Báo cáo khoa học: Nuclear aggregates of polyamines are supramolecular structures that play a crucial role in genomic DNA protection and conformation potx

Nuclear aggregates of polyamines are supramolecular
structures that play a crucial role in genomic DNA
protection and conformation
Luciano D’Agostino
1
, Massimiliano di Pietro
1
and Aldo Di Luccia
2
1 Department of Clinical and Experimental Medicine, ‘Federico II’ University of Naples, Italy
2 Department of Animal Production, University of Bari, Italy
Polyamines interact with DNA phosphate groups by
means of nonspecific electrostatic bonds [1]. This inter-
action has been shown to result in the protection of
small DNA molecules from common damaging agents,
such as ionizing radiation and reactive oxygen species
[2,3]. Polyamines in solution with polynucleotides have
also been shown to inhibit the activity of endonucle-
ases, including DNase I [4–7]. The protective ability of
polyamines is attributable not only to the formation of
a steric barrier against DNA-damaging agents, but
also to their property to condense the DNA. In fact,
polyamines, like other cations, induce DNA condensa-
tion as a consequence of the inhibition of > 90% of
DNA negative charges [8]. Analogous in vivo experi-
ments have demonstrated that spermine and, to a
lesser extent, spermidine, prevent DNA fragmenta-
tion and the onset of apoptosis. Protection from
enzymatic cleavage appears to be the result of a modi-
fied chromatin arrangement, rather than inhibition of
the endonuclease activity [9].

Condensation of DNA in the presence of poly-
amines has also been proposed to be instrumental in
genome packaging [10]. This should be regarded of
crucial importance if we consider that the total length
of cellular DNA is  1 m, whereas the size of the nuc-
leus is in the range of several micrometers [11]. How-
ever, condensation should not be considered as a static
state, as the elasticity is a mechanical property of the
DNA, indispensable to cellular processes such as repli-
cation and transcription [12,13]. For these reasons the
DNA strands in vivo, at the same time, must be pack-
aged and protected, but not restrained.
The structural impact of polyamines on DNA is also
supported by the evidence that these compounds
induce, on polynucleotides, a transition from the right-
oriented B-form to the left-handed Z-form [14,15].
Such an effect might be important for DNA physiol-
ogy, as a tight connection occurs between transcrip-
tional activity on DNA and the acquisition of a
Z-form [16].
Keywords
DNA conformation; DNA protection;
molecular aggregates; polyamines; supra-
molecular chemistry
Correspondence
L. D’Agostino, MD, Facolta
`
di Medicina
‘Federico II’, Edificio-6, Via S. Pansini, 5,
80131 Naples, Italy

Fax: +39 081746 2707
Tel: +39 081746 2707
E-mail:
(Received 4 April 2005, accepted 19 May
2005)
doi:10.1111/j.1742-4658.2005.04782.x
In a previous study we showed that natural polyamines interact in the
nuclear environment with phosphate groups to form molecular aggregates
[nuclear aggregates of polyamines (NAPs)] with estimated molecular mass
values of 8000, 4800 and 1000 Da. NAPs were found to interact with
genomic DNA, influence its conformation and interfere with the action of
nucleases. In the present work, we demonstrated that NAPs protect naked
genomic DNA from DNase I, whereas natural polyamines (spermine, sper-
midine and putrescine) fail to do so. In the context of DNA protection,
NAPs induced noticeable changes in DNA conformation, which were
revealed by temperature-dependent modifications of DNA electrophoretic
properties. In addition, we presented, for NAPs, a structural model of
polyamine aggregation into macropolycyclic compounds. We believe that
NAPs are the sole biological forms by which polyamines efficiently protect
genomic DNA against DNase I, while maintaining its dynamic structure.
Abbreviation
NAP, nuclear aggregate of polyamines.
FEBS Journal 272 (2005) 3777–3787 ª 2005 FEBS 3777
A body of evidence also indicates a role for poly-
amines in the regulation of gene expression [17] and in
the cell cycle. A correlation has been shown between
increased concentrations of spermine and spemidine in
the nucleus and the induction of mitosis [18–20].
Moreover, alterations in the polyamine biosynthetic
pathway affect the correct progression of the cell cycle,

particularly the S-phase [21].
Temperature is an additional factor capable of
affecting DNA conformation. It has been reported
that (a) an increase of a few °C is associated with a
reduction in the overstretching forces of the DNA
strands [22,23], (b) that the DNA melting tempera-
ture is directly correlated with the chain length of
interacting polyamines [24–27] and (c) that a trans-
ition of the DNA chain from a dispersed coil state
to a condensed-collapsed state parallels an increase
in temperature [28]. These findings draw attention to
the relationship existing between temperature and
stabilization, aggregation, elasticity and conforma-
tional transition of the DNA, all phenomena closely
linked to DNA protection and influenced by poly-
amines.
Recently we described new compounds with mole-
cular mass values of  1000, 4800 and 8000 Da, named
nuclear aggregates of polyamines (NAPs), whose
molecular structure is based on the ionic interaction
between polyamines and phosphate groups. These
compounds were isolated from the nuclei of several cell
types. In vitro aggregation experiments demonstrated
that by mixing polyamines (spermine, spermidine and
putrescine) in phosphate buffer it is possible to gener-
ate compounds with molecular mass identical to the
NAPs extracted from cells [29]. This finding suggested
that NAPs can form naturally in the nuclear environ-
ment, where phosphates are particularly abundant.
The positive net charge of NAPs allows interaction

with the negatively charged DNA phosphates. We
have shown previously that these compounds influence
DNA conformation and protect DNA from Exonuc-
lease III and DNase I [29].
The compound with a molecular mass of 4800 Da,
which was suggested to induce supercoiled DNA
forms, is clearly associated with cell replication, being
recovered in large quantities in the nuclei of cells
in S-phase and absent in non-replicating cells. Experi-
mental evidence indicates the NAP with the lowest
molecular mass (1000 Da) functions as a precursor for
the 4800 Da form of NAP. The concentration of the
8000 Da form of NAP in the nucleus did not vary
throughout the phases of the cell cycle.
In this study, based on the investigation (by gel elec-
trophoresis) of the interaction between polyamines and
genomic naked DNA, we compared the protective
efficacy of NAPs with that of single polyamines
against DNase I. We demonstrated that only NAPs
efficiently impede nuclease cleavage, suggesting that
they are the biologically effective forms by which poly-
amines protect the genomic DNA from endonucleases.
We propose a model of molecular organization of
polyamines into NAPs extrapolated from our previous
and present experimental evidence.
NAPs were previously named according to their
molecular mass. However, as the molecular mass was
only an approximate value, estimated by GPC analysis
and therefore different from that calculated by the
molecular formulas presented here, we now adopt, in

the present study, an alternative nomenclature based
on size. Therefore, < 1000 NAP, 4800 NAP and 8000
NAP will subsequently be named s-NAP, m-NAP and
l-NAP (small-, medium- and large-sized), respectively.
Results and Discussion
Single polyamines fail to protect naked genomic
DNA from DNase I
To assess the protective role of NAPs against endonuc-
leases and compare it with that of single polyamines,
we carried out electrophoretic assays of genomic DNA
treated with DNase I. Single polyamines or NAPs were
allowed to interact with genomic DNA before expo-
sure to phosphodiesterasic endonuclease. Assuming
that the tested compounds, interacting with DNA, pre-
vent DNA degradation, the protective effect can be
demonstrated by a higher molecular mass of the DNA
molecules migrating into the gel.
We first tested DNA degradation by DNase I upon
incubation with increasing concentrations of single
polyamines, starting from a concentration of 1 lm
polyamines, which is comparable to the concentration
of polyamines forming NAPs in the extractive elution.
As shown in Fig. 1A, a 1 lm concentration of poly-
amines did not protect from DNase I, and no noticeable
increase in DNA protection was observed with 50 or
150 lm single polyamine concentrations. Increasing the
concentration of single polyamines up to 600 lm resul-
ted in no clear impediment to DNA cleavage (Fig. 1B).
At 600 lm spermine, a peculiar effect was observed,
namely the paradoxical facilitation of DNase I, result-

ing in the complete degradation of DNA (Fig. 1B, lane
c). This phenomenon, which probably depends on the
electrostatic nature of the polyamines–DNA inter-
action, is in accordance with previously published
results indicating a biphasic behavior of DNA structure
in water solution with increasing concentrations of
Polyamine aggregates and DNA L. D’Agostino et al.
3778 FEBS Journal 272 (2005) 3777–3787 ª 2005 FEBS
spermine [30]. The authors of this study observed that
spermine induces DNA condensation and precipitation
as a consequence of the progressive neutralization of
the negative charges of DNA. This phenomenon was
attributed to the organization of DNA into a liquid-
crystalline ordered structure determined by the interac-
tion of a single polyamine with several negative DNA
charges. However, DNA resolubilization occurred
when spermine was supplemented in excess. DNA res-
olubilization was attributed to the osmotic stress gen-
erated by the high concentration of polyamines, which
drives these cations into the nonpolar DNA phase,
and to the decreased number of DNA-binding sites
per polyamine, making the DNA more hydrophilic.
Analogously, in our experimental model, high levels of
spermine in solution rendered the genomic DNA extre-
mely sensitive to the action of DNase I. We believe
that this is a result of the resolubilization of DNA and
the massive exposure of the phosphodiester bonds to
the active site of the nuclease, probably because of
repulsive effects exerted by polyamines present in the
nonpolar phase of the DNA on polyamines linked to

the backbone phosphates.
As single polyamines did protect naked genomic
DNA from DNase I, we investigated whether natural
aggregates of polyamine, such as NAPs, might possess
this ability. As shown in Fig. 2 (left panel), all NAPs
protected DNA from degradation, although the migra-
tion pattern of DNA preincubated with l-NAP differed
from that of DNA preincubated with either m-NAP or
s-NAP.
As polyamines were not only ineffective in DNA
protection, but even detrimental for DNA integrity
at higher concentrations, aggregation into NAPs may
reflect the need to keep the concentration of intra-
nuclear polyamines at low levels and under stringent
control. In fact, other studies have demonstrated that
an excess of polyamines may result in the perturbation
of vital functions dependent on DNA integrity and
conformation [31], whereas their drastic decrease under
the lower threshold can impede cell mitosis and ⁄ or
trigger the mitochondria-mediated apoptotic pathway
[32]. In order to accomplish this tight regulation that
hampers an excessive increase of polyamines in the
A
B
Fig. 1. Electrophoresis of genomic DNA preincubated with single
polyamines and then exposed to DNase I. (A) Electrophoretic
migration, at 37 °C, of genomic DNA preincubated with three differ-
ent concentrations (1, 50 and 150 l
M) of spermine (lanes c, c¢ and
c¢¢), spermidine (lanes d, d¢ and d¢¢) or putrescine (lanes e, e¢ and

e¢¢) and then exposed to DNase I. Whole genomic DNA (lane a)
and DNase I-digested genomic DNA (lane b) were controls. (B)
Electrophoretic migration, at 37 °C, of genomic DNA preincubated
with 600 l
M spermine (lane c), spermidine (lane d) or putrescine
(lane e) and exposed to DNase I. Controls were in lanes a (whole
genomic DNA) and b (DNA exposed to DNase I). Identical results
were obtained at a migration temperature of 40 °C (data not
shown).
Fig. 2. Nuclear aggregates of polyamines (NAPs) protect genomic
DNA from DNase I and, at the same time, influence DNA conforma-
tion. The electrophoretic migration at 37 °C (left) and 40 °C (right)
of genomic DNA preincubated at 37 °C with small-size NAP (s-NAP;
lanes c and c¢), medium-size NAP (m-NAP; lanes d and d¢) or large-
size NAP (l-NAP; lanes e and e¢) and exposed to DNase I. Controls
were the whole genomic DNA (lane a) and the DNA fully digested
by DNase I (lane b). The DNA 1 kb ladder marker was in lane m.
L. D’Agostino et al. Polyamine aggregates and DNA
FEBS Journal 272 (2005) 3777–3787 ª 2005 FEBS 3779
nucleus, cells are also provided with enzymes that
interconvert and catabolize polyamines [33,34]. A key
enzyme for polyamine catabolism is diamine oxidase,
which is able to bind the DNA and oxidize DNA-
bound polyamines [35]. The fact that this enzyme is
particularly evident in differentiated enterocytes [36–
38], which are involved in the uptake and distribution
of polyamines [39] to the entire organism via the intes-
tinal bloodstream, suggests that it probably has the
strategic function of coping with the flow of poly-
amines potentially harmful to the DNA of intestinal

cells. We believe that NAPs are an important part of
this physiological scenario.
NAPs protect DNA in the context of structural
elasticity
The data shown in Fig. 2 (left panel) indicated not
only that NAPs preserve naked genomic DNA from
DNase I-dependent degradation with an efficacy much
greater than that of single polyamines, but also that
the migration patterns of NAP–DNA complexes differ
substantially. Namely, the DNA preincubated with
l-NAP showed a diffuse migration pattern, whereas the
DNA interacting with s-NAP and m-NAP migrated in
a compact form similar to that of naked DNA (lane
a), but significantly faster. Hence, we wondered whe-
ther such a change in DNA migration properties was
caused by a difference in the protection ability of sin-
gle NAPs or by conformational changes induced in
DNA by the interaction with NAPs. As temperature
has been shown to be a variable that dynamically
influences DNA conformation in terms of elasticity
and condensation status [28], we varied, within physio-
logical ranges, the temperature of the electrophoretic
run. We believed that this modification of the experi-
mental conditions, taking place after inactivation of
the endonuclease, could influence migration changes
based on DNA conformation, but not on the differen-
tial degradation of nucleic acids. By raising the run-
ning temperature from 37 °Cto40°C, we observed a
mirroring change of the DNA electrophoretic patterns
(Fig. 2, right panel).

Many aspects of the experiments described in Fig. 2
are worthy of discussion. First, upon incubation with
each NAP, DNA, although exposed to the endonuc-
lease, showed migration features that, at least in one
of the thermal conditions applied, were not dissimilar
to that of control DNA. This result undoubtedly dem-
onstrates that all NAPs, independently of their
molecular mass and net charge, completely protect
DNA from DNase I. We believe that the protection
occurs as a result of steric hindrance of DNase I access
to the DNA phosphodiester bond even though we
cannot exclude that modification of the DNA conden-
sation status might play an additional role. Theoretic-
ally, the possibility exists that the protection of DNA
by NAPs depends on modification of the catalytic
properties of DNase I, rather than by preventing
access of DNase I to the DNA phosphodiester bonds.
However, the latter seems to be the likeliest possibility,
as the protection of DNA from nucleases is a general
property of NAPs, regardless of the type of nuclease
tested (NAPs have been shown to prevent exonuclease
III – another type of nuclease – degradation of DNA
[29]). Furthermore, it has already been suggested that
spermine prevents in vivo endonuclease activity as con-
sequence of a modified degree of chromatin accessibil-
ity to the enzyme [9].
Second, all NAPs increased the electrophoretic speed
of genomic DNA, up to induce a diffuse migration
pattern, which was determined by each NAP selec-
tively at a given temperature (either 37 or 40 °C). As

temperature modifications always followed DNase I
inactivation, we were able to exclude any interference
of this environmental change with the enzymatic activ-
ity. Therefore, we concluded that, in the context of
constant DNA protection, NAPs interfere with the
DNA condensation status in a temperature-dependent
manner. We believe that enhancement of the migration
speed is attributable to DNA decondensation and
strand elongation, which facilitates the penetration of
DNA into the gel matrix [40,41].
A third aspect concerns the relationship between
DNase I and DNA conformation. Our experiments
suggest that the incubation of NAPs–DNA complexes
with DNase I was a decisive factor in the above men-
tioned conformational effects, as NAPs alone deter-
mined only slight modifications in the running
properties of DNA [29]. It has already been shown
that single polyamines, mainly spermine, can modulate
the DNA-binding properties of proteins, either increas-
ing or diminishing their affinity for DNA [42]. This
ability, to modulate the DNA-binding properties of
proteins, was analyzed in relation to the conformational
effects of polyamines on DNA and was found to be
directly dependent on the degree of their positive
charge. Accordingly, we hypothesize that, in the pres-
ence of NAPs, DNase I, while prevented from acting
as a nuclease as a result of steric inhibition, interacts
with NAPs–DNA complexes and, in turn, cooperates
with NAPs to modify DNA arrangement. Ionic forces
may drive the interaction between DNase I and

NAPs–DNA complexes. In fact, under the experi-
mental conditions applied (inactivation by EDTA and
the electrophoretic run performed at pH 8), the
Polyamine aggregates and DNA L. D’Agostino et al.
3780 FEBS Journal 272 (2005) 3777–3787 ª 2005 FEBS
enzyme acquires a negative net charge and can there-
fore bind the positively charged NAPs.
In contrast to polyamines, the effects of NAPs on
DNA condensation status do not seem to depend
mainly on DNA charge neutralization. By virtue of
their net charge, single polyamines have been shown to
influence the conformation of high M
r
DNA [28,41].
Namely, T4 DNA in buffer solution acquired an elon-
gated coiled conformation, whereas the progressive
neutralization of the global DNA charge by interacting
polyamines determined the acquisition of a compact-
folded conformation, which showed a slower electro-
phoretic mobility [41]. Differently, the modification of
the electrostatic properties of DNA induced by NAPs
does not appear to be a primary factor driving the
change of condensation status, as the different migra-
tion patterns were obtained without altering the con-
centration of NAPs in the solution. The marked DNA
electrophoretic changes indicate, in our opinion, that
NAPs efficiently preserve DNA elasticity and can
modulate the degree of DNA strand elongation, which
is measured by mobility on the gel. These findings sug-
gest a possible role for NAPs in the in vivo nuclear

environment: NAPs-dependent modification of the
DNA condensation status might play a role in the
regulation of chromatin complexation onto histones.
The elasticity of the DNA was correlated to both
its interaction with polyamines [43] and temperature.
Melting experiments demonstrated that polyamines
stabilize the DNA structure with an ability that is a
function of the polyamine chain length [24–26]. More-
over, the melting entropy of DNA was determined by
measuring the overstretching force of single molecules
of DNA [22,23]. This transitional force decreases with
the increase of temperature from 11 to 52 °C, thus
indicating that the stability of the DNA double helix is
a temperature-dependent phenomenon and that DNA
melting occurs during the overstretching transition.
However, the maintenance of an appropriate func-
tional morphology of the DNA seems to require more
complex mechanisms. Studies of cation interactions
indicate that the size of the DNA grooves depends on
the number of charges present on the DNA backbone.
In fact, the repulsion of phosphate groups across the
minor groove makes it widen, whereas the neutraliza-
tion of the phosphate groups reduces the groove width
[44,45]. Even though the groove’s flexibility is crucial,
its collapse [46] should be considered a detrimental
event that can be more efficiently prevented by the
interaction with the l-NAPs rather than with the small
sized polyamines. Our conviction is strengthened by
the fact that the collapse of high M
r

DNA to toroidal
and spheroidal structures has been reported in the
presence of multivalent cations, including spermidine
and spermine [46]. Recent DNA thermodynamic stud-
ies also support this belief, as they indicate that the
distension of strands caused by temperature increases
widen the grooves [22,23], so permitting the interaction
of larger moieties.
Therefore, two implications can be inferred from
our results (a) the preservation of the DNA integrity is
fully assured by NAPs along with the modification of
the folding state of the DNA and (b) a few degrees of
temperature increase, a normal occurrence in living
cells, is able to drive significant conformational chan-
ges in the presence of NAPs and DNase I. Both of
these events imply that NAPs carry out their defensive
function against DNase I having constantly full acces-
sibility into the DNA grooves.
For all of these reasons, NAPs, compounds with an
optimal mass ⁄ charge ratio, represent supramolecular
structures able to determine a broader impact on
DNA structure and physiology than single polyamines.
A model of polyamine aggregation into NAPs
As a last step of the present study, we sought to pro-
pose a structural model of polyamine organization into
NAPs in accordance with the experimental data pro-
duced to date about NAPs biochemistry (summarized
in Table 1), and the theoretical principles of macro-
molecule self-assembly (see the Experimental proce-
dures).

The NAPs were drawn as macro(poly)cyclic com-
pounds on the basis of the following assumptions
(a) the attraction of opposite charges of polyamines
and phosphate represents the driving force of self-
assembly, (b) the intercalation of a phosphate anion
between the N-terminal ends of two polyamines per-
mits the formation of a cyclical structure character-
ized by a minimal repulsive force as a consequence
of thermodynamic and kinetic stability and selectiv-
ity, (c) ion water solvatation takes part in the supra-
molecular aggregation, conferring high flexibility to
ionic bonds, (d) amine nitrogen of polyamines, com-
pletely protonated at physiological pH, cannot parti-
cipate in the formation of hydrogen bonds, whereas
phosphate groups are able to form hydrogen bonds,
and (e) hydrogen bonds among phosphate groups
stabilize adjacent polycyclic units into tridimensional
supramolecular structures.
The binding of phosphate groups to polyamines to
form cyclic supramolecules can overcome the mere
phenomenon of attraction of charges to imply the pro-
cess of molecular recognition, already described more
than 20 years ago [47]. Our previous NMR studies
L. D’Agostino et al. Polyamine aggregates and DNA
FEBS Journal 272 (2005) 3777–3787 ª 2005 FEBS 3781
showed that phosphate groups which interact with
long polyamines (spermidine and spermine), are able
to determine molecular rearrangements in their struc-
ture [29]. These modifications might include expression
of enhanced flexibility on the major axis of poly-

amines, which favours their bending, and would be
instrumental to the formation of cyclic supra-molecules.
A cyclical structure of NAPs has already been sugges-
ted in view of their absorbance peak at 280 nm [29],
which is compatible with an electron delocalization
typical of molecules with p fi p bonds, such as poly-
ene systems. Moreover, it has already been postulated
that macropoycyclic compounds possess a structure
favourable for maximizing and optimizing functional
molecular activities. Such molecules are usually large
(macro), and may therefore contain central cavities and
possess numerous bridges and connections (polycyclic)
[47]. Importantly, the formation of polyamine-based
macrocyclic compounds has already been described,
either as a spontaneous biological event [48], or as a
result of in vitro synthetic experiments [49].
The s-NAP, whose molecular mass, extrapolated
from the simplest formula, is  1000 Da, is composed
of two spermines, one spermidine and one putrescine.
As a result, we can predict its structure to be a single
tetrameric ring formed by four polyamines linked by
four phosphate groups through ionic bonds (Fig. 3A).
We have previously shown that, in synchronized Caco-
2 cells stimulated to replicate by gastrin, the diminu-
tion of the s-NAP pool was accompanied by
increased levels of m-NAP. This observation raised the
hypothesis that s-NAPs have the property to aggregate
into larger molecules (i.e. m-NAPs) [29]. Further sup-
port of this hypothesis came from the detection of
compounds with intermediate molecular mass values

(ranging from 1000 to 4800 Da) in the above
mentioned in vitro aggregation studies [29]. Further-
more, previous biochemical studies indicated that
m-NAP conserves the same spermine ⁄ spermidine ⁄
putrescine ratio (2 : 1 : 1) of s-NAP. For all of these
reasons, and because the molecular mass of m-NAP
was estimated to be 4800 Da [29], we proposed its
structure to consist of five 4-polyamine monomers con-
nected by hydrogen bonds (Fig. 3B).
The largest NAP (l-NAP) has a polyamine ratio of
1 : 1 : 1 [29]. In contrast to m-NAP, we did not isolate
a pool of monomers for the formation of l-NAP,
therefore its structural model results are more specula-
tive. However, following the criterion of analogy with
the smaller compounds (s-NAP and m-NAP), we pre-
dict that its structure may originate from the aggrega-
tion, by hydrogen bonds, of five 6-polyamine rings (i.e.
two spermines, two spermidines and two putrescines)
linked by phosphate groups (Fig. 3C). We predicted
6-polyamine rings in the structure as (a) 3-polyamine
rings are likely to require an excessive degree of poly-
amine bending and a high energetic level, which would
give rise to a less favourable structure, and (b) larger
rings (of 9-polyamine units or larger) would be extre-
mely large for fitting into the DNA grooves where
NAPs are believed to interact.
The primum movens in NAPs–DNA aggregation is
the charge attraction between DNA phosphates and
the amino groups of polyamines. As the amino groups
of polyamines are already engaged in ionic bonds with

the phosphates of NAPs, secondary amino groups
are those available to establish interstrand interaction
with the backbone phosphates. In accordance with a
recently proposed model of spermine interstrand com-
plexation along the major groove, we believe that the
interaction of NAPs rings with DNA is then stabilized
by intra major groove bonds with DNA bases. They
Table 1. Experimental data supporting nuclear aggregates of polyamines (NAPs) modelling. Put, putrescine; Spd, spermidine; Spm, spermine;
Ph, phosphate group.
s-NAP m-NAP l-NAP
Spontaneous aggregation in vitro
a
Yes Yes Yes
Pick of absorbance at 280 nm
b
Yes Yes Yes
Simplest formula
c
Put-Ph-Spd-Ph-(Spm)
2
Put-Ph-Spd-Ph-(Spm)
2
Put-Ph-Spd-Ph-Spm
Estimated molecular mass (Da)
d
 1000  4800  8000
Calculated molecular mass (Da)
e
1035.1 5175.5 9552.15
Number of monomers 1 5 ? (5)

g
Estimated diameter of each ring
f
 15 A
˚
 15 A
˚
 25 A
˚
Compatibility with major groove dimensions Yes Yes Yes
Proposed interacting DNA form ? (A-DNA)
g
Z-DNA ? (B-DNA)
g
a
Gel permeation chromatographic analysis of 25 lM polyamines (Spm, Spd and Put) dissolved in phosphate buffer (pH 7.2).
b
UV spectro-
photometric detection at 280 nm.
c
Defined on the basis of the molar concentration of polyamines forming NAPs.
d
Gel permeation chroma-
tographic analysis.
e
On formulae shown in Fig. 3.
f
Diameters can vary owing to the flexibility of the electrostatic interactions linking
polyamines and phosphate groups.
g

Speculative hypotheses.
Polyamine aggregates and DNA L. D’Agostino et al.
3782 FEBS Journal 272 (2005) 3777–3787 ª 2005 FEBS
can be either of the hydrophobic type (between the
CH
2
group of thymine and the methylene groups of
spermine) and ⁄ or the ion-dipole type (between the sec-
ondary amino groups of polyamines and purine-N7
or thymine-O4 residues) [1,50]. The insertion of NAP
monomers into the DNA grooves forms the basis of
the recognition process occurring between the two
supramolecular structures (i.e. DNA and NAPs). In
our model, the adaptation of both l-NAP and m-NAP
to the DNA shape was hypothesized to be favoured by
bidirectional movements of the arms of an arch-like
structure (Fig. 3B,C). Such an event is made possible
by the hydrogen bonds between phosphates belonging
to contiguous rings that confer great flexibility to the
macropolycyclic structures of NAPs. It could be
argued that NAPs, whose structural integrity relies on
weak interactions (electrostatic and hydrogen bonds),
might disaggregate once in contact with DNA. How-
ever the results of electrophoretic experiments allowed
us to exclude such a possibility. In fact, the loss of
NAPs’ integrity would leave, in solution, single poly-
amines (spermine, spermidine, and polyamines), which
we showed do not possess any relevant DNA protect-
ive activity. Therefore, in order to protect DNA from
DNase I, NAPs must maintain their structural integ-

rity. Moreover, their ability to influence DNA confor-
mation is a further indirect sign that NAPs must be
not disaggregated when in contact with DNA.
It is also conceivable that NAPs interacting with
DNA must stabilize their bond to the double helix by
creating a solid structure around it. NAPs would
greatly strengthen the alignment along the DNA longi-
tudinal axis through the formation of hydrogen bonds
between phosphate groups of adjacent molecules
(Fig. 4D,E). According to this model, NAPs would
thereby form a supra-molecular tunnel capable of
enveloping the entire DNA. The final effect would be
the formation of an external scaffolding that protects
the DNA by masking the sugar-phosphate backbone,
as indicated by the evidence of protection against
DNase I and exonuclease III [29].
An additional property, namely conformational,
might be ascribed to the differences, in terms of size
Fig. 3. Structural models of nuclear aggregates of polyamines
(NAPs). (A) Small-size NAP (s-NAP). In accordance with the simp-
lest formula indicating a spermine (Sm) ⁄ spermidine (Sd) ⁄ putrescine
(P) ratio of 2 : 1 : 1, polyamines were represented as terminally
linked by phosphate groups to form a single cyclical structure (grey
disks, carbon atoms; blue disks, nitrogen atoms; yellow disks,
phosphate atoms; red disks, oxygen atoms; white disks, hydrogen
atoms). (B) Medium-size NAP (m-NAP). This NAP is represented as
a polymer of five s-NAPs linked by hydrogen bonds (green tri-
angles). The white arrows indicate the possible opening ⁄ closure
movements that allow the adaptation of NAPs to DNA grooves.
The closure of the arch (resting state) may occur when the com-

pound is in phosphate buffer solution. (C) Large-size NAP (l-NAP).
This NAP is represented as a polymer of five 6-polyamine units,
linked by hydrogen bonds, according to the simplest formula indica-
ting an Sm ⁄ Sd ⁄ P ratio of 1 : 1 : 1.
L. D’Agostino et al. Polyamine aggregates and DNA
FEBS Journal 272 (2005) 3777–3787 ª 2005 FEBS 3783
and shape, among the single NAPs. Previously, we
have shown that m-NAP can enhance the electropho-
retic mobility of genomic DNA [29]. Furthermore, we
showed by spectrophotometry that m-NAP has the
property to increase the absorbance at 260 nm of
genomic DNA, whereas other NAPs failed to do so.
These results suggest that the m-NAP interacting with
DNA might determine structural rearrangements char-
acterized by base extrusion, an event occurring in the
transition to the left-oriented conformation [16]. For
this reason, we predicted a model in which m-NAP
favours DNA transition to the Z-form (Fig. 4C), a
DNA form characterized by a narrower diameter, more
elongated strands and outward exposure of bases.
Our previous experiments suggest that the s-NAP
works both as a functional NAP, binding the DNA as
such, and as a precursor of the m-NAP. Although the
aggregation of s-NAPs can occur independently of the
DNA structure, the possibility exists that m-NAPs
can be built up directly in loco, through the sequential
aggregation of three small units, to two s-NAPs
already bound to DNA. Thereby, the progressive for-
mation of compounds with an increasing number of
monomers (up to five) would force, with a type of

wedge-like progression (Fig. 4B), the DNA grooves to
widen and simultaneously determine the transition
towards the Z-form [16], which then proceeds along
the two strands in a zip-like manner. Another import-
ant consequence of s-NAP complexation into m-NAP
is the strong increase of electrostatic forces that the
latter compound exerts on DNA. It is known that elec-
trostatic forces play a greater role in the A–Z trans-
ition than they do in the B–Z transition, because the
difference in the linear charges density is greater
between the A and the Z forms than between the B
and the Z forms [51]. For this reason, we constructed
a model in which s-NAP interacts with A-DNA and,
among the non-Z fi Z transitional possibilities, we
chose the A fi Z possibility (Fig. 4A,C). Additionally,
because the A-DNA major groove is narrower than
the B-DNA major groove, we hypothesized, by virtue
of size compatibility criteria, a preferential interaction
of the s-NAP with A-DNA (Fig. 4A). In fact, we eval-
uated the diameters of the monomers to be  15 A
˚
for
s-NAP and m-NAP and  25 A
˚
for l-NAP.
We do not have clear evidence for proposing an
interactive model for l-NAP with a specific DNA type.
However, as l-NAP is the most widely represented
compound in the nuclei of quiescent and replicating
cells, and the B-DNA is the most common DNA form

[52], a specific role for l-NAP in the protection and
conformation of B-DNA can be suggested. However,
Fig. 4. Interaction of single nuclear aggre-
gates of polyamines (NAPs) with different
DNA forms. (A) Small-size NAP (s-NAP)
interacting with A-DNA. Grey rings repre-
sent the polyamine backbone. Red dots
indicate the phosphate groups. To simplify
interpretation of the figures, the NAPs
phosphates facing the DNA groove were
omitted. A-DNA has a groove width more
suitable than other DNA forms for interac-
tion with this NAP. (B) Progressive forma-
tion of medium-size NAP (m-NAP). The
addition of s-NAP units to two s-NAPs
already bound to DNA (up to five) can allow
the formation of m-NAP directly onto the
DNA. This may favour the transition to the
Z-DNA, through the progressive widening of
DNA strands and the exposure of bases. (C)
m-NAP interaction with Z-DNA. Z-DNA sta-
bilization by the m-NAP arch-like structure
was represented as a result of the distan-
cing of consecutive A-DNA major grooves.
(D) Perspective view of s-NAPs connected
by hydrogen bonds. Aggregation of more
s-NAPs units can allow the formation of a
tunnel-like envelope around the DNA. (E)
Perspective view of m-NAPs connected by
hydrogen bonds. A 3D m-NAP tunnel

structure enveloping the DNA is suggested.
Polyamine aggregates and DNA L. D’Agostino et al.
3784 FEBS Journal 272 (2005) 3777–3787 ª 2005 FEBS
it should be mentioned that whatever the DNA con-
formation, DNA protection is fully assured by each
interacting NAP.
Studies conducted (based on CD and ⁄ or Raman
spectroscopy), to date, on the interaction between
polyamines and DNA, analysed DNA molecules much
smaller than the genomic DNA used in the present
study, testing polyamine ⁄ DNA ratios of 1 : 10 to 1 : 1,
which was quite different from those used in our
experimental setting (NAP ⁄ DNA ratio of 1 : 5000)
[1,50,53]. For this reason, we are convinced that these
methodological approaches cannot be easily extrapola-
ted to the NAPs setting. Therefore, much work must
be carried out to clarify several aspects of our model.
However, a recent review from Medina and coworkers
stated that ‘despite the great amount of experimental
and theoretical works carried out up to now, it is not
possible to give an undoubted explanation about how
the polyamines bind to DNA’ [54]. In this context, we
believe that our work, although not fully exhaustive in
all the aspects approached, might shed novel light on
the matter.
In conclusion, we demonstrated that NAPs are able
to preserve the genomic DNA from DNase I-dependent
degradation, with an efficacy extraordinarily greater
than single polyamines. Furthermore, we showed that
DNA, while preserved in its integrity by single NAPs,

undergoes temperature-sensitive conformational chan-
ges, which are indicative of a preserved DNA elasticity.
We believe that NAPs are the sole biologically active
forms by which polyamines physiologically interact with
and protect genomic DNA, given that these quasi-stable
molecular aggregates, natural examples of supramolecu-
lar chemistry, are able to reach the maximum effect with
the minimum effort.
Experimental procedures
Human genomic DNA was isolated from peripheral blood
leukocytes donated by M. di Pietro. DNA was extracted
and purified in phenol ⁄ chloroform and then resuspended in
Tris ⁄ EDTA (TE) buffer. NAPs were extracted from the
nuclei of preconfluent Caco-2 cells and purified by gel per-
meation chromatography, as previously described [29].
Genomic DNA (4 lg per 2.5 lL of phosphate buffer)
was incubated for 6 min at 37 °C with 4.5 lL of l-, m- or
s-NAP (mean polyamine concentration: 0.25 ngÆlg
)1
DNA)
or water solutions of single polyamines (putrescine, sper-
midine and spermine) at a concentration of 1, 50, 150 or
600 lm. The mixture was then exposed to DNase I
(RQ1RNase-free DNase; Promega, Milan, Italy) at a con-
centration of 0.025 UÆlg
)1
DNA. Briefly, 1 lL of the
DNase I solution was added to 1 lL of the reaction buffer
solution (400 mm Tris ⁄ HCl, pH 8, 100 mm MgSO
4

and
10 mm CaCl
2
) and then mixed with NAP–DNA or poly-
amine–DNA solutions. The enzyme action was stopped
after 30 min of incubation at 37 °C by adding 1 lLof
20 mm EDTA, pH 8. Samples were then loaded onto a
1Æ5% (w ⁄ v) ultrapure DNA grade agarose gel. Electrophor-
esis of DNA was carried out for 1 h in an HE 100 supersub
(Amersham Pharmacia Biotech, Uppsala, Sweden), at a
constant temperature of 37 or 40 °C (controlled by a peri-
staltic pump system), by applying an electric field strength
of 11.1 VÆcm
)1
in Tris ⁄ borate ⁄ EDTA buffer. Each gel was
then photographed by using a Polaroid MP-4 L camera.
NAPs modelling was carried out, producing molecular
structures that were in strict accordance with biochemical
data and theoretical rationales.
All biochemical data were collected from analytical, elec-
trophoretic and NMR studies shown in our previously pub-
lished work [29] and in the present study. Theoretical
rationales were derived from the universally accepted prin-
ciples of the supramolecular chemistry based on the self-
assembly by means of weak interactions [47,55–57]. A
multistep aggregation plan was developed as follows (a) a
cyclic and planar structure representing the single base
module, (b) base modules combined in polycyclic planar
structures (ladder), (c) the polycyclic modules developed 3D
into tunnel-like structures. The compatibility between the

NAPs dimensions, evaluated on formulas, and the currently
accepted DNA grooves size was the requisite for the inter-
action scheme of NAPs with DNA forms.
Acknowledgements
We are grateful to Dr Alma Contegiacomo and Dr
Luigi Gomez-Paloma for helpful suggestions and
advice, to Mr Paolo Mastranzo for technical assistance
and to Mr Stefano D’Agostino for his contribution in
artwork production. This work was supported by a
research grant from the Campania Region.
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