independently varied (5, 10, 24 or 48 mm), keeping constant the concen- tration of the other two (24 mm). GPC-HPLC separation of ivNAPs was carried out on a Gilson modular chroma- tographer, model 152 A (Gilson Inc., Middleton, WI, USA), equipped with a Superose 12 prepacked HR 10 ⁄ 30 column (GE Healthcare, Uppsala, Sweden), which has an optimum for separation in the range 1–300 kDa. The col- umn was equilibrated with 50 mm sodium phosphate buffer containing 150 mm NaCl (pH 7.2), and calibration was car- ried out using a protein standard mixture according to the instructions of the column manufacturer. Fifty microliters of polyamine–phosphate solution was diluted in an equal volume of equilibration buffer and loaded onto the column. Elution was performed with the same buffer at a constant flow rate of 0.4 mLÆmin )1 , and effluents were monitored at 280 nm. The GPC peaks (the ivNAPs) were manually col- lected and stored at 4 °C until being used. To quantify the polyamines that formed the ivNAPs, RP- HPLC peak areas of derivatized polyamines with dansyl chloride (Sigma) were referred to calibration curves obtained by derivatizing the single standard polyamines (aliquots ranging between 0.125 and 0.5 lg for Put and Spd, and between 0.5 and 3 lg for Spm). Each standard sample was run in triplicate, and the mean value was used. Derivatization was carried out on ivNAPs obtained from 48 mm solutions of polyamines by adapting protocols already described [28]. Aliquots (125 lL) of GPC eluted peaks (the ivNAPs) or aliquots of the standard polyamine solution (1 mgÆmL )1 ) were diluted to 250 lL with a 50 mm sodium phosphate solution, previously filtered. After sam- ple alkalinization, performed by vigorous vortexing with 40 lLof2m NaOH and 60 lL of saturated NaHCO 3 solu- tion, 250 lLof10mgÆmL )1 dansyl chloride in acetone was added. Derivatization was left to proceed for 15 min at room temperature, and finally stopped with 20 lL of 33% NH 4 OH. The reaction mixture was diluted with 380 lLof 0.1 m sodium acetate containing 50% (v ⁄ v) acetonitrile. A. Di Luccia et al. ivNAPs FEBS Journal 276 (2009) 2324–2335 ª 2009 The Authors Journal compilation ª 2009 FEBS 2333 [...]... flow rate of 1 mLÆmin)1 by application of a linear gradient of solvent A (50–90% in 30 min), where solvent A was acetonitrile and solvent B was 0.1 m sodium acetate The effluent was monitored by UV detection at k = 254 nm Chromatographic peaks were integrated using the software provided with the HPLC instrument The determination of polyamine concentrations in the GPC peaks allowed us to calculate the chromatographic... when frozen and thawed, the in uence of NaCl on the stability of ivNAPs was investigated Namely, ivNAPs were produced by dissolving 24 mm polyamines in 50 mm phosphate buffer (pH 7.2), and isolated by GPC using the same mobile phase The isolated GPC fractions formed cloudy precipitates as consequence of their freezing and defrosting To determine whether the chemical composition of precipitates was ascribable... D’Agostino L, di Pietro M & Di Luccia A (2005) Nuclear aggregates of polyamines are supramolecular compounds that play a crucial role in genomic DNA protection and conformation FEBS J 272, 3777–3787 4 D’Agostino L, di Pietro M & Di Luccia A (2006) Nuclear aggregates of polyamines IUBMB Life 58, 75–82 5 Sumper M, Lorenz S & Brunner E (2003) Biomimetic control of size in the polyamine-directed formation of. .. This kind of GPC was performed in order to obtain as ‘pure’ as possible ivNAPs and thus to prevent a possible inhibition of DNase I by NaCl [18,19] Human genomic DNA (1.25 lg) was incubated with 11 lL of either l-ivNAP, m-ivNAP, s-ivNAP or single polyamines (10 mm Spd, 6.1 mm Spm, or 2 mm Put), as control, in a 12.25 lL final volume of phosphate buffer (50 mm, pH 7.2) for 6 min at 37 °C These polyamine... magnification The ivNAP crystallite–DNA interaction was evaluated by fluorescence microscopy Briefly, 10 lL of the single defrosted crystallized fractions (l-ivNAP, m-ivNAP, or s-ivNAP) were preincubated with 1.25 lg of genomic DNA and H2O in a final volume of 20 lL for 10 min at 37 °C Subsequently, 100 ng of EtBr was added, and the solution was gently vortexed and incubated in the dark for 5 min Samples... lg) preincubated with each ivNAP or polyamine solution in a final volume of 16 lL, and then incubated at 37 °C for 30 min The enzymatic reaction was stopped by adding 1.6 lL of 20 mm EDTA (pH 8) Electrophoresis of digested genomic DNA or kHINDIII molecular weight marker (Sigma-Aldrich) was carried out in a Sub GT system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at a constant temperature of 37 °C,... analyzed by RP-HPLC Samples were centrifuged at 14 800 g at 4 °C, for 20 min (Microfuge, Heraeus Instruments, Germany) The collected precipitates were washed twice with 50 mm phosphate buffer at 4 °C, and redissolved in 250 lL of 50 mm sodium phosphate buffer containing 40 lL of 2 m NaOH and 60 lL of a saturated solution of NaHCO3 Polyamines were derivatized with dansyl chloride as previously described,... allowed us to calculate the chromatographic recovery in 1 mL of equimolar 48 mm polyamines dissolved in 50 mm sodium phosphate buffer solution To study the protective effect of ivNAPs on the genomic DNA against DNase I digestion, highly concentrated and freshly prepared ivNAPs were used GPC peaks were collected from equimolar 48 mm single polyamines in 50 mm sodium phosphate (pH 7.2) solutions eluted... were used since they correspond to the highest polyamine concentration values found in the ivNAPs The protective effect of ivNAPs on genomic DNA was tested, as previously described [1,3], in the presence of DNase I (RQ1RNase-free DNase; Promega) Briefly, DNase I (0.06 UÆlg)1 DNA) was added to the reaction buffer solution (400 mm Tris ⁄ HCl, pH 8, 100 mm MgSO4, 10 mm CaCl2) and mixed with the genomic... Sumper M (2004) Biomimetic synthesis of silica nanospheres depends on the aggregation and phase separation of polyamines in aqueous solution Phys Chem Chem Phys 6, 854–857 14 Ciferri A & Kudaibergenov S (2007) Natural and synthetic polyampholytes, Part.1: Theory and basic structures Macromol Rapid Commun 28, 1953–1968 15 Dickerson RE (1969) Molecular Thermodynamics, Benjamin/Cummings Pub Co., Menlo Park, . FEBS in vitro model in order to investigate some of the fea- tures of the polyamine–phosphate interactions. Specifi- cally, we examined the role played by each polyamine in the self-assembly of in. named nuclear aggregates of polyamines (NAPs). Interestingly, other authors have described the phosphate-induced self-assembly of polyamines in a different biological setting [5]. Polyamine and. by NaCl in conferring stability on these supramolecular aggregates is a rough indication of the degree of difference in complexity between the in vitro and in vivo nuclear settings. For instance,