Aldo Di Luccia1,2,*, Gianluca Picariello1,*, Giuseppe Iacomino1,*, Annarita Formisano1,Luigi Paduano3and Luciano D’Agostino1,4 1 Institute of Food Sciences, National Research Council CNR
Trang 1Aldo Di Luccia1,2,*, Gianluca Picariello1,*, Giuseppe Iacomino1,*, Annarita Formisano1,
Luigi Paduano3and Luciano D’Agostino1,4
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.
Trang 2in 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 vitroaggregation 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
Trang 3The 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-assemmetasta-bled 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 )
Spd (m M )
Spm (m M )
Trang 4changes 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.
Trang 5Composition 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 detecfrac-tion 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 m M 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)
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
Trang 6of 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 vitroNAPs 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 NaH2PO4 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
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.
Trang 7The 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, H2O 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 NaH2PO4⁄
Na2HPO4buffer) 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 physchem-ical 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)
Trang 8solutions 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)
2 (t)
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
2 (t)
2 (t)
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 m M 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 + H2O (11 lL).
Trang 9potential 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
A
NAP + DNA + EtBr
NAP + EtBr
B
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.
Trang 10Altogether, 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 4C 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 lL of 2 m NaOH and 60 lL of saturated NaHCO3 solu-tion, 250 lL of 10 mgÆmL)1dansyl chloride in acetone was added Derivatization was left to proceed for 15 min at room temperature, and finally stopped with 20 lL of 33%
NH4OH The reaction mixture was diluted with 380 lL of 0.1 m sodium acetate containing 50% (v⁄ v) acetonitrile