Poly(L-lysine)-graft-dextran Copolymer
Promotes Pyrimidine Motif Triplex DNA Formation at Physiological
pH
THERMODYNAMIC AND KINETIC STUDIES*
Hidetaka
Torigoe
§,
Anwarul
Ferdous¶,
Hiromitsu
Watanabe¶,
Toshihiro
Akaike¶, and
Atsushi
Maruyama§¶
From the
Gene Bank, Tsukuba Life Science Center, The
Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai,
Tsukuba, Ibaraki 305-0074, and ¶ Department of Biomolecular
Engineering, Tokyo Institute of Technology, 4259 Nagatsuda, Midori,
Yokohama 226-8501, Japan
 |
ABSTRACT |
Extreme instability of pyrimidine motif triplex
DNA at physiological pH severely limits its use for artificial control
of gene expression in vivo. Stabilization of the pyrimidine
motif triplex at physiological pH is therefore of great importance in improving its therapeutic potential. To this end, isothermal titration calorimetry interaction analysis system and electrophoretic mobility shift assay have been used to explore the thermodynamic and kinetic effects of our previously reported triplex stabilizer, poly
(L-lysine)-graft-dextran (PLL-g-Dex) copolymer, on pyrimidine motif triplex
formation at physiological pH. Both the thermodynamic and kinetic
analyses have clearly indicated that in the presence of the
PLL-g-Dex copolymer, the binding constant of the pyrimidine
motif triplex formation at physiological pH was about 100 times higher
than that observed without any triplex stabilizer. Of importance, the
triplex-promoting efficiency of the copolymer was more than 20 times
higher than that of physiological concentrations of spermine, a
putative intracellular triplex stabilizer. Kinetic data have also
demonstrated that the observed copolymer-mediated promotion of the
triplex formation at physiological pH resulted from the considerable
increase in the association rate constant rather than the decrease in
the dissociation rate constant. Our results certainly support the idea
that the PLL-g-Dex copolymer could be a key material and may eventually lead to progress in therapeutic applications of the
antigene strategy in vivo.
 |
INTRODUCTION |
In recent years, triplex DNA has attracted considerable interest
because of its possible biological functions in vivo and its
wide variety of potential applications, such as regulation of gene
expression, site-specific cleavage of duplex DNA, mapping of genomic
DNA, and gene-targeted mutagenesis (1-3). A triplex is usually formed
through the sequence-specific interaction of a single-stranded
homopurine or homopyrimidine triplex-forming oligonucleotide
(TFO)1 with the major groove
of the homopurine-homopyrimidine stretch in duplex DNA (1-5). In the
purine motif triplex, a homopurine TFO binds antiparallel to the
homopurine strand of the target duplex by reverse Hoogsteen hydrogen
bonding to form A·A:T (or T·A:T) and G·G:C triplets (1-5). On
the other hand, in the pyrimidine motif triplex, a homopyrimidine TFO
binds parallel to the homopurine strand of the target duplex by
Hoogsteen hydrogen bonding to form T·A:T and C+·G:C
triplets (1-5). Because the cytosine bases in a homopyrimidine TFO are
to be protonated to bind with the guanine bases of the G:C duplex, the
formation of the pyrimidine motif triplex needs an acidic pH condition
and is thus extremely unstable at physiological pH (6-10). The extreme
instability of the pyrimidine motif triplex at physiological pH
severely limits its use for artificial control of gene expression
in vivo. Stabilization of the pyrimidine motif triplex at
physiological pH is therefore of great importance in improving its
therapeutic potential. Numerous efforts such as the replacement of
cytosine bases in a homopyrimidine TFO with 5-methylcytosine (9,
11-13) or other chemically modified bases (14-18), the conjugation of
different DNA intercalators to TFO (19, 20) and/or the use of
polyamines such as spermine or spermidine as triplex stabilizers (21)
have been made to improve the stability of the pyrimidine motif triplex
at physiological pH. However, in some cases, modification strategies
lessened the overall binding affinity of the TFO or increased its
nonspecific interaction with DNA (2, 18).
We have previously reported that poly
(L-lysine)-graft-dextran (PLL-g-Dex)
copolymer (poly (L-lysine) with grafts of hydrophilic dextran chains; Fig. 1a) not only significantly increased
the thermal stability of the triplex involving poly(dA)·2poly(dT) (22) but also stabilized the pyrimidine motif triplex DNA at neutral pH
using a 30-bp homopurine-homopyrimidine target duplex from rat
1(I)
collagen gene promoter and an unmodified cytosine-rich TFO (23, 24).
However, the mechanistic explanation for the PLL-g-Dex
copolymer-mediated triplex stabilization was not clearly understood.
Therefore, we have further extended our previous study to address this
issue in the context of the thermodynamic and kinetic effects of the
PLL-g-Dex copolymer on pyrimidine motif triplex formation at
neutral pH. The thermodynamic and kinetic effects of the copolymer on
pyrimidine motif triplex formation between a 23-bp
homopurine-homopyrimidine target duplex (Pur23A·Pyr23T) (Fig.
1b) and its specific 15-mer homopyrimidine TFO (Pyr15T) (Fig. 1b) have been analyzed by isothermal titration
calorimetry (ITC) (25, 26), interaction analysis system (IAsys)
(27-29), and electrophoretic mobility shift assay (EMSA) (23, 24). Results from the three independent lines of experiments have clearly indicated the effect of the copolymer in promoting pyrimidine motif
triplex formation at neutral pH. In the presence of the copolymer, the
binding constant for pyrimidine motif triplex formation at neutral pH
was about 100 times higher than that observed with TFO alone. Moreover,
the triplex-promoting efficiency of the copolymer was more than 20 times higher than that of the physiological concentration (about 1 mM) of spermine, a putative intracellular triplex
stabilizer (30). Kinetic data have also demonstrated that the major
contribution for the copolymer-mediated promotion of triplex formation
resulted from the considerable increase in the association rate
constant rather than the decrease in the dissociation rate constant.
The ability of the PLL-g-Dex copolymer to promote pyrimidine
motif triplex formation at physiological pH would support further
progress in therapeutic applications of the antigene strategy in
vivo.
 |
MATERIALS AND METHODS |
Preparation of Oligonucleotides--
We synthesized 23-mer
complementary oligonucleotides for target duplex Pur23A and Pyr23T
(Fig. 1b) and the 15-mer homopyrimidine TFO Pyr15T (Fig.
1b) on an ABI DNA synthesizer using the solid-phase cyanoethyl phosphoramidite method and purified them with reverse-phase high performance liquid chromatography on a Wakosil DNA column. 5'-Biotinylated Pyr23T (denoted as Bt-Pyr23T) was prepared from biotin
phosphoramidite. Two different nonspecific TFOs, Pyr15NS-1 and
Pyr15NS-2 (Fig. 1b), were synthesized as mentioned above and purchased from Grainers Japan Co. (Tokyo, Japan), respectively. The
concentration of all oligonucleotides was determined by UV absorbance.
Complementary strands Pur23A and Pyr23T were annealed by heating at up
to 90 °C, followed by a gradual cooling to room temperature. The
annealed sample was applied on a hydroxyapatite column (KOKEN Inc.) to
remove unpaired single strands. The concentration of the duplex DNA
(Pur23A·Pyr23T) was determined by UV absorption considering the DNA
concentration ratio of 1 OD = 50 µg/ml, with a
Mr of 15180. The purified oligonucleotide
solutions were dialyzed (molecular weight cutoff = 500) extensively
against Buffer A (10 mM sodium cacodylate-cacodylic acid
(pH 6.8), 200 mM sodium chloride, and 20 mM
magnesium chloride) with or without the triplex stabilizer (0.84 mM spermine or 0.038 mM PLL-g-Dex copolymer).
Preparation of the PLL-g-Dex Copolymer--
The
PLL-g-Dex copolymer (number average molecular weight = 7.9 × 104) (Fig. 1a) was prepared by a
reductive amination reaction between poly (L-lysine) and
dextran T-10, as described in detail previously (22, 31). The purified
copolymer solution was dialyzed (molecular weight cutoff = 1000)
extensively against Buffer A.
ITC--
Isothermal titration experiments were carried out on a
MCS ITC system (Microcal Inc.) essentially as described previously (25,
26). Briefly, the TFO and Pur23A·Pyr23T duplex DNA solutions were
prepared by extensive dialysis against Buffer A with or without the
triplex stabilizer. The TFO solution in Buffer A with or without the
triplex stabilizer was injected 20 times in 5-µl increments at 10-min
intervals into the Pur23A·Pyr23T duplex solution without changing the
reaction conditions. The heat for each injection was subtracted by the
heat of dilution of the injectant, which was measured by injecting the
TFO into Buffer A with or without the triplex stabilizer. Each
corrected heat was divided by the number of moles of TFO injected and
analyzed with Microcal Origin software supplied by the manufacturer.
IAsys--
Kinetic experiments were performed on an IAsys
instrument (Affinity Sensors Cambridge Inc.), where a real-time
biomolecular interaction was measured with a laser biosensor (27-29).
The resonant layer of a cuvette was washed with 200 µl of 10 mM acetate buffer (pH 4.6) and then activated with 200 µl
of a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and
N-hydroxysuccinimide solution. The activated surface was
washed again with 10 mM acetate buffer (pH 4.6), and
streptavidin in 10 mM acetate buffer (pH 4.6) was
immobilized on the surface. After blocking the remaining reactive
groups with 1 M ethanolamine (pH 8.5), the cuvette was
washed extensively with 10 mM acetate buffer (pH 4.6) and
then with 20 mM hydrochloric acid to remove the loosely
associated protein. The cuvette was washed with Buffer A, and Bt-Pyr23T
(1.2 µM in Buffer A) was added to bind with the streptavidin on the surface. After washing the cuvette with the same
buffer, complementary oligonucleotide Pur23A (1.2 µM in
Buffer A) was added to hybridize with Bt-Pyr23T. After extensive
washing and equilibrating of the Bt-Pyr23T·Pur23A-immobilized surface with Buffer A with or without the triplex stabilizer for more than 30 min, the TFO in 200 µl of Buffer A with or without the triplex
stabilizer was injected over the immobilized Bt-Pyr23T·Pur23A duplex,
and then triplex formation was monitored for 30 min. This was followed
by washing the cuvette with Buffer A with or without the triplex
stabilizer, and the dissociation of the preformed triplex was monitored
for an additional 20 min. Finally, 100 mM Tris-HCl (pH 8.0)
was injected for 3 min for a complete break of the Hoogsteen hydrogen
bonding between the TFO and Pur23A, during which the Bt-Pyr23T·Pur23A
duplex may be partially denatured. The Bt-Pyr23T·Pur23A duplex was
regenerated by injecting 1.2 µM Pur23A. The resulting
sensorgrams were analyzed with Fastfit software supplied by the
manufacturer to calculate the kinetic parameters.
EMSA--
EMSA experiments were performed essentially as
described previously, with slight modifications (23, 24). In 9 µl of
reaction mixture, 32P-labeled duplex (~10,000 cpm; ~1
ng) was mixed with increasing concentrations of Pyr15T and the
nonspecific oligonucleotide Pyr15NS-2 in either the absence or presence
of the triplex stabilizer, (spermine or the PLL-g-Dex
copolymer) in a buffer containing 50 mM Tris acetate (pH
7.0), 100 mM sodium chloride, and 10 mM
magnesium chloride. Pyr15NS-2 was added to achieve equimolar
concentrations of TFO in each lane as well as to minimize the adhesion
of the DNA (target duplex and Pyr15T) to plastic surfaces during
incubation and subsequent losses during processing. After a 6-h
incubation at 37 °C, 2 µl of 50% glycerol solution containing
bromphenol blue and 1 µl of thymus DNA (6 µg) were added without
changing the pH and salt concentrations of the reaction mixtures.
Thymus DNA was added to chase the ionic interaction between DNA (target duplex and TFO) and the copolymer as described previously (23, 24).
Samples were then directly loaded onto a 15% native polyacrylamide gel
prepared in buffer (50 mM Tris acetate, pH 7.0, and 10 mM magnesium chloride), and electrophoresis was performed
at 8 V/cm for 16 h at 4 °C. The percentage of the formed
triplex was estimated as described previously (23, 24).
 |
RESULTS |
Thermodynamic Effect of PLL-g-Dex Copolymer on the Pyrimidine Motif
Triplex Formation at Neutral pH--
We have examined the
thermodynamics of pyrimidine motif triplex formation between the 23-bp
target duplex Pur23A·Pyr23T and its specific 15-mer TFO, Pyr15T (Fig.
1b), at 25 °C and pH 6.8 by
ITC under three different conditions: 1) Buffer A (10 mM
sodium cacodylate-cacodylic acid at pH 6.8 containing 200 mM sodium chloride and 20 mM magnesium
chloride), 2) Buffer A + 0.84 mM spermine, and 3) Buffer A + 0.038 mM PLL-g-Dex copolymer. The
concentration of amino groups in 0.84 mM spermine is
equivalent to that in 0.038 mM PLL-g-Dex
copolymer. Fig. 2a compares
the ITC profiles of the initial three injections for triplex formation
between Pyr15T and Pur23A·Pyr23T at 25 °C and pH 6.8 with TFO
alone or in the presence of spermine or the PLL-g-Dex
copolymer. The magnitudes of the exothermic peaks in the presence of
0.038 mM PLL-g-Dex copolymer were much larger
than those observed with TFO alone. Triplex formation in the presence
of 0.038 mM PLL-g-Dex copolymer reached to
equilibrium within 10 min after each injection of Pyr15T. On the other
hand, the magnitudes of the exothermic peaks in the presence of 0.84 mM spermine were indistinguishable from those observed with
TFO alone. Fig. 2b shows a 200-min ITC profile for triplex
formation in the presence of the PLL-g-Dex copolymer. An
exothermic heat pulse was observed after each injection of Pyr15T into
Pur23A·Pyr23T. The magnitude of each peak decreased gradually
with each new injection, and a small peak was still observed at a molar
ratio of [Pyr15T]/[Pur23A·Pyr23T] = 2. The area of the small peak
was equal to the heat of dilution measured in a separate experiment by
injecting Pyr15T into the same buffer (data not shown). The area under
each peak was integrated, and the heat of dilution of Pyr15T was
subtracted from the integrated values. The corrected heat was divided
by the number of moles injected, and the resulting values were plotted
as a function of the molar ratio of [Pyr15T]/[Pur23A·Pyr23T], as
shown in Fig. 2c. The resulting titration plot was fitted to
a sigmoidal curve by using a nonlinear least-squares method. The
binding constant (Ka) and the enthalpy change
(
H) were obtained from the fitted curve (25, 26). The
Gibbs free energy change (
G) and the entropy change
(
S) were calculated from the equation
G =
RTlnKa =
H
T
S. The titration
plots with TFO alone or in the presence of spermine are also shown in
Fig. 2c. The thermodynamic parameters under these conditions
were obtained from these plots in the same way.

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 1.
a, structural formula of
PLL-g-Dex copolymer. b, oligonucleotide sequences
of the target duplex Pur23A·Pyr23T, the specific TFO Pyr15T, and the
nonspecific TFOs Pyr15NS-1 and Pyr15NS-2.
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2.
Thermodynamic analyses of pyrimidine
motif triplex formation at neutral pH by ITC. a, the ITC
profiles of the initial three injections for triplex formation between
Pyr15T and Pur23A·Pyr23T at 25 °C and pH 6.8 in the absence or
presence of the triplex stabilizer. The Pyr15T solution (120 µM in Buffer A; see "Materials and Methods") in the
absence or presence of the triplex stabilizer (0.84 mM
spermine or 0.038 mM PLL-g-Dex copolymer) was
injected in 5-µl increments into 5 µM Pur23A·Pyr23T
solution in the same buffer. Injections occurred over 10 s at
10-min intervals. The concentration of amino groups in 0.84 mM spermine is equivalent to that in 0.038 mM
PLL-g-Dex copolymer. The profile in the presence of spermine
is indistinguishable from that observed in the absence of the triplex
stabilizer. b, a total ITC profile for triplex formation
between Pyr15T and Pur23A·Pyr23T in the presence of the
PLL-g-Dex copolymer. The Pyr15T solution was injected 20 times into the Pur23A·Pyr23T solution. Other experimental conditions
were the same as those described in a. c,
titration plots in the absence or presence of the triplex stabilizer
against the molar ratio of [Pyr15T]/[Pur23A·Pyr23T]. The data
were fitted using a nonlinear least-squares method.
|
|
Table I summarizes the thermodynamic
parameters of triplex formation at 25 °C and pH 6.8 obtained from
ITC under the three different conditions. The signs of both
H and
S were negative under all reaction
conditions. Because an observed negative
S was
unfavorable for triplex formation, triplex formation was driven by a
large negative
H under each condition. The magnitude of the
H of pyrimidine motif triplex formation in the
presence of 0.038 mM PLL-g-Dex copolymer was 2.5 times larger than those observed with TFO alone or in the presence of
0.84 mM spermine, consistent with the ITC profiles in Fig.
2a. The Ka of pyrimidine motif triplex
formation was increased 2.6 times or 95.9 times by the addition of 0.84 mM spermine or 0.038 mM PLL-g-Dex
copolymer, respectively. Although the concentration of amino groups in
0.84 mM spermine and 0.038 mM
PLL-g-Dex copolymer is equivalent, an increase in
Ka by the addition of 0.038 mM
PLL-g-Dex copolymer was much higher than that by the
addition of 0.84 mM spermine.
View this table:
[in this window]
[in a new window]
|
Table I
Thermodynamic parameters for triplex formation between the 15-mer TFO
Pyr15T and the 23-bp duplex Pur23A · Pyr23T at 25 °C and pH
6.8 in 10 mM sodium cacodylate-cacodylic acid, 200 mM sodium chloride, and 20 mM magnesium
chloride with or without the triplex stabilizer obtained from ITC
measurements
The concentration of Pyr15T is 120 µM in the syringe.
Pyr15T is injected 20-times in 5-µl increments into Pur23A · Pyr23T. The concentration of Pur23A · Pyr23T is 5 µM in the cell. The obtained values are the average of at
least three ITC experiments.
|
|
Kinetic Effect of PLL-g-Dex Copolymer on Pyrimidine Motif Triplex
Formation at Neutral pH--
To understand the putative mechanism
involved in the tremendous increase in Ka of
pyrimidine motif triplex formation in the presence of the
PLL-g-Dex copolymer (Table I), we have assessed the kinetic
parameters for the association and dissociation of Pyr15T with
Pur23A·Pyr23T at 25 °C and pH 6.8 by IAsys. Fig. 3a compares the IAsys
sensorgrams representing the triplex formation and dissociation
involving 2.0 µM of the specific (Pyr15T) or nonspecific
(Pyr15NS-1) TFOs (Fig. 1b) and the immobilized
Bt-Pyr23T·Pur23A at 25 °C and pH 6.8 in either the absence
or presence of the triplex stabilizer. The injection of Pyr15T alone
(Pyr15T in no stabilizer) over the immobilized Bt-Pyr23T·Pur23A
caused an increased response, and the injection of Pyr15T and spermine
(Pyr15T in 0.84 mM spermine) slightly increased the
response. However, the response was substantially changed when Pyr15T
and the PLL-g-Dex copolymer were injected (Pyr15T in 0.038 mM copolymer). It is important to note that the negligible
response observed when Pyr15NS-1 was injected with the
PLL-g-Dex copolymer (Pyr15NS-1 in 0.038 mM
copolymer) indicates that the specificity of triplex formation was
preserved in the presence of the copolymer. Taken together, it
unambiguously indicates that the PLL-g-Dex copolymer
significantly increased the association rate constant of triplex
formation, and its triplex-promoting efficacy was remarkably higher
than that of spermine. We have measured a series of association and
dissociation curves at the various concentrations of Pyr15T in the
presence of the PLL-g-Dex copolymer to obtain the kinetic
parameters. An increase in the concentration of Pyr15T led to a gradual
change in the response of the association curves as shown in Fig.
3b. The on-rate constant (kon) was
obtained from the analysis of each association curve. Fig.
3c shows a plot of kon against the
concentrations of Pyr15T. The resultant plot was fitted to a straight
line (r2 = 0.98) using a linear least-squares
method. The association rate constant (kassoc)
was determined from the slope of the fitted line (27-29). On the other
hand, the off-rate constant (koff) was obtained
from the analysis of each dissociation curve (Fig. 3a; data
not shown). Because koff is usually independent
of the concentration of the injected solution, the dissociation rate
constant (kdissoc) was determined by averaging
koff for several concentrations (27-29). Ka was calculated from the equation
Ka = kassoc/kdissoc. The
kinetic parameters with TFO alone and in the presence of spermine were
obtained in the same way.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 3.
Kinetic analyses of pyrimidine motif
triplex formation at neutral pH by IAsys. Analysis of triplex
formation by IAsys is described in detail under "Materials and
Methods." a, typical IAsys sensorgrams for triplex
formation at 25 °C and pH 6.8 after injecting 2.0 µM
specific TFO alone (Pyr15T in Buffer A without the triplex stabilizer)
or in the presence of spermine (Pyr15T in Buffer A with 0.84 mM spermine) or the PLL-g-Dex copolymer (Pyr15T
in Buffer A with 0.038 mM PLL-g-Dex copolymer)
into the Bt-Pyr23T·Pur23A-immobilized cuvette are shown. A solution
containing 2.0 µM nonspecific TFO, Pyr15NS-1, and the
copolymer (Pyr15NS-1 in Buffer A with 0.038 mM
PLL-g-Dex copolymer) was also injected into the
Bt-Pyr23T·Pur23A-immobilized cuvette as a control experiment.
b, a series of IAsys sensorgrams for triplex formation
between Pyr15T and Pur23A·Pyr23T at 25 °C and pH 6.8 in the
presence of 0.038 mM PLL-g-Dex copolymer. The
Pyr15T solution, which was diluted in Buffer A to achieve the
indicated final concentrations, was injected with 0.038 mM PLL-g-Dex copolymer into the
Bt-Pyr23T·Pur23A-immobilized cuvette. The binding of Pyr15T to
Bt-Pyr23T·Pur23A was monitored as the response against time.
c, measured kon values of triplex
formation in b were plotted against the respective
concentrations of Pyr15T. The plot was fitted to a straight line
(r2 = 0.98) using a linear least-squares
method.
|
|
Table II summarizes the kinetic
parameters of triplex formation at 25 °C and pH 6.8 obtained from
IAsys under the three different conditions. Although the relative
values of Ka were consistent with those obtained
from ITC under the three conditions (Table I), the magnitudes of
Ka calculated from the ratio of kassoc/kdissoc in Table
II were slightly smaller than those in Table I. The precise source of
this discrepancy is not clearly understood. However, at least two
possible reasons can interpret this. First, in IAsys, the target duplex
Pur23A·Pyr23T is immobilized on the matrix surface. Therefore, the
triplex formation in IAsys, rather than that in solution in ITC, may be
accompanied by a larger negative change in entropy, which in turn leads
to loss in
G and thereby Ka. Second,
triplex formation on the matrix surface might be affected by the
molecular exclusion effect due to the accumulation of oligonucleotides
and the polymer on the matrix surface. Of course, further analysis is
needed to provide a precise explanation for this observation.
View this table:
[in this window]
[in a new window]
|
Table II
Kinetic parameters for triplex formation between the 15-mer TFO Pyr15T
and the 23-bp duplex Pur23A · Pyr23T at 25 °C and pH 6.8 in
10 mM sodium cacodylate-cacodylic acid, 200 mM
sodium chloride, and 20 mM magnesium chloride with or
without the triplex stabilizer obtained from IAsys measurements
Several different concentrations of Pyr15T were injected over the
immobilized Bt-Pyr23T · Pur23A. The concentrations of the
injected Pyr15T were between 2 and 40 µM for None,
between 2 and 20 µM for 0.84 mM spermine, and
between 0.5 and 2 µM for 0.038 mM
PLL-g-Dex copolymer.
|
|
The kassoc of pyrimidine motif triplex formation
increased 3.6 times or 45.6 times by the addition of 0.84 mM spermine or 0.038 mM PLL-g-Dex
copolymer, respectively. Once again, we have found a more significant
increase in kassoc by adding 0.038 mM PLL-g-Dex copolymer rather than 0.84 mM spermine, although the concentration of their amino
groups is almost equal. In contrast, when the dissociation rate
constant (kdissoc) of pyrimidine motif triplex
formation was compared, a 1.1 times or 1.5 times lower kdissoc was obtained by the addition of 0.84 mM spermine or 0.038 mM PLL-g-Dex
copolymer, respectively. Thus, the much larger Ka in
the presence of the PLL-g-Dex copolymer resulted mainly from the increase in kassoc rather than the decrease
in kdissoc.
Electrophoretic Mobility Shift Assay of Pyrimidine Motif Triplex
Formation at Neutral pH--
Finally, we have investigated pyrimidine
motif triplex formation between Pyr15T and Pur23A·Pyr23T in the
absence or presence of the triplex stabilizer at pH 7.0 by EMSA. Fig.
4 shows that in the absence of the
triplex stabilizer (None), Pyr15T had a very poor binding
affinity for Pur23A·Pyr23T; therefore, a stable triplex was formed
only at higher concentrations of Pyr15T. The addition of 5 µM nonspecific oligonucleotides (Pyr15NS-2 (see lane 1 for None) or Pyr15NS-1 (data not shown))
failed to form a triplex, suggesting a sequence-specific interaction of
Pyr15T with Pur23A·Pyr23T to form a triplex. The addition of 1.0 mM spermine (Spermine) with Pyr15T did indeed
slightly increase (~2 times) the binding affinity (see lane
5 for None and Spermine). It is important
that the addition of 1.9 µM (1/20 concentration of that used in ITC and IAsys) PLL-g-Dex copolymer
(Polymer) along with Pyr15T significantly increases triplex
formation. Note that whereas no triplex was detected when 0.02 µM TFO was incubated in either the absence of the triplex
stabilizer or the presence of 1.0 mM spermine (see
lane 2 for None and Spermine), more
than 60% of the triplex was formed in the presence of the
PLL-g-Dex copolymer (see lane 2 for
Polymer), although the charge ratio of (amino groups)stabilizer to (phosphate groups)DNA in
the presence of the PLL-g-Dex copolymer was 25 times lower
than that in the presence of spermine (see the figure legends). The
Ka of pyrimidine motif triplex formation in the
presence of the PLL-g-Dex copolymer was about 100 times
higher than that in the absence of the triplex stabilizer or even in
the presence of spermine. The tremendously higher triplex-promoting
efficiency of the PLL-g-Dex copolymer observed by ITC (Fig.
2a; Table I) and IAsys (Fig. 3a; Table II) was
further supported by EMSA.

View larger version (71K):
[in this window]
[in a new window]
|
Fig. 4.
EMSA of pyrimidine motif triplex formation at
neutral pH in either the absence or presence of the triplex
stabilizer. Triplex formation was initiated by adding
32P-labeled Pur23A·Pyr23T duplex (~1 ng) with the
indicated final concentrations of the specific TFO Pyr15T. Nonspecific
oligonucleotide Pyr15NS-2 was added to adjust the equimolar
concentrations of TFO (5.0 µM) in each lane. The reaction
mixtures in the buffer (50 mM Tris acetate, pH 7.0, 100 mM sodium chloride, and 10 mM magnesium
chloride) without the triplex stabilizer (None) or with 1.0 mM spermine (the charge ratio of (amino
groups)spermine/(phosphate groups)DNA was 50)
or with 1.9 µM PLL-g-Dex copolymer (the charge
ratio of (amino groups)copolymer/(phosphate
groups)DNA was 2) were incubated for 6 h at 37 °C
and then electrophoretically separated on a 15% native polyacrylamide
gel. The positions of the duplex (D) and triplex
(T) are indicated.
|
|
 |
DISCUSSION |
Using the ITC study, we have previously reported that the
Ka,
H, and
S values of
pyrimidine motif triplex formation between Pyr15T and Pur23A·Pyr23T
at pH 4.8 were 9 × 107 M
1,
83.8 kcal mol
1, and
245 cal mol
1
K
1, respectively (26). The Ka observed
for the same triplex formation at pH 6.8 without the triplex stabilizer
(Table I) is more than 100 times lower than that at pH 4.8 (26), which is consistent with the previously reported results (6-10) showing that
neutral pH is unfavorable for pyrimidine motif triplex formation involving C+·GC triads. In contrast, the
Ka at pH 6.8 in the presence of the
PLL-g-Dex copolymer (Table I) is similar in magnitude to
that at pH 4.8 (26), indicating that the copolymer tremendously promotes pyrimidine motif triplex formation at pH 6.8 up to a similar
level at pH 4.8.
The
H measured by ITC reflects a contribution from the
protonation of cytosine bases and the deprotonation of the buffer (26)
in addition to a contribution from the hydrogen bonding and base
stacking involved in the triplex formation. Because the pKa value of cytosine in a homopyrimidine
oligonucleotide is 4.4 (13) and Pyr15T contains five cytosines,
approximately 1.4 and 0.02 cytosines of Pyr15T are protonated in
solution at pH 4.8 and pH 6.8, respectively. Therefore, the residual
3.6 and 4.98 cytosines need to be protonated for effective pyrimidine motif triplex formation. After correcting for the deprotonation enthalpy change of the buffer (
0.47 kcal mol
1) and the
protonation enthalpy change of cytosine (
2.8 kcal mol
1)
(32), the corrected
H at pH 4.8 in the absence of the
copolymer and at pH 6.8 in the presence of the copolymer was about
72.0 and
71.6 kcal mol
1, respectively. These values
are consistent with each other, suggesting that pyrimidine motif
triplex formed almost stoichiometrically even at pH 6.8 in the presence
of the copolymer (Table I).
The
S measured by ITC includes a positive entropy change
from a release of structured water upon triplex formation, an entropy change accompanying the counterion condensation effect described below
(33, 34), and a major contribution of a negative entropy change from
the conformational restraint of TFO upon triplex formation (26). The
magnitudes of
S at pH 4.8 in the absence of the copolymer (
245 cal mol
1 K
1) (26) and at pH 6.8 in
the presence of the copolymer (
262 cal mol
1
K
1) (Table I) are quite similar, which also supports
effective pyrimidine motif triplex formation at pH 6.8 in the presence
of the copolymer (Table I). In contrast to stoichiometric triplex formation in the presence of the copolymer, triplex formation without
the triplex stabilizer (Table I) and in the presence of spermine (Table
I) is less stoichiometric, because the magnitudes of
H
and
S under these conditions are considerably lower than those at pH 4.8 (26).
Because the Ka of pyrimidine motif triplex formation
at pH 6.8 increased in the presence of spermine or the
PLL-g-Dex copolymer (Tables I and II; Fig. 4), both of them
have the ability to stabilize the triplex, but to a different extent.
The counterion condensation (CC) model developed by Manning (33) and
elaborated by Record et al. (34) provides reasonable
descriptions of the electrostatic interaction of counterions with DNA.
The association of TFO with the duplex to form a triplex results in an
increase in the linear charge density of DNA. Because the extent of CC per phosphate charges is a function of the linear charge density of DNA
(33, 34), triplex formation facilitates the CC in the vicinity of the
triplex from bulk solution (35, 36). However, the extent of the CC,
which is entropically unfavorable for triplex formation, should be
reduced in the presence of spermine or PLL-g-Dex copolymer
because the association of these polycationic (or oligocationic) materials with DNA is a counterion release process (37).
Whereas the concentration of amino groups in 0.038 mM
PLL-g-Dex copolymer is equivalent to that in 0.84 mM spermine, the Ka in the presence of
the former was much larger than that seen in the presence of the latter
(Tables I and II). Furthermore, as shown by EMSA (Fig. 4), triplex
formation was still promoted while a 1/20 concentration (1.9 µM) of the copolymer was used. The difference in the
promoting efficacy between spermine and the copolymer probably results
from their association constant with polynucleotides. An association
constant of a cationic substance with polynucleotides increases with
the valency of the cation (37, 38). Compared with spermine, which bears
only four cationic charges, the polycationic copolymer associates more
stably with polynucleotides. Indeed, the electrostatically
equivalent amount of the copolymer (the charge ratio of (amino
groups)copolymer to (phosphate groups)DNA was 1) completely abrogated the electrophoretic migration of 20-mer oligonucleotides into polyacrylamide gels (31), confirming a stoichiometric and stable complex formation between the copolymer and
the oligonucleotide. Furthermore, our previous melting curve analysis
also supports the stable association of the copolymer with
polynucleotides at physiological ionic strength (31). Because the
association of a polycationic (or oligocationic) substance with
polyanions, such as polynucleotides, is largely driven by entropic gain
from counterion release (37, 38), the more stable association of the
copolymer rather than spermine should be accompanied by a larger
counterion release. Thus, the stable association of the copolymer would
considerably reduce the extent of the CC and isolate triplex formation
from the CC effect. Isolation from the CC effect causes a net increase
in
S upon triplex formation, providing a favorable
component to
G and Ka, although the
apparent
S observed by ITC in the presence of the
copolymer (Table I) is decreased compared with that observed in the
absence of triplex stabilizers (Table I) or in the presence of spermine (Table I) due to the negative conformational entropy change from the
conformational restraint of TFO upon the stoichiometric triplex formation discussed above (26).
Despite such a strong association of the copolymer, it is unique that
the copolymer allows polynucleotides to recognize and associate with
each other on the basis of the base sequences (22, 31). As shown in the
present (Fig. 3a) and previous (23) studies, little
association of nonspecific oligonucleotides with the target duplex was
observed. This result further supports the idea that the copolymer does
not hinder the sequence specificity of triplex formation. The abundant
dextran moieties of the copolymer seem to play an important role in the
maintenance of sequence-specific recognition of polynucleotides.
Polycationic homopolymer, such as poly L-lysine and
polyarginine, formed insoluble complexes with polynucleotides (39). The
higher-order structure of the polynucleotides was considerably affected
in these complexes (40, 41). The sequence-specific recognition and
association between polynucleotides was disturbed by these polycations
(31). In contrast, the copolymer containing hydrophilic dextran chains in addition to poly L-lysine formed soluble complexes with
polynucleotides (22) and did not disorder the higher-order structure of
polynucleotides, such as poly(dA)·poly(dT) duplex,
poly(dA)·2poly(dT) triplex, and bovine thymus DNA (22, 31). The
maintenance of the solubility and the higher-order structure of
polynucleotides may be important for conserving the sequence-specific
interaction between polynucleotides.
In addition to the influence of the copolymer upon the CC effect, other
mechanisms might be involved in the enhancement of triplex formation by
the copolymer. The copolymer is designed to consist of 90 weight % dextran and 10 weight % poly L-lysine. The polynucleotides
associated with the copolymer may be forced to merge into the
dextran-rich phase, which is low in dielectric constant. Such an
environment under a low dielectric constant may enhance hydrogen
bonding between TFO and the target duplex to promote triplex formation.
Indeed, it has been reported that triplex stabilized by polyamines was
further stabilized by the addition of an organic solvent to aqueous
media (42).
Kinetic data have demonstrated that the copolymer considerably
increases the kassoc of pyrimidine motif triplex
formation (Table II). The increase in the kassoc
is a plausible kinetic reason to explain the remarkable gain in the
Ka at neutral pH by the copolymer. Both our group
(26) and others (43) have previously proposed a model in which
triplexes form along nucleation-elongation processes; in a nucleation
step, only a few base contacts of the Hoogsteen hydrogen bonds may be
formed between TFO and the target duplex, and this may be followed by
an elongation step, in which Hoogsteen base pairings progress to
complete triplex formation. Both groups (26, 43) have also suggested
that the observed Ka, which is the ratio of
kassoc/kdissoc, may
largely reflect a rapid equilibrium of the nucleation step, which is
probably the rate-limiting process of triplex formation. In this sense, the copolymer is considered to accelerate the nucleation step by
reducing the extent of the CC to increase the Ka in pyrimidine motif triplex formation.
The present study has clearly demonstrated that the
PLL-g-Dex copolymer promotes pyrimidine motif triplex
formation at neutral pH. The triplex-promoting effect of the
PLL-g-Dex copolymer does not affect the sequence specificity
of triplex formation. We conclude that the design of polycationic
copolymer with grafts of hydrophilic side chains (i.e.
comb-type copolymer) is certainly a promising strategy for the
promotion of triplex formation and may eventually lead to progress in
therapeutic applications of the antigene strategy in
vivo.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge M. Aoyagi and Y. Kanaya of Nissei Sangyo Co., Ltd. for generous help with the IAsys measurements.
 |
FOOTNOTES |
*
This work was supported in part by Grants-in-Aid 08249247 (to H. T.) and 09750967 (to A. M.) from the Ministry of Education, Science, Sports and Culture of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed. Tel.: 81-298-36-9082;
Fax: 81-298-36-9080; E-mail: torigoe{at}rtc.riken.go.jp (H. T.). Tel.:
81-45-924-5809; Fax: 81-45-924-5815; E-mail: amaruyam{at}bio.titech.ac.jp (A. M.).
 |
ABBREVIATIONS |
The abbreviations used are:
TFO, triplex-forming
oligonucleotide;
PLL-g-Dex, poly
(L-lysine)-graft-dextran;
ITC, isothermal
titration calorimetry;
IAsys, interaction analysis system;
EMSA, electrophoretic mobility shift assay;
kon, on-rate constant;
kassoc, association rate
constant;
koff, off-rate constant;
kdissoc, dissociation rate constant;
CC, counterion condensation;
bp, base pair.
 |
REFERENCES |
-
Mirkin, S. M.,
and Frank-Kamenetskii, M. D.
(1994)
Annu. Rev. Biophys. Biomol. Struct.
23,
541-576[CrossRef][Medline]
[Order article via Infotrieve]
-
Frank-Kamenetskii, M. D.,
and Mirkin, S. M.
(1995)
Annu. Rev. Biochem.
64,
65-95[CrossRef][Medline]
[Order article via Infotrieve]
-
Soyfer, V. N.,
and Potaman, V. N.
(1996)
Triple-Helical Nucleic Acids, Springer-Verlag New York, Inc., New York
-
Sun, J.-S.,
and Helene, C.
(1993)
Curr. Opin. Struct. Biol.
3,
345-356
-
Sun, J.-S.,
Garestier, T.,
and Helene, C.
(1996)
Curr. Opin. Struct. Biol.
6,
327-333[CrossRef][Medline]
[Order article via Infotrieve]
-
Lee, J. S.,
Johnson, D. A.,
and Morgan, A. R.
(1979)
Nucleic Acids Res.
6,
3073-3091[Abstract]
-
Lyamichev, V. I.,
Mirkin, S. M.,
and Frank-Kamenetskii, M. D.
(1985)
J. Biomol. Struct. Dyn.
3,
327-338[Medline]
[Order article via Infotrieve]
-
Frank-Kamenetskii, M. D.
(1992)
Methods Enzymol.
211,
180-191[Medline]
[Order article via Infotrieve]
-
Singleton, S. F.,
and Dervan, P. B.
(1992)
Biochemistry
31,
10995-11003[Medline]
[Order article via Infotrieve]
-
Shindo, H.,
Torigoe, H.,
and Sarai, A.
(1993)
Biochemistry
32,
8963-8969[Medline]
[Order article via Infotrieve]
-
Lee, J. S.,
Woodsworth, M. L.,
Latimer, L. J. P.,
and Morgan, A. R.
(1984)
Nucleic Acids Res.
12,
6603-6614[Abstract]
-
Povsic, T. J.,
and Dervan, P. B.
(1989)
J. Am. Chem. Soc.
111,
3059-3061
-
Xodo, L. E.,
Manzini, G.,
Quadrifoglio, F.,
van der Marel, G. A.,
and van Boom, J. H.
(1991)
Nucleic Acids Res.
19,
5625-5631[Abstract]
-
Ono, A.,
Ts'o, P. O. P.,
and Kan, L.-S.
(1991)
J. Am. Chem. Soc.
113,
4032-4033
-
Krawczyk, S. H.,
Milligan, J. F.,
Wadwani, S.,
Moulds, C.,
Froehler, B. C.,
and Matteucci, M. D.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
3761-3764[Abstract]
-
Koh, J. S.,
and Dervan, P. B.
(1992)
J. Am. Chem. Soc.
114,
1470-1478
-
Jetter, M. C.,
and Hobbs, F. W.
(1993)
Biochemistry
32,
3249-3254[Medline]
[Order article via Infotrieve]
-
Ueno, Y.,
Mikawa, M.,
and Matsuda, A.
(1998)
Bioconjugate Chem.
9,
33-39[CrossRef][Medline]
[Order article via Infotrieve]
-
Sun, J. S.,
Giovannangeli, C.,
Francois, J. C.,
Kurfurst, R.,
MontenayGarestier, T.,
Asseline, U.,
Saison-Behmoaras, T.,
Thuong, N. T.,
and Helene, C.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
6023-6027[Abstract]
-
Mouscadet, J.-F.,
Ketterle, C.,
Goulaouic, H.,
Carteau, S.,
Subra, F.,
Le Bret, M.,
and Auclair, C.
(1994)
Biochemistry
33,
4187-4196[Medline]
[Order article via Infotrieve]
-
Hampel, K. J.,
Crosson, P.,
and Lee, J. S.
(1991)
Biochemistry
30,
4455-4459[Medline]
[Order article via Infotrieve]
-
Maruyama, A.,
Katoh, M.,
Ishihara, T.,
and Akaike, T.
(1997)
Bioconjugate Chem.
8,
3-6[CrossRef][Medline]
[Order article via Infotrieve]
-
Ferdous, A.,
Watanabe, H.,
Akaike, T.,
and Maruyama, A.
(1998)
Nucleic Acids Res.
26,
3949-3954[Abstract/Free Full Text]
-
Ferdous, A.,
Watanabe, H.,
Akaike, T.,
and Maruyama, A.
(1998)
J. Pharm. Sci.
87,
1400-1405[CrossRef][Medline]
[Order article via Infotrieve]
-
Wiseman, T.,
Williston, S.,
Brandts, J. F.,
and Lin, L.-N.
(1989)
Anal. Biochem.
179,
131-137[Medline]
[Order article via Infotrieve]
-
Kamiya, M.,
Torigoe, H.,
Shindo, H.,
and Sarai, A.
(1996)
J. Am. Chem. Soc.
118,
4532-4538[CrossRef]
-
Cush, R.,
Cronin, J. M.,
Stewart, W. J.,
Maule, C. H.,
Molloy, J.,
and Goddard, N. J.
(1993)
Biosens. Bioelectron.
8,
347-353[CrossRef]
-
Edwards, P. R.,
Gill, A.,
Pollard-Knight, D. V.,
Hoare, M.,
Buckle, P. E.,
Lowe, P. A.,
and Leatherbarrow, R. J.
(1995)
Anal. Biochem.
231,
210-217[CrossRef][Medline]
[Order article via Infotrieve]
-
Bates, P. J.,
Dosanjh, H. S.,
Kumar, S.,
Jenkins, T. C.,
Laughton, C. A.,
and Neidle, S.
(1995)
Nucleic Acids Res.
23,
3627-3632[Abstract]
-
Thomas, T.,
and Thomas, T. J.
(1993)
Biochemistry
32,
14068-14074[Medline]
[Order article via Infotrieve]
-
Maruyama, A.,
Watanabe, H.,
Ferdous, A.,
Katoh, M.,
Ishihara, T.,
and Akaike, T.
(1998)
Bioconjugate Chem.
9,
292-299[CrossRef][Medline]
[Order article via Infotrieve]
-
Manzini, G.,
Xodo, L. E.,
Gasparotto, D.,
Quadrifoglio, F.,
van der Marel, G. A.,
and van Boom, J. H.
(1990)
J. Mol. Biol.
213,
833-843[Medline]
[Order article via Infotrieve]
-
Manning, G. S.
(1978)
Q. Rev. Biophys.
11,
179-246[Medline]
[Order article via Infotrieve]
-
Record, M. T., Jr.,
Anderson, C. F.,
and Lohman, T. M.
(1978)
Q. Rev. Biophys.
11,
103-178[Medline]
[Order article via Infotrieve]
-
Singleton, S. F.,
and Dervan, P. B.
(1993)
Biochemistry
32,
13171-13179[Medline]
[Order article via Infotrieve]
-
Bond, J. P.,
Anderson, C. F.,
and Record, M. T., Jr.
(1994)
Biophys. J.
67,
825-836[Abstract]
-
Mascotti, D. P.,
and Lohman, T. M.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
3142-3146[Abstract]
-
Record, M. T., Jr.,
Lohman, T. M.,
and de Haseth, P.
(1976)
J. Mol. Biol.
107,
145-158[Medline]
[Order article via Infotrieve]
-
Olins, D. E.,
Olins, A. L.,
and von Hippel, P. H.
(1967)
J. Mol. Biol.
24,
157-176[Medline]
[Order article via Infotrieve]
-
Tsuboi, M.
(1967)
in
Conformation of Biopolymers (Ramachandran, G. N., ed), Vol. II, pp. 689-702, Academic Press, New York
-
Haynes, M.,
Garrett, R. A.,
and Gratzer, W. B.
(1970)
Biochemistry
9,
4410-4416[Medline]
[Order article via Infotrieve]
-
Moser, H. E.,
and Dervan, P. B.
(1987)
Science
238,
645-650[Medline]
[Order article via Infotrieve]
-
Rougee, M.,
Faucon, B.,
Mergny, J. L.,
Barcelo, F.,
Giovannangeli, C.,
Garestier, T.,
and Helene, C.
(1992)
Biochemistry
31,
9269-9278[Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.