Effective chiral discrimination of tetravalent polyamines on the compaction of single DNA molecules

Article abstract of DOI:10.1002/anie.201209144

Normal doughnuts or lots of little ones? Among four stereoisomers of a tetravalent polyamine, the isomer pictured was the most potent at inducing DNA compaction, whereby the DNA molecules adopted a unique morphology of minitoroid clusters. The nonchiral polyamine spermine induced the formation of typical, larger toroidal structures. Copyright

Full text of DOI:10.1002/anie.201209144

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DOI: 10.1002/anie.201209144
DNA Structure
Effective Chiral Discrimination of Tetravalent Polyamines on the
Compaction of Single DNA Molecules**
Yuko Yoshikawa,* Naoki Umezawa,* Yuki Imamura, Toshio Kanbe, Nobuki Kato,
Kenichi Yoshikawa, Tadayuki Imanaka, and Tsunehiko Higuchi*
Numerous in vitro studies have shown that the binding of
natural and synthetic polyamines to DNA induces the
condensation/compaction of DNA.[1–6] This phenomenon is
of interest in biology and chemistry, since genomic DNA is
often found in different degrees of condensation and requires
polyamines for the adoption and stabilization of its compact
structures.[1,7] Among naturally occurring polyamines, sper-
mine (4 +) is much more potent at promoting DNA
compaction than putrescine (2 +) and spermidine (3 +); this
observation indicates that the valence strongly influences
DNA compaction.[4] Several systematic studies on tetravalent
polyamines have shown that their influence on DNA com-
paction is dependent on the geometrical arrangement of
positively charged amino groups.[6,8,9] As well as such geo-
metric effects, chiral effects on DNA compaction have also
been reported. Nayvelt et al. studied the influence of the
chirality of stereoisomers of a-methylated spermine ana-
logues on DNA condensation by light-scattering experiments,
and showed that there is a small but definite difference in the
threshold concentration required for DNA condensation; the
maximum difference was approximately 20%.[10]
Herein, we describe a method for the highly sensitive
chiral discrimination of newly synthesized stereoisomers of
Scheme 1. Chemical structures of spermine and spermine analogues
tetravalent (4 +) polyamines on the basis of their ability to
1, 2, 3, and 4.
induce compaction in a single molecule of DNA. The
polyamines we used are shown in Scheme 1. Since many
polyamine receptor sites, such as DNA, are chiral, we
envisioned that the introduction of stereogenic centers into
a polyamine backbone would affect the activity of the
polyamine. The simple incorporation of trans-cyclopentane
rings into the spermine backbone provided chirality and
substantial rigidity. Furthermore, a p-methoxyphenyl group
was introduced as a chromophore for the detection of UV
absorption during HPLC purification and the convenient
determination of concentration. These polyamines were
synthesized by a method similar to a previously reported
procedure (see the Supporting Information).[11–13] Each sper-
mine analogue contained two trans-cyclopentane units with
a total of four stereogenic centers (Scheme 1).
To monitor the changes in the higher-order structure of
DNA at the single-molecule level, we used a large genomic
DNA molecule, T4 GT7 phage DNA (166 kbp, 57 mm). It has
been established that such large DNA molecules undergo
a discrete conformational transition from an expanded coil
state to a compact state upon the addition of various
condensing agents.[4,14,15] We observed the large DNA in the
presence and absence of chiral polyamines by fluorescence
microscopy. As DNA is a long, thin molecule and shows
significant intramolecular and translational Brownian
motions in bulk solution, we adapted an inverted wide-field
fluorescence microscope for the observations with a relatively
[*] Prof. Dr. Y. Yoshikawa, Prof. Dr. T. Imanaka
Department of Biotechnology, College of Life Sciences
Ritsumeikan University, Kusatsu 525-8577 (Japan)
E-mail: yyoshi@fc.ritsumei.ac.jp
Dr. N. Umezawa, Y. Imamura, Dr. N. Kato, Prof. Dr. T. Higuchi
Graduate School of Pharmaceutical Sciences
Nagoya City University, Nagoya 467-8603 (Japan)
E-mail: umezawa@phar.nagoya-cu.ac.jp
higuchi@phar.nagoya-cu.ac.jp
Prof. Dr. T. Kanbe
Laboratory of Medical Mycology, School of Medicine
Nagoya University, Nagoya 464-0064 (Japan)
Prof. Dr. K. Yoshikawa
Faculty of Life and Medical Sciences
Doshisha University, Kyotanabe 610-0394 (Japan)
[**] This research was supported in part by Grants-in-Aid for Scientific
Research from the Japan Science and Technology Agency CREST
program, and from the Japan Society for the Promotion of Science
(20249006, 22510123, 23240044, 23659058, and 24590142).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201209144.
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high time resolution, that is, 30 frames per
second, which is not possible with a confocal
microscope. Representative fluorescence
images of individual DNA molecules in
aqueous solution and the corresponding
quasi-3D profiles of fluorescence intensity
are shown in Figure 1a,b. As shown in Fig-
ure 1a, individual DNA molecules existed in
a coil conformation in the buffer solution.
Upon the addition of 1 to the solution of the
DNA, individual DNA molecules underwent
a structural transition from a coil state to
a compact-globule state via a partial-globule
state, in which swollen and shrunken parts
coexist in a single DNA molecule. Figure 1c
shows the distribution of the long-axis length
of the DNA. It is clear that 1 causes the
folding transition from a coil to a compact
state in a dose-dependent manner.
Figure 2 shows a comparison of the size
distribution of the DNA after the addition of
each of the four stereoisomers at different
concentrations; the histogram in the top left
corner corresponds to the highest concen-
tration of 1 in Figure 1c. The results clearly
indicate that 1 (R,S–R,S) is more efficient at
promoting DNA compaction than the other
stereoisomers. For example, all of the DNA
molecules adopt a fully compact state with
1 (R,S–R,S) at a concentration of 5 mm. In
contrast, essentially no compact DNA mol-
ecules exist in the presence of the other three
stereoisomers at this concentration: 2, 3, and
4 require concentrations higher than 20 mm to
induce all of the DNA molecules to adopt
a compact state. These results imply that the
concentration of 1 required to induce DNA
compaction is about 4 times less than that
required for the three other stereoisomers. In
related studies on the effect of di- and
tetraammonium cations with two stereogenic
centers on the higher-order structure of T4
DNA molecules, divalent stereoisomers
showed a distinct difference in their potency
for inducing DNA compaction, whereas
there was essentially no difference for tetra-
valent stereoisomers.[16,17] A theoretical study
on chiral discrimination effects upon DNA
compaction induced by the above-mentioned
ammonium cations suggested that chiral
sensitivity can be detected more readily in
the case of divalent cations than for tetrava-
lent cations because the latter are all highly
potent at inducing DNA compaction.[18] In
contrast, the present results clearly show that
there is a definite difference in the abilities of
tetravalent chiral polyamines with four ste-
reogenic centers to promote DNA compac-
tion.
Figure 1. Change in the higher-order structure of T4 DNA in the presence of 1 (0–5 mm).
a,b) Fluorescence microscopy images of individual DNA molecules, which exhibit intra-
molecular and translational Brownian motions in bulk solution, and the corresponding
quasi-3D profiles of the fluorescence intensity. c) Distribution of the long-axis length of the
DNA and assignment of the conformational characteristics observed in the DNA images:
coil, partial-globule, and compact-globule states of single DNA molecules. The DNA
concentration was 0.1 mm in nucleotide units. The illustration shows a schematic represen-
tation of a fluorescent image of the DNA. Each image was captured at a rate of 30 frames
per second.
Figure 2. Distribution of the long-axis length of the DNA in solution at various concen-
trations of 1, 2, 3, and 4.
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We used circular dichroism (CD) spectroscopy to further
interpret the effects of chirality on the conformation of DNA.
Herein, we do not show CD spectra of long T4 DNA: T4
DNA tended to precipitate at the DNA concentration
detectable by CD. Since polyamines were found to bind
preferentially to GC-rich DNA,[19,20] we recorded and ana-
lyzed CD spectra of poly(dG-dC)·poly(dG-dC) (ca. 1 kbp) in
the presence and absence of the four stereoisomers or
spermine (Figure 3). A positive Cotton effect at around
280 nm in the CD spectrum corresponds to base stacking, and
a negative Cotton effect at around 250 nm corresponds to
To evaluate the binding affinity of these chiral isomers for
DNA, we performed H NMR titration experiments. Poly-
1
(dG-dC)·poly(dG-dC) was again used to avoid precipitation:
T4 DNA was prone to precipitation under the conditions for
1
NMR titration. Figure 4 shows the H NMR signals of the
polyamines at around d = 2.3–2.4 and 2.7 ppm in the presence
of poly(dG-dC)·poly(dG-dC) at different concentrations. The
signals at d = 2.3–2.4 and 2.7 ppm were assigned to the
hydrogen atoms of the cyclopentane rings and the benzyl
hydrogen atoms next to the chromophore, respectively. We
1
did not observe DNA signals in the H NMR spectra of the
Figure 4. Titration of chiral polyamines (100 mm) with poly(dG-dC)·
poly(dG-dC) DNA. 1H NMR spectra in D2O: a) at d=2.3–2.4 ppm,
b) at d=2.6–2.7 ppm. Decrease upon titration with poly(dG-dC)·poly-
1
(dG-dC) DNA in the relative integral of the H NMR signal: c) at
d=2.3–2.4 ppm, d) at d=2.7 ppm.
mixtures of the polyamines and DNA. The intensity of the
1H NMR signals observed for the chiral polyamines
decreased as the DNA concentration increased, whereas the
widths of the signals remained essentially constant. This trend
indicates that the bound fraction of the chiral polyamines is
not visible in the 1H NMR spectrum, and that the line width,
T2, remains essentially constant for unbound polyamines.
Thus, we can evaluate the fraction of unbound chiral
polyamines from the change in signal intensity (Figure 4a,b).
The difference in the slopes of the regression lines in
Figure 4c,d provides information on the binding affinity of
the chiral polyamines for DNA. The results show that 1 and 4
exhibit preferential binding to DNA. The binding of 1 and 4 to
DNA is about twice as strong as that observed for the other
pair of enantiomers, 2 and 3. This stronger binding by a factor
of 2 implies that the difference in the binding free energy, DG,
is given by Equation (1):
Figure 3. CD spectra of poly(dG-dC)·poly(dG-dC) DNA at various
concentrations of 1, 2, 3, 4, and spermine. The DNA concentration
was 50 mm. The polyamine/nucleotide molar ratios were 0, 0.8, and
1.2.
helicity.[21] Upon the addition of 1 (40 mm) to a solution of
poly(dG-dC)·poly(dG-dC), the intensity of a band corre-
sponding to a negative Cotton effect increased significantly,
which indicates a change in helicity (Figure 3a). For 1 at
60 mm, there was no detectable signal in the CD spectrum, and
precipitation was observed (Figure 3a). For 2, 3, and 4,
relatively small changes in the CD spectrum were observed at
both 40 and 60 mm (Figure 3b,c,d), which indicates that 1 has
more influence on the secondary structure. In contrast,
spermine induced a B-to-Z transition in the secondary
structure of poly(dG-dC)·poly(dG-dC) DNA, as character-
ized by both a positive signal at 265 nm and a negative signal
at 295 nm (Figure 3e). These results suggest that the chiral
polyamines interact with DNA in a different manner to
spermine.
DG ¼ R T ln2 ffi 1:7 kJ molÀ1
ð1Þ
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in which R is the gas constant (R = 8.314 JKÀ1 molÀ1) and T is
the absolute temperature (T= 293 K).
observed in the presence of 2 and 3 (see the Supporting
Information). There is no apparent difference in the mor-
phology of these structures, even though the threshold
concentrations required for DNA compaction are different.
In contrast, typical toroids (60–100 nm in diameter)[1–3,22] were
observed for spermine-bound compact DNA (Figure 5c).
Thus, the chiral tetravalent polyamines caused significant
changes in the toroid size and overall morphology of compact
DNA.
Interestingly, Shen et al. reported the similar formation of
minitoroids when they inserted multiple A-tract sequences
into DNA polymers; in this case, DNA compaction was
induced by hexamine cobalt(III).[23] An A-tract is a segment
that is formed by a number of consecutive adenine residues in
a double-helix DNA molecule, and the incorporation of
a single A-tract produces a bend as large as 208 in the helical
axis of DNA.[23–25] They suggested that curved DNA inserts
not only resulted in the generation of a much smaller toroid,
but also limited the length of DNA segments contained in
each toroid.[23] Our results demonstrate that chiral polyamines
promote both the generation of many nucleation centers and
the formation of minitoroid clusters in the compaction of
DNA without any A-tract inserts (Figure 5). Therefore, we
can expect that the binding of chiral isomers to DNA induces
substantial bending rigidity and produces an effect similar to
the insertion of curved DNA.
As we demonstrated through a single-molecule observa-
tion, isomer 1 (R,S–R,S) is more potent at inducing DNA
compaction than the other isomers: the threshold concen-
tration of 1 is one quarter of that found for the other isomers
(Figure 2). A similar tendency was observed for the efficacy at
inducing changes in the secondary structure of DNA, namely,
1 is more effective than the other polyamines (Figure 3). On
the other hand, however, 1H NMR titrations showed that the
binding potential of the pair of enantiomers 1 and 4 was twice
as high as that of the other pair of enantiomers 2 and 3
(Figure 4). These findings clearly indicate that the ability to
induce DNA compaction cannot be explained by simple
electrostatic binding models. The distinct difference between
enantiomers 1 and 4 in their potency for inducing DNA
compaction may be directly related to the change in the
bending rigidity of double-stranded DNA.
On the basis of the difference in the threshold concen-
trations of 1 (cR,S–R,S) and the other isomers (cothers) for the
induction of DNA compaction, the difference in the chemical
potential, Dm, of these isomers can be expressed by Equa-
tion (2):
Dm ¼ ÀR TðlncR,SÀR,SÀlncothersÞ ffi 3:4 kJ molÀ1
ð2Þ
On the other hand, the difference in binding free energy
between the two pairs of enantiomers (1 and 4 versus 2 and 3)
is 1.7 kJmolÀ1, as calculated from Equation (1). Thus, it
becomes apparent that the energy difference for the induction
of DNA compaction (3.4 kJmolÀ1) is twice as large as that
found for the simple electrostatic interaction (1.7 kJmolÀ1).
By considering these results together with the difference
between 1 and 4, we can conclude that “induced-fit” chiral
recognition, rather than an electrostatic interaction, primarily
contributes to the difference in the ability to induce DNA
compaction.
We also evaluated the hydrodynamic radius, RH, of the
compact DNA through quantitative analysis of the Brownian
motion by fluorescence microscopy (see the Supporting
Information). It was found that the RH value with 1 was
111 Æ 45 nm, whereas the RH value with spermine was 53 Æ
16 nm. This result corresponds well with the TEM observa-
tions (Figure 5).
In summary, we have synthesized novel stereoisomers of
tetravalent (4 +) polyamines and examined their ability to
induce the compaction of single DNA molecules. Isomer
1 (R,S–R,S) is more potent at inducing both DNA compaction
and changes in the secondary structure. These results suggest
that a cooperative transition of the secondary and higher-
order structures of single DNA molecules was induced by
chiral polyamines. Minsky suggested previously that the
secondary conformation may affect the higher-order structure
of DNA.[26] Furthermore, TEM investigations of the compact
DNA revealed that chiral polyamines induced the formation
of unique assemblies of multiple minitoroidal structures.
Since all genomic DNA molecules in living cells are very
large, structural transitions in the higher-order structure are
believed to play a key role in the mechanism of the self-
regulation of genetic activity.[15] The present results highlight
the importance of studies on the chiral effect of small ligands
on the higher-order structure of giant DNA molecules.
To gain further insight into the influence of chirality on
DNA compaction, we examined compact DNA molecules by
transmission electron microscopy (TEM). Figure 5 shows
examples of TEM images of T4 DNA compacted by 1 (Fig-
ure 5a), its enantiomer 4 (Figure 5b), and spermine (Fig-
ure 5c). Assemblies of multiple minitoroidal structures
(20 nm in diameter) were observed for DNA compacted by
both 1 and 4 (Figure 5a,b). A similar morphology was also
Received: November 15, 2012
Revised: January 26, 2013
Published online: February 25, 2013
Figure 5. TEM images of compacted T4 DNA in the presence of a) 1
(5 mm), b) 4 (20 mm), and c) spermine (5 mm). Scale bar: 100 nm. The
DNA concentration was 0.1 mm.
Keywords: amines · chirality · DNA structures ·
fluorescence microscopy · single-molecule studies
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