From the Tsukuba Life Science Center, Institute of
Physical and Chemical Research (RIKEN), Ibaraki 305-0074, Japan and
¶ Graduate School of Pharmaceutical Sciences, Osaka University,
Osaka 565-0871, Japan
Received for publication, August 25, 2000, and in revised form, October 5, 2000
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ABSTRACT |
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Extreme instability of pyrimidine motif triplex
DNA at physiological pH severely limits its use in an artificial
control of gene expression in vivo. Stabilization of the
pyrimidine motif triplex at physiological pH is, therefore, crucial in
improving its therapeutic potential. To this end, we have investigated
the thermodynamic and kinetic effects of our previously reported
chemical modification, 2'-O,4'-C-methylene
bridged nucleic acid (2',4'-BNA) modification of triplex-forming
oligonucleotide (TFO), on pyrimidine motif triplex formation at
physiological pH. The thermodynamic analyses indicated that the
2',4'-BNA modification of TFO increased the binding constant of the
pyrimidine motif triplex formation at neutral pH by ~20 times. The
number and position of the 2',4'-BNA modification introduced into the
TFO did not significantly affect the magnitude of the increase in the
binding constant. The consideration of the observed thermodynamic
parameters suggested that the increased rigidity itself of the
2',4'-BNA-modified TFO in the free state relative to the unmodified TFO
may enable the significant increase in the binding constant at neutral
pH. Kinetic data demonstrated that the observed increase in the binding
constant at neutral pH by the 2',4'-BNA modification of TFO resulted
from the considerable decrease in the dissociation rate constant. Our
results certainly support the idea that the 2',4'-BNA modification of
TFO could be a key chemical modification and may eventually lead to
progress in therapeutic applications of the antigene strategy in
vivo.
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 homopurine-homopyrimidine stretch in duplex DNA (1-5). 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). On the other
hand, 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).
Because the cytosine bases in a homopyrimidine TFO must 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 (6-8). Instead, the
pH-independent formation of the purine motif triplex is available at
physiological pH. However, the purine motif triplex formation is
severely inhibited by physiological concentrations of certain monovalent cations, especially K+. Undefined association
between K+ and the guanine-rich homopurine TFO has been
applied to explain the inhibitory effect (9, 10). Therefore,
stabilization of the pyrimidine motif triplex at physiological pH is of
great importance in improving its therapeutic potential to artificially
control gene expression in vivo. Numerous efforts such as
the replacement of cytosine bases in a homopyrimidine TFO with
5-methylcytosine (7, 11-13) or other chemically modified bases
(14-18), the conjugation of different DNA intercalators to TFO (19,
20), and 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.
We first synthesized and developed a new class of chemical
modifications of nucleic acids, bridged nucleic acid (BNA), such as
2'-O,4'-C-methylene BNA (2',4'-BNA; Fig.
1a; Refs. 22-27)2
and 3'-O,4'-C-methylene BNA (3',4'-BNA; Refs.
30-32). The 2',4'-BNA modification of TFO increased the thermal
stability of the pyrimidine motif triplex DNA at neutral pH using a
homopurine-homopyrimidine target duplex and its specific cytosine-rich
TFO (25). However, the mechanistic explanation for the 2',4'-BNA
modification-mediated triplex stabilization was not clearly understood.
Here, therefore, we have further extended our previous study to
explore thermodynamic and kinetic effects of the 2',4'-BNA modification
on the pyrimidine motif triplex formation at neutral pH. The
thermodynamic and kinetic effects of the 2',4'-BNA modification on the
pyrimidine motif triplex formation between a 23-base pair
homopurine-homopyrimidine target duplex (Pur23A·Pyr23T; Fig.
1b) and its specific 15-mer unmodified or 2',4'-BNA-modified
homopyrimidine TFO (Pyr15T, Pyr15BNA7-1, Pyr15BNA7-2, Pyr15BNA5-1, and
Pyr15BNA5-2; Fig. 1b) have been analyzed by electrophoretic
mobility shift assay (EMSA; Refs. 33, 34), UV melting, isothermal
titration calorimetry (ITC; Refs. 34-40), and interaction analysis
system (IAsys; Refs. 34, 39-43). Results from these independent lines
of experiments have clearly indicated the significant effect of the
2',4'-BNA modification to promote the pyrimidine motif triplex
formation at neutral pH. The binding constant at neutral pH for the
pyrimidine motif triplex formation with the 2',4'-BNA-modified TFO was
~10-20 times larger than that observed with the corresponding
unmodified TFO. Kinetic data have also demonstrated that the
contribution for the increase in the binding constant by the 2',4'-BNA
modification of TFO resulted from the considerable decrease in the
dissociation rate constant. The ability of the 2',4'-BNA modification
of TFO to promote the pyrimidine motif triplex formation at
physiological pH would support further progress in therapeutic
applications of the antigene strategy in vivo.
Preparation of Oligonucleotides--
We synthesized 23-mer
complementary oligonucleotides for the target duplex Pur23A and Pyr23T
(Fig. 1b), a 15-mer unmodified homopyrimidine TFO specific
for the target duplex Pyr15T (Fig. 1b), and a nonspecific
homopyrimidine oligonucleotide, Pyr15NS (Fig. 1b), on an
Applied Biosystems DNA synthesizer using the solid-phase cyanoethyl
phosphoramidite method and purified them with a reverse-phase high
performance chromatography on a Wakosil DNA column. The 15-mer
2',4'-BNA-modified homopyrimidine TFOs Pyr15BNA7-1, Pyr15BNA7-2,
Pyr15BNA5-1, and Pyr15BNA5-2 (Fig. 1b) were synthesized and
purified as described previously (22, 28). 5' biotinylated Pyr23T
(denoted Bt-Pyr23T) was prepared using biotin phosphoramidite. 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 optical density unit = 50 µg/ml,
with an Mr of 15180.
EMSA--
EMSA experiments were performed essentially as
described previously (34). In 9 µl of reaction mixture,
32P-labeled duplex (~1 ng) was mixed with increasing
concentrations of the specific TFO (Pyr15T, Pyr15BNA7-1, Pyr15BNA7-2,
Pyr15BNA5-1, or Pyr15BNA5-2) and the nonspecific oligonucleotide
(Pyr15NS) in a buffer containing 50 mM Tris acetate, pH
7.0, 100 mM sodium chloride, and 10 mM
magnesium chloride. Pyr15NS was added to achieve equimolar
concentrations of TFO in each lane as well as to minimize adhesion of
the DNA (target duplex and TFO) to plastic surfaces during incubation
and subsequent losses during processing. After 6 h of incubation
at 37 °C, 2 µl of 50% glycerol solution containing bromphenol
blue was added without changing the pH and salt concentrations of the
reaction mixtures. 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.
UV Melting--
UV melting experiments were carried out on a
Jasco Ubest-30 spectrophotometer equipped with an EHC-363 Peltier-type
cell holder controlled by a TPU-436 temperature programmer. UV melting
profiles were measured in buffer A (10 mM sodium
cacodylate-cacodylic acid, pH 6.8, containing 200 mM NaCl
and 20 mM MgCl2) at a scan rate of
0.5 °C/min at 260 nm. The first derivative was calculated from the
UV melting profile. The peak temperatures in the derivative curve were
designated the melting temperatures (Tms). Cell
path length was 1 cm. The triplex DNA concentration used was 1 µM.
Circular Dichroism (CD) Spectroscopy--
CD spectra at
room temperature were recorded in buffer A on a Jasco J-720
spectropolarimeter interfaced with a microcomputer. Cell path length
was 1 cm. The triplex DNA concentration used was 4 µM.
ITC--
Isothermal titration experiments were carried out on an
MCS ITC system (Microcal Inc.), essentially as described previously (34, 38, 39). The TFO and Pur23A·Pyr23T duplex DNA solutions were
prepared by extensive dialysis against buffer A or buffer B (10 mM sodium cacodylate-cacodylic acid, pH 5.8, containing 200 mM NaCl and 20 mM MgCl2). The TFO
solution in buffer A or buffer B was injected 20 times in 5-µl
increments and 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 the same buffer. Each
corrected heat was divided by the moles of the TFO injected and
analyzed with Microcal Origin software supplied by the manufacturer.
IAsys--
Kinetic experiments by resonant mirror method were
performed on an IAsys Plus instrument (Affinity Sensors Cambridge
Inc.), essentially as described previously, in which a real-time
biomolecular interaction was measured with a laser biosensor (34, 39). The resonant layer of a cuvette was washed with 80 µl of 10 mM acetate buffer, pH 4.6, and then activated with 80 µl
of a mixture of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and
N-hydroxysuccinimide solution. The activated surface was
again washed with 10 mM acetate buffer, pH 4.6, and
streptavidin in 10 mM acetate buffer, pH 4.6, was
immobilized to the surface. After blocking the remaining reactive groups with 1 M ethanolamine, pH 8.5, the cuvette was
extensively washed with 10 mM acetate buffer, pH 4.6, and
then with 20 mM HCl 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, the
complementary oligonucleotide Pur23A (1.2 µM in buffer A)
was added to hybridize with Bt-Pyr23T. After extensive washing and
equilibrating the Bt-Pyr23T·Pur23A-immobilized surface with buffer A
for >30 min, the TFO in 80 µl of buffer A was injected over the
immobilized Bt-Pyr23T·Pur23A duplex, and then the triplex formation
was monitored for 30 min. This was followed by washing the cuvette with
buffer A, and the dissociation of the preformed triplex was monitored
for an additional 20 min. Finally, 10 mM NaOH, pH 12, was
injected for 3 min for 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.
Electrophoretic Mobility Shift Assay of Pyrimidine
Motif Triplex Formation at Neutral pH--
The pyrimidine motif
triplex formation of the target duplex (Pur23A·Pyr23T; Fig.
1b) with unmodified (Pyr15T;
Fig. 1b) or 2',4'-BNA-modified (Pyr15BNA7-1,
Pyr15BNA7-2, Pyr15BNA5-1, or Pyr15BNA5-2; Fig. 1b) TFO was
examined at pH 7.0 by EMSA (Fig. 2). The
total oligonucleotide concentration ([specific TFO (Pyr15T, Pyr15BNA7-1, Pyr15BNA7-2, Pyr15BNA5-1, or Pyr15BNA5-2; Fig.
1b)] + [nonspecific oligonucleotide (Pyr15NS; Fig.
1b)]) was kept constant at 1 µM to minimize
loss of DNA during processing. Although incubation with 1 µM Pyr15NS alone did not cause a shift in electrophoretic migration of the target duplex (see Fig. 2, lane 1 for Pyr15T), those with Pyr15T, Pyr15BNA7-1, Pyr15BNA7-2, Pyr15BNA5-1,
or Pyr15BNA5-2 at particular concentration caused retardation of the
duplex migration owing to triplex formation (33). The
Kd of triplex formation was determined from the
concentration of the TFO, which caused half of the target duplex to
shift to the triplex (33). The Kd of the triplex
with Pyr15T was estimated to be ~1.0 µM. In contrast,
the Kd of the triplex with Pyr15BNA7-1, Pyr15BNA7-2,
Pyr15BNA5-1, or Pyr15BNA5-2 was ~0.06 µM, indicating that the 2',4'-BNA modification of TFO increased the binding affinity of the pyrimidine motif triplex formation at neutral pH by ~20 times.
The increase in the Ka by the 2',4'-BNA modification was similar in magnitude among the four modified TFOs.
Spectroscopic Characterization of Pyrimidine Motif Triplex at
Neutral pH--
The thermal stability of the pyrimidine motif
triplex with unmodified (Pyr15T) or 2',4'-BNA-modified
(Pyr15BNA7-1, Pyr15BNA7-2, Pyr15BNA5-1, or Pyr15BNA5-2) TFO was
investigated at pH 6.8 by UV melting (Fig.
3 and Table
I). All the triplexes showed
two-step melting. The first transition at a lower temperature,
Tm1, was the melting of the triplex to a duplex
and a TFO, and the second transition at a higher temperature,
Tm2, was the melting of the duplex (Fig. 3).
Although the Tm2 was almost identical among all the triplexes, the Tm1 for Pyr15BNA7-1,
Pyr15BNA7-2, Pyr15BNA5-1, or Pyr15BNA5-2 was significantly higher than
that for Pyr15T (Table I), indicating that the 2',4'-BNA modification
of TFO increased the thermal stability of the pyrimidine motif triplex
at neutral pH.
To further characterize the triplexes involving unmodified (Pyr15T) or
2',4'-BNA-modified (Pyr15BNA7-1, Pyr15BNA7-2, Pyr15BNA5-1, or
Pyr15BNA5-2) TFO, CD spectra of the triplexes were measured at
25 °C and pH 6.8 (Fig. 4). The overall
shape of the CD spectra was similar among all the profiles. A negative
band in the short-wavelength (210-220-nm) region was observed for all
the profiles, confirming the triplex formation involving each TFO
(44, 45). The intensity of the negative short-wavelength (210-220-nm)
band for the triplexes involving Pyr15BNA7-1, Pyr15BNA7-2, Pyr15BNA5-1,
or Pyr15BNA5-2 was larger than that observed for the triplex
involving Pyr15T, indicating that all the pyrimidine motif triplexes
involving the 2',4'-BNA-modified TFO had more aspects of the A
conformation than that involving the unmodified TFO (45).
Thermodynamic Analyses of Pyrimidine Motif Triplex Formation at
Neutral pH by ITC--
We examined the thermodynamic parameters of the
pyrimidine motif triplex formation between a 23-base pair target duplex
(Pur23A·Pyr23T) and its specific 15-mer unmodified (Pyr15T) or
2',4'-BNA-modified (Pyr15BNA7-1, Pyr15BNA7-2, Pyr15BNA5-1, or
Pyr15BNA5-2) TFO at 25 °C and pH 6.8 by ITC. To investigate the
pH dependence of the pyrimidine motif triplex formation, the
thermodynamic parameters of the triplex formation between
Pur23A·Pyr23T and Pyr15T were also analyzed at 25 °C and pH 5.8 by
ITC. Fig. 5a compares the ITC profiles of
the initial three injections for the binding of Pur23A·Pyr23T with
Pyr15T or Pyr15BNA7-1 at pH 6.8 and with Pyr15T at pH 5.8. The
magnitudes of the exothermic peaks for Pyr15BNA7-1 at pH 6.8 and for
Pyr15T at pH 5.8 were larger than those observed for Pyr15T at pH 6.8. Fig. 5b shows an ITC profile over 200 min for triplex formation with
Pyr15BNA7-1 at pH 6.8. An exothermic heat pulse was observed after each
injection of Pyr15BNA7-1 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 [Pyr15BNA7-1]/[Pur23A·Pyr23T] = 2. The area of the small peak was equal to the heat of dilution
measured in a separate experiment by injecting Pyr15BNA7-1 into the
same buffer (data not shown). The area under each peak was integrated,
and the heat of dilution of Pyr15BNA7-1 was subtracted from the
integrated values. The corrected heat was divided by the moles of
injected solution, and the resulting values were plotted as a
function of a molar ratio of [Pyr15BNA7-1]/[Pur23A·Pyr23T], as
shown in Fig. 5c. The resultant titration plot was fitted to
a sigmoidal curve by a nonlinear least-squares method. The binding
constant, Ka, and the enthalpy change,
Table II summarizes the thermodynamic
parameters for the pyrimidine motif triplex formation with all the TFOs
at 25 °C and pH 6.8 and those with Pyr15T at 25 °C and pH 5.8, obtained from ITC. The signs of both Kinetic Analyses of Pyrimidine Motif Triplex Formation at Neutral
pH by IAsys--
To examine the putative mechanism involved in the
increase in Ka of the pyrimidine motif triplex
formation by the 2',4'-BNA modification (Fig. 2 and Table II), we
assessed the kinetic parameters for the association and dissociation of
TFO (Pyr15T, Pyr15BNA7-1, Pyr15BNA7-2, Pyr15BNA5-1, or Pyr15BNA5-2) with Pur23A·Pyr23T at 25 °C and pH 6.8 by IAsys. Fig.
6a compares the sensorgrams
representing the triplex formation and dissociation involving 90 µM of the specific TFO (Pyr15T or Pyr15BNA7-1). The injection of Pyr15T over the immobilized Bt-Pyr23T·Pur23A caused an
increase in response. The response was substantially delayed on
the injection of Pyr15BNA7-1, indicating that the 2',4'-BNA modification decreased the association rate constant of the triplex equilibrium. In contrast, the change in the dissociation curve with
time for Pyr15BNA7-1 was much smaller than that for Pyr15T. The result
clearly indicated that the 2',4'-BNA modification of TFO remarkably
decreased the dissociation rate constant of the triplex equilibrium.
The similar profiles were obtained for Pyr15BNA7-2, Pyr15BNA5-1, and
Pyr15BNA5-2.
To understand the kinetic parameters more quantitatively, we analyzed a
series of association and dissociation curves at the various
concentrations of TFO. As shown in Fig. 6b, an increase in
the concentration of the 2',4'-BNA-modified TFO (Pyr15BNA7-1) led to a
gradual change in the response of the association curves. The
kon was obtained from the analysis of each
association curve. Fig. 6c shows a plot of
kon against the Pyr15BNA7-1 concentrations. The
resultant plot was fitted to a straight line by a linear least-squares method. The association rate constant (kassoc)
and the dissociation rate constant
(kdissoc) were determined from the
slope and the intercept of the regression line, respectively (41-43).
The kinetic parameters for the triplex formation with Pyr15BNA7-2,
Pyr15BNA5-1, and Pyr15BNA5-2 were obtained in the same way.
Ka was calculated from the equation,
Ka = kassoc/kdissoc.
Because the change of the dissociation curves with time was very small for Pyr15BNA7-1, Pyr15BNA7-2, Pyr15BNA5-1, and Pyr15BNA5-2 (Fig. 6a; data not shown), that is, the dissociation was
very slow, we could not directly determine the
kdissoc from the dissociation curves. Thus, the
kdissoc obtained from the association curves was
presented for Pyr15BNA7-1, Pyr15BNA7-2, Pyr15BNA5-1, and Pyr15BNA5-2, although its S.D. was relatively large (Table
III). On the other hand, although the
kassoc for Pyr15T was obtained in the same way
as described above, the kdissoc for Pyr15T was
determined in the following way owing to the much faster dissociation.
The off-rate constant (koff) was obtained from
the analysis of each dissociation curve (Fig. 6a; data not
shown). Because koff is usually independent of
the concentration of the injected solution, the
kdissoc was determined by averaging
koff for several concentrations (41-43).
Ka was calculated from the equation
Ka = kassoc/kdissoc.
Table III summarizes the kinetic parameters for the pyrimidine motif
triplex formation with all the TFOs at 25 °C and pH 6.8, obtained
from IAsys. The magnitudes of Ka calculated from the
ratio of kassoc to
kdissoc (Table III) were consistent with those
obtained from ITC (Table II). The 2',4'-BNA modification of TFO
increased the Ka for the pyrimidine motif triplex formation at neutral pH, which supported the results of EMSA (Fig. 2)
and ITC (Table II). The kdissoc of the triplex
formation decreased ~40-70 times by the 2',4'-BNA modification of
TFO. In contrast, when the kassoc of the triplex
formation was compared, 1-1.3 times smaller
kassoc was obtained by the 2',4'-BNA
modification of TFO, which opposes the increase in
Ka. Thus, the much larger Ka by
the 2',4'-BNA modification resulted from the decrease in
kdissoc. The kinetic effect to increase the
Ka by the 2',4'-BNA modification was similar among
the four modified TFOs.
The Ka of the pyrimidine motif triplex
formation with Pyr15T at pH 5.8 was 20 times larger than that observed
with Pyr15T at pH 6.8 (Table II), which is consistent with the
previously reported results that the neutral pH is unfavorable for the
pyrimidine motif triplex formation involving C+·GC triads
(6-8). The Ka of the triplex formation with Pyr15BNA7-1, Pyr15BNA7-2, Pyr15BNA5-1, or Pyr15BNA5-2 at pH 6.8 was 10-20 times larger than that observed with Pyr15T at pH 6.8 (Table
II). The increase in Ka at pH 6.8 by the 2',4'-BNA modification of TFO was supported by the results of EMSA (Fig. 2) and
IAsys (Table III). In addition, the 2',4'-BNA modification of TFO
increased the thermal stability of the pyrimidine motif triplex at pH
6.8 (Table I). These results indicate that the 2',4'-BNA modification
of TFO considerably promotes the pyrimidine motif triplex formation at
neutral pH.
The Although the Ka and The increase in the Ka by the 2',4'-BNA modification
was similar in magnitude among the four modified TFOs (Fig. 2 and
Tables II and III), indicating that the number and position of the
2',4'-BNA modification did not significantly affect the magnitude of
the increase in the Ka at neutral pH. The rigidity
itself of the 2',4'-BNA-modified TFO may be more important to achieve
the increase in the Ka at neutral pH than the
variation of the number and position of the 2',4'-BNA modification. Thus, other modification strategies to gain the increased rigidity of
TFO may also be useful to increase the Ka at neutral pH.
Kinetic data have demonstrated that the 2',4'-BNA modification of TFO
considerably decrease the kdissoc of the
pyrimidine motif triplex formation (Table III). The decrease in the
kdissoc is a plausible kinetic reason to explain
the remarkable gain in the Ka at neutral pH by the
2',4'-BNA modification (Fig. 2 and Tables II and III). Both our group
(38) and others (49) have previously proposed a model that 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 (38, 49) have also suggested that the observed Ka, which is the ratio of
kassoc to kdissoc, may
mostly reflect rapid equilibrium of the nucleation step, which is
probably the rate-limiting process of the triplex formation. In this
sense, the 2',4'-BNA modification of TFO is considered to slow the
collapse of the nucleation intermediate using the rigidity of TFO to
increase the Ka of the pyrimidine motif triplex formation.
The present study has clearly demonstrated that the 2',4'-BNA
modification of TFO promotes pyrimidine motif triplex formation at
neutral pH. We conclude that the design of TFO to bridge different positions of sugar moiety with the alkyl chain to gain the increased rigidity of TFO is certainly a promising strategy for the promotion of
triplex formation under physiological condition and may eventually lead
to progress in therapeutic applications of the antigene strategy in vivo.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
a, nucleotide unit of 2',4'-BNA.
b, oligonucleotide sequences of the target duplex
(Pur23A·Pyr23T), the specific TFOs (Pyr15T, Pyr15BNA7-1, Pyr15BNA7-2,
Pyr15BNA5-1, and Pyr15BNA5-2), and the nonspecific oligonucleotide
(Pyr15NS).
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Fig. 2.
EMSA of the pyrimidine motif triplex
formation with the specific TFO (Pyr15T, Pyr15BNA7-1, Pyr15BNA7-2,
Pyr15BNA5-1, or Pyr15BNA5-2) at neutral pH. Triplex formation was
initiated by adding 32P-labeled Pur23A·Pyr23T duplex
(~1 ng) with the indicated final concentrations of the specific TFO
(Pyr15T, Pyr15BNA7-1, Pyr15BNA7-2, Pyr15BNA5-1, or Pyr15BNA5-2). The
nonspecific oligonucleotide (Pyr15NS) was added to adjust the equimolar
concentrations of TFO (1 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) were incubated for 6 h at 37 °C, and then
electrophoretically separated on a 15% native polyacrylamide gel at
4 °C. The positions of the duplex (D) and triplex
(T) are indicated.
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Fig. 3.
UV melting profiles of the pyrimidine motif
triplex with the specific TFO (Pyr15T, Pyr15BNA7-1, Pyr15BNA7-2,
Pyr15BNA5-1, or Pyr15BNA5-2) at neutral pH. The triplexes in
buffer A were melted at a scan rate of 0.5 °C/min with detection at
260 nm. Cell path length was 1 cm. The triplex DNA concentration used
was 1 µM.
Melting temperatures of the triplexes between a 15-mer TFO (Pyr15T,
Pyr15BNA7-1, Pyr15BNA7-2, Pyr15BNA5-1, or Pyr15BNA5-2) and a 23 base pair target duplex (Pur23A · Pyr23T)
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Fig. 4.
CD spectra of the pyrimidine motif triplex
with the specific TFO (Pyr15T, Pyr15BNA7-1, Pyr15BNA7-2,
Pyr15BNA5-1, or Pyr15BNA5-2) at neutral pH. The triplexes at
25 °C and pH 6.8 in buffer A were measured in the wavelength range
of 210-320 nm. Cell path length was 1 cm. The triplex DNA
concentration used was 4 µM.
H, were obtained from the fitted curve (37). The Gibbs
free energy change,
G, and the entropy change,
S, were calculated from the equation,
G =
RTlnKa =
H
T
S (37) where R is gas
constant and T is temperature. The titration plots for
Pyr15T at pH 5.8 and pH 6.8 are also shown in Fig. 5c. The
thermodynamic parameters for Pyr15T at pH 5.8 and 6.8 were obtained
from the titration plots in the same way. The ITC profiles and the
titration plots for Pyr15BNA7-2, Pyr15BNA5-1, and Pyr15BNA5-2 at pH 6.8 were almost the same as those observed for Pyr15BNA7-1 at pH 6.8 (data
not shown). The thermodynamic parameters for Pyr15BNA7-2, Pyr15BNA5-1,
and Pyr15BNA5-2 at pH 6.8 were obtained in the same way.
View larger version (23K):
[in a new window]
Fig. 5.
Thermodynamic analyses of the pyrimidine
motif triplex formation with Pyr15T or Pyr15BNA7-1 at pH 6.8 and with
Pyr15T at pH 5.8 by ITC. a, ITC profiles of the initial
three injections for the binding of Pur23A·Pyr23T to Pyr15T or
Pyr15BNA7-1 at 25 °C and pH 6.8 and to Pyr15T at 25 °C and pH
5.8. The TFO solution (120 µM in buffer A or buffer B;
see "Materials and Methods") 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. b,
total ITC profile for the triplex formation between Pyr15BNA7-1 and
Pur23A·Pyr23T. The Pyr15BNA7-1 solution was injected 20 times into
the Pur23A·Pyr23T solution. Other experimental conditions were the
same as in a. c, titration plots against the
molar ratio of [TFO]/[Pur23A·Pyr23T]. The data were fitted by a
nonlinear least squares method.
H and
S were negative under all the conditions. Because an
observed negative
S was unfavorable for the triplex formation, the triplex formation was driven by a large negative
H under each condition. The magnitudes of the negative
H of the triplex formation for Pyr15T at pH 5.8 and for
Pyr15BNA7-1, Pyr15BNA7-2, Pyr15BNA5-1, or Pyr15BNA5-2 at pH 6.8 were 2.5 or 1.6-1.7 times larger than that observed for Pyr15T at pH
6.8, consistent with the ITC profiles in Fig. 5a. The
Ka for Pyr15T at pH 5.8 was ~20 times larger than
that observed for Pyr15T at pH 6.8, confirming, like others (6-8),
that neutral pH is unfavorable for the pyrimidine motif triplex
formation involving C+·G:C triads. In addition, the
Ka for Pyr15BNA7-1, Pyr15BNA7-2, Pyr15BNA5-1, or
Pyr15BNA5-2 at pH 6.8 was 10-20 times larger than that observed for
Pyr15T at pH 6.8, indicating that the 2',4'-BNA modification of TFO
increased the Ka of the pyrimidine motif triplex
formation at neutral pH by 10-20 times. The increase in the
Ka by the 2',4'-BNA modification of TFO was similar in magnitude among the four modified TFOs.
Thermodynamic parameters for the triplex formation between a 15-mer TFO
(Pyr15T, Pyr15BNA7-1, Pyr15BNA7-2, Pyr15BNA5-1, or Pyr15BNA5-2) and
a 23-base pair duplex (Pur23A · Pyr23T) at 25 °C, obtained
from ITC
View larger version (17K):
[in a new window]
Fig. 6.
Kinetic analyses of the pyrimidine motif
triplex formation with Pyr15T or Pyr15BNA7-1 at pH 6.8 by IAsys.
a, typical sensorgrams for the triplex formation at 25 °C
and pH 6.8 after injecting 90 µM specific TFO (Pyr15T or
Pyr15BNA7-1) in buffer A into the Bt-Pyr23T·Pur23A-immobilized
cuvette. b, series of sensorgrams for the triplex formation
between Pyr15BNA7-1 and Pur23A·Pyr23T at 25 °C and pH 6.8. The
Pyr15BNA7-1 solution, diluted in buffer A to achieve the indicated
final concentrations, was injected into the
Bt-Pyr23T·Pur23A-immobilized cuvette. The binding of Pyr15BNA7-1 to
Bt-Pyr23T·Pur23A was monitored as the response against time.
c, measured kon values of the triplex
formation in b were plotted against the respective
concentrations of Pyr15BNA7-1. The plot was fitted to a straight line
(r2 = 0.97) by a linear least squares
method.
Kinetic parameters for the triplex formation between a 15-mer TFO
(Pyr15T, Pyr15BNA7-1, Pyr15BNA7-2, Pyr15BNA5-1, or Pyr15BNA5-2) and
a 23-base pair duplex (Pur23A · Pyr23T)
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
H on the triplex formation measured by ITC reflects a
major contribution from the hydrogen bonding and the base stacking involved in the triplex formation (38, 46-48). On the other hand, the
S on the triplex formation measured by ITC includes a
positive entropy change from release of structured water on the triplex formation and a major contribution of a negative conformational entropy
change from the conformational restraint of TFO involved in the triplex
formation (38, 46-48). Because the formed triplex structure involving
Pyr15T at pH 5.8 and that involving Pyr15T at pH 6.8 is the same, the
magnitude of
H and
S on the triplex formation measured by ITC could be the same between the two conditions. However, unexpectedly, the magnitudes of
H and
S for Pyr15T at pH 6.8 were significantly smaller than
those observed for Pyr15T at pH 5.8 (Table II). When the
H and
S are calculated from the fitting
procedure of ITC, the heat observed by ITC is divided not by the
effective concentration really involved in the triplex formation but by
the apparent concentration added to the triplex formation (37). The
calculation does not take it into consideration what percentage of the
added concentration is really effectively involved in the triplex
formation. Thus, if the triplex formation is less stoichiometric under
a certain condition, the magnitudes of
H and
S for the less stoichiometric triplex formation estimated by ITC are smaller than those observed for the more stoichiometric triplex formation under another condition. Therefore, the significantly smaller magnitudes of
H and
S for Pyr15T at
pH 6.8 relative to those for Pyr15T at pH 5.8 (Table II) suggest that
the triplex formation with Pyr15T at pH 6.8 was significantly less
stoichiometric than that with Pyr15T at pH 5.8, which was also
supported by the significantly smaller magnitudes of
Ka and
G for Pyr15T at pH 6.8 (Table
II). In contrast, the Ka and
G for Pyr15T at pH 5.8 and those for the 2',4'-BNA modified TFOs at pH 6.8 were quite similar (Table II), suggesting that the triplex formation
under the two conditions was similarly quite stoichiometric. We
conclude that the triplex formation with Pyr15T at pH 6.8 was significantly less stoichiometric than that with Pyr15T at pH 5.8 and
that with the 2',4'-BNA-modified TFOs at pH 6.8. Thus, to discuss the
promotion mechanism of the triplex formation by the 2',4'-BNA
modification, the comparison of the
H and
S
between Pyr15T at pH 6.8 and 2',4'-BNA-modified TFOs at pH 6.8 is not valid because of the significantly reduced stoichiometry for Pyr15T at
pH 6.8. The comparison of the
H and
S
between Pyr15T at pH 5.8 and 2',4'-BNA-modified TFOs at pH 6.8 with
similar stoichiometry will provide a reasonable promotion mechanism for
the triplex formation by the 2',4'-BNA modification, as discussed below.
G for Pyr15T at pH
5.8 and those for the 2',4'-BNA-modified TFOs at pH 6.8 were quite
similar (Table II), the ingredients of
G, that is,
H and
S, were obviously different from each
other. The magnitudes of the negative
H and
S for the 2',4'-BNA-modified TFOs at pH 6.8 were smaller
than those observed for Pyr15T at pH 5.8 (Table II). The hydrogen
bonding and the base stacking involved in the triplex formation are
usually considered the major sources of the negative
H on
the triplex formation (38, 46-48). Thus, the difference in
H for the stoichiometric triplex formations between
Pyr15T at pH 5.8 and the 2',4'-BNA-modified TFOs at pH 6.8 (Table II)
suggests that the hydrogen bonding and the base stacking of the triplex
with the 2',4'-BNA-modified TFOs are significantly different from those
with the corresponding unmodified TFO. In fact, the CD spectra show
that the triplexes with the 2',4'-BNA-modified TFO had the A-like
conformation (Ref. 45 and Fig. 4). The A-like conformation by the
2',4'-BNA modification of TFO may result in the difference in the
negative
H between the unmodified and 2',4'-BNA-modified
TFOs. On the other hand, the negative
S on the triplex
formation is mainly contributed by a negative conformational entropy
change attributable to the conformational restraint of TFO involved in
the triplex formation (38, 46-48). Therefore, the smaller magnitude of
the negative
S for the 2',4'-BNA modified TFOs at pH 6.8 relative to that for Pyr15T at pH 5.8 (Table II) suggests that the
2',4'-BNA-modified TFO in the free state is more rigid than the
corresponding unmodified TFO. The increased rigidity of the 2',4'-BNA
modified TFO in the free state relative to the corresponding unmodified
TFO causes the smaller entropic loss on the triplex formation with the
2',4'-BNA-modified TFO at neutral pH, which provides a favorable
component to the
G and leads to the increase in the
Ka of the triplex formation at neutral pH. We
conclude that the increased rigidity of the 2',4'-BNA-modified TFO in
the free state may be one of the factors that increases the
Ka of the pyrimidine motif triplex formation at
neutral pH.
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ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Dr. Hiroshi Nakanishi and Dr. Koko Mizumachi for generous consideration concerning the use of the CD spectropolarimeter and IAsys Plus instrument, respectively.
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FOOTNOTES |
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* This research was supported in part by Ministry of Education, Science, Sports, and Culture of Japan Grants-in Aid 08249247 and 12217158 (to H. T.) and 09557201 and 12217081 (to T. I.).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: Tsukuba Life Science Center, Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan. Tel.: 81-298-36-9082; Fax: 81-298-36-9080; E-mail: torigoe@rtc.riken.go.jp.
To whom correspondence should be addressed: Graduate School of
Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka
565-0871, Japan. Tel.: 81-6-6879-8200; Fax: 81-6-6879-8204; E-mail:
imanishi@phs.osaka-u.ac.jp.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M007783200
2 After our report on the first synthesis of 2',4'-BNA monomers, Wengel's group demonstrated some properties of 2',4'-BNA (28, 29).
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ABBREVIATIONS |
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The abbreviations used are: TFO, triplex-forming oligonucleotide; BNA, bridged nucleic acid; 2', 4'-BNA, 2'-O,4'-C-methylene bridged nucleic acid; 3', 4'-BNA, 3'-O,4'-C-methylene bridged nucleic acid; EMSA, electrophoretic mobility shift assay; CD, circular dichroism; ITC, isothermal titration calorimetry; IAsys, interaction analysis system; Pur, purine; Pyr, pyrimidine; Bt, biotinylated; NS, nonspecific; Tm, melting temperature; kon, on-rate constant; kassoc, association rate constant; koff, off-rate constant; kdissoc, dissociation rate constant.
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REFERENCES |
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1. | Mirkin, S. M., and Frank-Kamenetskii, M. D. (1994) Annu. Rev. Biophys. Biomol. Struct. 23, 541-576[CrossRef][Medline] [Order article via Infotrieve] |
2. | Frank-Kamenetskii, M. D., and Mirkin, S. M. (1995) Annu. Rev. Biochem. 64, 65-95[CrossRef][Medline] [Order article via Infotrieve] |
3. | Soyfer, V. N., and Potaman, V. N. (1996) Triple-Helical Nucleic Acids , Springer-Verlag, New York |
4. | Sun, J.-S., and Helene, C. (1993) Curr. Opin. Struct. Biol. 3, 345-356 |
5. | Sun, J.-S., Garestier, T., and Helene, C. (1996) Curr. Opin. Struct. Biol. 6, 327-333[CrossRef][Medline] [Order article via Infotrieve] |
6. | Frank-Kamenetskii, M. D. (1992) Methods Enzymol. 211, 180-191[Medline] [Order article via Infotrieve] |
7. | Singleton, S. F., and Dervan, P. B. (1992) Biochemistry 31, 10995-11003[Medline] [Order article via Infotrieve] |
8. | Shindo, H., Torigoe, H., and Sarai, A. (1993) Biochemistry 32, 8963-8969[Medline] [Order article via Infotrieve] |
9. | Milligan, J. F., Krawczyk, S. H., Wadwani, S., and Matteucci, M. D. (1993) Nucleic Acids Res. 21, 327-333[Abstract] |
10. | Cheng, A.-J., and Van Dyke, M. W. (1993) Nucleic Acids Res. 21, 5630-5635[Abstract] |
11. | Lee, J. S., Woodsworth, M. L., Latimer, L. J. P., and Morgan, A. R. (1984) Nucleic Acids Res. 12, 6603-6614[Abstract] |
12. | Povsic, T. J., and Dervan, P. B. (1989) J. Am. Chem. Soc. 111, 3059-3061 |
13. | 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] |
14. | Ono, A., Ts'o, P. O. P., and Kan, L.-S. (1991) J. Am. Chem. Soc. 113, 4032-4033 |
15. | 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] |
16. | Koh, J. S., and Dervan, P. B. (1992) J. Am. Chem. Soc. 114, 1470-1478 |
17. | Jetter, M. C., and Hobbs, F. W. (1993) Biochemistry 32, 3249-3254[Medline] [Order article via Infotrieve] |
18. | Ueno, Y., Mikawa, M., and Matsuda, A. (1998) Bioconj. Chem. 9, 33-39[CrossRef][Medline] [Order article via Infotrieve] |
19. | Sun, J. S., Giovannangeli, C., Francois, J. C., Kurfurst, R., Montenay-Garestier, T., Asseline, U., Saison-Behmoaras, T., Thuong, N. T., and Helene, C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6023-6027[Abstract] |
20. | 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] |
21. | Hampel, K. J., Crosson, P., and Lee, J. S. (1991) Biochemistry 30, 4455-4459[Medline] [Order article via Infotrieve] |
22. | Obika, S., Nanbu, D., Hari, Y., Morio, K., In, Y., Ishida, T., and Imanishi, T. (1997) Tetrahedron Lett. 38, 8735-8738[CrossRef] |
23. | Obika, S., Nanbu, D., Hari, Y., Andoh, J., Morio, K., Doi, T., and Imanishi, T. (1998) Tetrahedron Lett. 39, 5401-5404[CrossRef] |
24. | Obika, S., Andoh, J., Sugimoto, T., Miyashita, K., and Imanishi, T. (1999) Tetrahedron Lett. 40, 6465-6468[CrossRef] |
25. | Imanishi, T., and Obika, S. (1999) J. Syn. Org. Chem. Jpn. 57, 969-980 |
26. | Obika, S., Hari, Y., Morio, K., and Imanishi, T. (2000) Tetrahedron Lett. 41, 215-219[CrossRef] |
27. | Obika, S., Hari, Y., Morio, K., and Imanishi, T. (2000) Tetrahedron Lett. 41, 221-224[CrossRef] |
28. | Singh, S. K., Nielsen, P., Koshkin, A. A., and Wengel, J. (1998) Chem. Commun. 455-456 |
29. | Koshkin, A. A., Singh, S. K., Nielsen, P., Rajwanshi, V. K., Kumar, R., Meldgaard, M., Olsen, C. E., and Wengel, J. (1998) Tetrahedron 54, 3607-3630[CrossRef] |
30. | Obika, S., Morio, K., Nanbu, D., and Imanishi, T. (1997) Chem. Commun. 1643-1644 |
31. | Obika, S., Morio, K., Hari, Y., and Imanishi, T. (1999) Chem. Commun. 2423-2424 |
32. | Obika, S., Morio, K., Hari, Y., and Imanishi, T. (1999) Bioorg. Med. Chem. Lett. 9, 515-518[CrossRef][Medline] [Order article via Infotrieve] |
33. | Lyamichev, V. I., Mirkin, S. M., Frank-Kamenetskii, M. D., and Cantor, C. R. (1988) Nucleic Acids Res. 16, 2165-2178[Abstract] |
34. |
Torigoe, H.,
Ferdous, A.,
Watanabe, H.,
Akaike, T.,
and Maruyama, A.
(1999)
J. Biol. Chem.
274,
6161-6167 |
35. | Langerman, N., and Biltonen, R. L. (1979) Methods Enzymol. 61, 261-286[Medline] [Order article via Infotrieve] |
36. | Biltonen, R. L., and Langerman, N. (1979) Methods Enzymol. 61, 287-318[Medline] [Order article via Infotrieve] |
37. | Wiseman, T., Williston, S., Brandts, J. F., and Lin, L.-N. (1989) Anal. Biochem. 179, 131-137[Medline] [Order article via Infotrieve] |
38. | Kamiya, M., Torigoe, H., Shindo, H., and Sarai, A. (1996) J. Am. Chem. Soc. 118, 4532-4538[CrossRef] |
39. | Torigoe, H., Shimizume, R., Sarai, A., and Shindo, H. (1999) Biochemistry 38, 14653-14659[CrossRef][Medline] [Order article via Infotrieve] |
40. | Torigoe, H., Ferdous, A., Watanabe, H., Akaike, T., and Maruyama, A. (1999) Nucleosides, Nucleotides & Nucleic Acids 18, 1655-1656 |
41. | Cush, R., Cronin, J. M., Stewart, W. J., Maule, C. H., Molloy, J., and Goddard, N. J. (1993) Biosens. Bioelectron. 8, 347-353[CrossRef] |
42. | 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] |
43. | 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] |
44. | 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] |
45. | Johnson, K. H., Gray, D. M., and Sutherland, J. C. (1991) Nucleic Acids Res. 19, 2275-2280[Abstract] |
46. | Edelhoch, H., and Osborne, J. C., Jr. (1976) Adv. Protein Chem. 30, 183-250[Medline] [Order article via Infotrieve] |
47. | Cheng, Y.- K., and Pettitt, B. M. (1992) Prog. Biophys. Mol. Biol. 58, 225-257[CrossRef][Medline] [Order article via Infotrieve] |
48. | Shafer, R. H. (1998) Prog. Nucleic Acids Res. Mol. Biol. 59, 55-94[Medline] [Order article via Infotrieve] |
49. | 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] |