From the Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel
Received for publication, January 23, 2001, and in revised form, February 12, 2001
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ABSTRACT |
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The thermodynamic parameters of DNA triplex
formation between oligonucleotides and double-stranded DNA segments
containing adenine runs (A-tracts) were investigated to explore
equilibrium structural effects exerted by flanking segments upon the
A-tracts. Results obtained from isothermal titration
calorimetry, temperature-dependent circular dichroism
(CD), and UV melting experiments indicate that A-tracts,
considered as a uniquely robust and inflexible DNA motif, can be
structurally perturbed by neighboring sequences in a way that
significantly affects the propensity of this motif to interact with
triplex-forming oligonucleotides. These contextual equilibrium effects,
which depend upon the composition and location of the flanking
sequences, are likely to apply not only to the interaction of A-tracts
with single-stranded DNA molecules but also to interactions with drugs
and proteins. As such, the current results refine the guidelines for
the design of triplex-forming oligonucleotides used for antigene
strategies. More generally, they substantiate the notion that
significant data might be encoded by structural DNA parameters.
The demonstrated in vivo existence of non-B-DNA
morphologies and the potential involvement of such altered structures
in the regulation and fine-tuning of cellular processes (1, 2) elicited
intensive studies on the factors that affect and modulate DNA
conformational transitions. It is well established that the physical
chemistry of these transitions is determined by an intricate array of
environmental conditions and intrinsic factors, including the ionic
strength, relative humidity, pH, and torsional strain. The actual
structure exhibited by a given DNA segment is, however, generally
considered to be dictated by the particular composition and sequential
order of the base pairs within this segment. Indeed, left-handed DNA
conformations, cruciforms, intramolecular triplexes, as well as bent or
macroscopically curved motifs are detected predominantly in non-random
DNA sequences such as alternating purine-pyrimidine segments, direct or
inverted repeats, and purine-rich tracts (2). Several studies have
indicated, however, that the kinetics of a conformational change, as
well as the features of the resulting DNA structure, may be influenced
or even dominated by sequences that are removed from those directly
participating in the transition.
The first evidence that structural information can be transmitted along
the DNA molecule was provided by the observation that the melting of a
DNA segment can be strongly affected by the base composition of a
contiguous region (3). The kinetics of cruciform extrusion (4), as well
as of the formation of intramolecular triple-stranded DNA motifs (5),
were found to be dominated by sequences flanking the region that
undergoes the transition. The effects were shown to operate over
relatively long distances and to be independent of polarity.
Right-to-left-handed transitions of alternating d(G-C)n and
d(A-T)n segments were also found to be significantly modulated
by regions flanking the alternating segment (6-8). A striking
demonstration of the long range nature of contextual factors is
provided by the crucial role displayed by these factors on DNA
packaging processes. The propensity of DNA molecules to condense was
shown to be substantially modulated by the presence of non-B-DNA motifs
such as Z-DNA or telomeres within the DNA (9-11), as well as by the
conformational junctions between such altered motifs and generic B-DNA
(12). Notably, these observations underline the ability of DNA
sequences to influence the kinetics of a structural transition that
occurs at a removed site. Equilibrium structural effects of DNA context have also been observed, albeit less frequently. Salient examples are
provided by structural changes induced by homopurine-homopyrimidine sequences on regions located 3' to the G tract (13), deformations exhibited by left-handed Z-DNA segments when flanked by AT-rich sequences (6), as well as the effects exerted by phased adenine tracts
on the morphology of condensed DNA structures (14).
To sustain conformational changes and to be affected by contiguous
regions, DNA segments must exhibit considerable structural flexibility.
In general, DNA sequences comply with this requirement, being supple,
flexible, and deformable. A conspicuous exception is provided by runs
of successive adenine bases, or A-tracts, which are characterized by an
unusual rigidity and hence an exceptional aversion toward
conformational changes (15-20). The distinctive structural features of
A-tracts and the potential effects of these features upon DNA-protein
interactions prompted us to examine the susceptibility of this motif to
contextual effects. Toward this end, we have exploited the propensity
of poly(dA)·poly(dT) segments to interact with a pyrimidine strand,
thus forming intermolecular triple-stranded structures (21, 22). It has
been reported recently that the ability of very short adenine tracts to
form a triple-stranded motif is abolished when the A-tract is flanked by GC-rich segments (23). By using isothermal titration calorimetry (ITC),1
temperature-dependent circular dichroism (CD), and UV
melting techniques, we show that the formation and stability of the
triplex motif dTm*dAn·dTn (where * designates third-strand binding, and · represents Watson-Crick base pairing) are
significantly influenced by sequences that flank the adenine tracts.
The nature of the influence depends upon the composition, location, and
number of the flanking sequences, at one or both ends of the A-tracts.
The results, assigned to long range equilibrium structural
modifications within the A-tract, demonstrate that even a particularly
robust DNA motif, generally considered to be "immune" to structural
deformations, is susceptible to contextual effects in a way that
markedly modulates its conformational, and hence chemical, properties.
As such, the observations underline the complex interplay between
conformational DNA motifs and provide an additional facet to the notion
that genetic information might be encoded within DNA structure.
Oligonucleotides--
Oligonucleotides containing thymines were
purchased from Sigma-Genosys and purified by preparative HPLC as the
5'-dimethoxytrityl derivatives. Oligonucleotides containing adenines
were purchased from Midland Certified Reagent Company (Midland, TX) and
purified by ion exchange HPLC. Oligonucleotide purity was analyzed by
HPLC and mass spectroscopy and found to be >95%. The concentration of
the oligonucleotides was determined by UV absorption using extinction
coefficient parameters from published nearest neighbor parameters (24).
The precision of this determination was further confirmed by ITC
experiments in which single-stranded species were titrated with their
complementary strands (under conditions refractory to triplex
formation) to give titration curves with a stoichiometry number of 1. The purified oligonucleotides were extensively dialyzed against 20 mM PIPES, pH 7.0. Stock solutions of the
double-stranded forms were prepared by mixing equimolar amounts of
complementary strands, heating to 90 °C, and cooling to room
temperature at a rate of 0.5 °C/min. Triple-stranded solutions for
CD and UV experiments were prepared by mixing the appropriate amounts
of stock solutions containing the single-stranded and the preformed
double-stranded DNA species. The resulting solutions were diluted to
the required concentration in 20 mM PIPES (pH 7.0), 1.0 M KCl (buffer A) for UV measurements and in 10 mM sodium phosphate (pH 7.0), 1.0 M NaCl
(buffer B) for CD studies. Triplex solutions were equilibrated at the
initial temperature of the particular experiment for 15 min or at
4 °C overnight; identical results were obtained from both
preparation conditions.
Isothermal Titration Calorimetry--
ITC experiments were
carried out on an MSC-ITC system (MicroCal Inc.).
Single-stranded solution (50 µM in buffer A) was injected 22 times in 5-µl increments at 3-min intervals into the isothermal cell containing the double-stranded targets (2.5 µM in
buffer A). Heats of dilution of the single-stranded species were
obtained separately by injecting the oligonucleotide into the buffer
and used to correct the data. Corrected heats were divided by the mole
number of the injectant and analyzed with Origin software supplied by
the manufacturer (25).
Circular Dichroism and UV Spectroscopies--
CD spectra were
recorded in the spectral range of 200-300 nm on an Aviv
spectropolarimeter, Model 202. Samples were allowed to equilibrate at
the particular temperature studied for 15 min; spectra were recorded in
a 1-cm light path cell. The thermal transitions revealed by the various
triplex motifs were obtained by measuring UV absorption at 260 nm on a
Cary 5E spectrophotometer (Varian) equipped with a Peltier temperature
controller. The temperature was measured at an accuracy of ±0.3 °C
by a probe that was placed in a buffer-containing cell. Melting
profiles were recorded at a heating and cooling rate of 0.5 °C/min.
The reported melting temperatures (Tm) are those at
which the derivative of absorbance versus temperature is maximal.
Thermodynamic Effects of Flanking Sequences on the Formation of the
Triplex Motif
Oligonucleotides represent a distinctive class of DNA-binding
species that recognize the major groove of the double helix by forming
Hoogsteen-type hydrogen bonds with Watson-Crick base pairs (26, 27).
The process attracted considerable interest because of the potential
application of triplex-forming oligonucleotides (TFOs) for targeting
specific DNA sites both in vitro and in vivo (28). We studied the thermodynamic parameters of triplex formation between a pyrimidine strand and various DNA targets specified in Table
I.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
List of duplex targets used in the study
Fig. 1a depicts a typical
isothermal titration profile obtained by 22 5-µl injections of the
18-mer TFO dT18 into the ITC cell containing the
double-stranded open-ended DNA target 18-0FE (Table I) at
20 °C. An exothermic heat pulse is detected following each
injection, whose magnitude progressively decreases until a plateau,
corresponding to the heat of dilution of the single-stranded species in
the buffer and indicating saturation, is reached. The heat evolved in
each injection was corrected for the heat of dilution, which was
determined separately by injecting the single-stranded oligonucleotide
into the buffer, and divided by the number of moles injected. The
resulting values were plotted as a function of the molar ratio between
the single-stranded species and the double-stranded segments and fitted
to a sigmoidal curve by using a nonlinear least squares method (25).
The equilibrium association constant (Ka) and the
enthalpy change (H) that characterize the formation of
the triple-stranded structure are directly obtained from the titration
curve; the Gibbs free energy change (
G) and the entropy
change (
S) of the process are then calculated from the
equation
G =
RTln Ka =
H
T
S.
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Titration curves obtained from the interaction between the
oligonucleotide dT18 and four double-stranded DNA targets,
18-0FE, 20-2(CGC)FE, 20-1FE/3', and 20-1FE/5', are shown in Fig.
1b. In all cases, titration reaches half-saturation near the
equimolar ratio, indicating a one-to-one binding to form the triplex
structures. The thermodynamic parameters derived from the curves are
summarized in Table II. Also included in
the table are the parameters exhibited by the interaction between
dT18 and 20-2(TAT)FE. As previously reported (22, 29, 30),
triplex formation reveals negative S and
H
values, indicating that the process is characterized by an unfavorable
entropy change and is, as such, driven by a large negative enthalpy
change. Conspicuous differences are, however, observed in the magnitude
of the thermodynamic parameters exhibited by the various processes.
Specifically, the interaction of the TFO with either 20-2(CGC)FE or
20-2(TAT)FE, which contain flanking sequences at both ends of the
A-tract, is associated with significantly smaller enthalpy changes than
those exhibited by the interaction between dT18 and the
double-stranded DNA target 18-0FE, in which no flanking ends are
present. The equilibrium association constants Ka
for the formation of the triplexes dT18*20-2(CGC)FE and
dT18*20-2(TAT)FE are 54 and 34 times smaller,
respectively, than the Ka of
dT18*18-0FE formation. Whereas the thermodynamic parameters of triplex formation are found to be sensitive to the presence and base pair composition of the flanking sequences (which are
GC- or AT-rich), they are unaffected by their sequential order. Specifically, we find that the formation of
dT18*20-2(CGC)FE and of dT18*20-2(GCG)FE
(Table I) exhibits identical thermodynamic features.
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Formation of triple-stranded structures containing one flanking
sequence at either the 3' or 5' end of the A-tract is characterized by
H and Ka values that are smaller than
those revealed by the interaction between dT18 and 18-0FE
but larger than those associated with the formation of
dT18*20-2(CGC)FE. Notably, the thermodynamic parameters
revealed by the interaction of dT18 with the targets
dT18·d(CGCA20CGC) or
d(GCGT20GCG)·dA18 (duplexes containing single-stranded, as opposed to double-stranded, flanking ends) are
identical to those exhibited by the interaction of dT18
with the open-ended double-stranded target 18-0FE (data not shown).
Reliance of Isothermal Titration Measurements
The accuracy of the ITC measurements critically depends upon
a precise identification of all species that are present in the calorimeter cell and of all processes that may occur as titration proceeds. The evaluation of the thermodynamic parameters exhibited by
triplex formation can be hampered by the presence of single-stranded DNA species in the ITC cell. These species may result from either dissociation or disproportionation of the double-stranded targets (22,
31): 2A + 2B 2AB
A + AB2 (where A and B represent dA18 and dT18, respectively, as well as other
single-stranded species that form the duplex targets). The presence of
single-stranded DNA molecules may lead to an erroneous interpretation
of the ITC results, because the titration curves would represent, in
such a case, the sum of both duplex and triplex formation. On the basis of previously reported observations (22), we submit that under the
experimental conditions used in the current study, the probability of
dissociation or disproportionation processes is low enough to render
the concentration of single-stranded species insignificant. It has
indeed been shown that for dA19·dT19
virtually all dA19 is in the duplex form below 300 K at
1.015 M NaCl (22). Notably, the tendency of double-stranded
molecules containing flanking sequences at the 3' or 5' end of
the A-tract to undergo melting and disproportionation reactions would
be even smaller. The absence of single-stranded species is further
implied by the observation that the titration of dT18 into
the ITC cell containing the various duplex targets results in a
monophasic titration curve. The presence of single-stranded molecules
would have resulted in the initial formation of double-stranded
species. Because the enthalpy change associated with the formation of
duplex forms is significantly larger than that exhibited by triplex
formation (22), a biphasic titration curve would have been observed.
Effects of Flanking Sequences on the Thermal Stability of the Triplex Motif
Circular Dichroism--
The ITC technique was used to obtain the
thermodynamic parameters that characterize the formation of various
triple-stranded species. We have conducted CD melting studies on
preformed triplex forms to assess their relative stability. Because the
ellipticities exhibited by double-stranded and triple-stranded DNA
motifs are conspicuously different (22), the spectra depicted in Fig.
2, a and b provide
a direct indication that under experimental conditions similar to those
used in the ITC experiments, a mixture of dT18 with either
18-0FE or 20-2(CGC)FE at an equimolar ratio reveals the formation of
the triplex forms as predominant products. The CD spectra of the
triple-stranded complexes dT18*18-0FE and
dT18*20-2(CGC)FE were recorded at different temperatures,
ranging between 10 and 50 °C (Fig. 2, c and d,
respectively). As temperature increases, both triplex species undergo
dissociation, gradually assuming ellipticity features characteristic of
the duplex form. The temperature-dependent patterns
revealed by the two complexes are, however, markedly different. Whereas
the mixture containing dT18 and 18-0FE still exhibits a
substantial contribution of a triple-stranded motif at 40 °C, the
ellipticities revealed by the mixture of dT18 and 20-2(CGC)FE indicate that already at 30 °C the duplex DNA form predominates and that at 40 °C the triplex is completely
dissociated.
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UV Melting Studies--
UV melting curves of the preformed
triplex forms dT18*18-0FE and
dT18*20-2(CGC)FE are shown in Fig.
3. The denaturation profile is biphasic,
as clearly indicated by the derivative plot (Fig. 3, inset).
The transition occurring at the lower temperature results from the
dissociation of the triple-stranded complexes into a duplex and a
single-stranded species, whereas the high temperature transition
corresponds to the melting of the DNA duplexes. As expected, the
20-2(CGC)FE duplex, which is stabilized by two CG-rich flanking ends,
melts into single strands at a higher temperature than that revealed by
the corresponding transition of 18-0FE duplex. In sharp contrast, the
dissociation of the dT18*20-2(CGC)FE into double- and
single-stranded species occurs at a significantly lower temperature
(27 °C) than the corresponding transition of dT18*18-0FE (38 °C). Thus, both the CD and UV melting
profiles indicate that the thermal stability of
dT18*20-2(CGC)FE is substantially lower than that of
dT18*18-0FE.
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We have carried out UV melting experiments on triplex structures obtained from the interaction between dT18 and double-stranded moieties containing one flanking sequence located at either the 3' or 5' end of the adenine tract (Table III). In both cases, the triplex motif melts into double- and single-stranded species at temperatures that are lower than the Tm of dT18*18-0FE and higher than the Tm of dT18*20-2(CGC)FE, in which two flanking ends are present. The position of the flanking sequence relative to the A-tract appears to significantly affect the Tm. dT18*20-1FE/5' undergoes melting transition at a lower temperature than dT18*20-1FE/3', thus indicating that a flanking sequence located at the 5' end of the A-tract destabilizes the triplex motif to a higher extent than does that positioned at the 3' end.
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To better understand the mechanism by which flanking sequences affect
the stability of the triplex motif, we have conducted UV melting
studies on triple-stranded structures in which the TFO strand is
further removed from the junction between the A-tract and the flanking
ends. We find that although the Tm of the triplex
dT18*26-2(CGC)FE is higher than that of
dT18*20-2(CGC)FE, it is still substantially lower than the
temperature at which dissociation of dT18*18-0FE occurs
(Table III). To further assess the range of the effects exerted by
flanking sequences, melting profiles exhibited by triplex structures
derived from the interaction of a shorter TFO, dT14, with
the various double-stranded targets were obtained. As expected, the
dissociation of triple-stranded structures containing dT14
occurs at a substantially lower temperature than that of the
corresponding triplex forms with dT18 as TFO (Table III).
Yet, although the dT14-containing triplex motifs are located further away from the junction sites between the A-tracts and
the flanking sequences than are the dT18 triplexes, the
destabilizing effects exerted by these flanking sequences remain. Thus,
a decrease of 11 °C is detected in the Tm of both
dT18*20-2(CGC)FE and dT14*20-2(CGC)FE
relative to dT18*18-0FE and dT14*18-0FE, respectively. Notably, the destabilizing effect of a flanking sequence
located at the 5' end of the A-tract in the dT14 triplexes is substantially larger than that exerted by a 3' flanking end, as was
found to be the case for the dT18-containing triplexes. Tm values exhibited by triplex species containing
single-stranded sequences at either one end or two ends of the A-tract
(i.e.
dT18*d(CGCA20)·dT18 or
dT18*d(CGCA20CGC)·dT18) are
identical to the Tm of dT18*18-0FE.
Notably, the UV melting profiles obtained either by heating or cooling
the samples were identical, thus indicating the reversibility of the process.
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DISCUSSION |
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Adenine tracts reveal unique structural features that differentiate this motif from all other DNA sequences. Specifically, base pairs within A-tracts are characterized by an unusually large propeller twist, which acts to substantially enhance purine-purine stacking interactions, to stabilize a hydration network along a particularly narrow minor groove, and to enable cross-strand bifurcated hydrogen bonds in the major groove (19). Altogether, these particular properties confer upon A-tracts an unusual rigidity that is manifested by the monomorphic structure exhibited by poly(dA)·poly(dT) fibers (15) as well as by the very limited conformational variation that is revealed by A-tracts in different crystalline structures (17). An extensively studied property of adenine runs is their ability to generate a permanent bend in the helical DNA trajectory (32), a structural feature considered to represent an important factor in the regulation of cellular processes in both prokaryotic and eukaryotic systems. Most of the models suggested to account for A-tract-dependent bends are based on the highly limited deformability of this motif and its unique structure that significantly differs from that revealed by generic B-type DNA segments (19). According to these models, DNA bending results from structural perturbations that are effected by the A-tracts upon flanking sequences that are manifested, in particular, in the junction sites between A-tracts and flanking generic B-DNA segments.
The observations reported in this study indicate that A-tracts are capable not only of producing contextual effects on flanking sequences but also of being significantly influenced by such sequences. This influence is manifested by the ability of flanking segments to modulate the interaction between adenine runs and single-stranded DNA molecules. Specifically, the presence of CG- or AT-rich flanking segments located at either one end or both ends of the A-tract is shown by isothermal titration calorimetry to attenuate the association constants of triplex formation, relative to the Ka revealed by the corresponding process involving an open-ended A-tract. Temperature-dependent CD and UV studies complement this observation by demonstrating that the thermal stability of the triplex motif is modulated by sequences that flank the A-tract target.
The thermodynamic parameters presented in Table II, along with the Tm values summarized in Table III, indicate that the open-ended 18-0FE segment is characterized by structural features that are optimally suited for the interaction with a TFO. This optimal conformation is clearly perturbed by the presence of one conformational junction at either end of the A-tract and even more so by the presence of junctions at both ends. The notion that the conformational perturbation within the A-tract motif is causally related to junctions between this motif and generic B-DNA helices is substantiated by the observation that single-stranded segments, which are located at either one end or two ends of the A-tract but do not form a structural junction, do not affect triplex formation or stability. Results obtained with a long A-tract target (26-2(CGC)FE) and a short TFO (dT14) clearly indicate that the structural perturbation induced by the junctions is not localized but rather transmitted over the adenine runs.
Although the nature of the structural effects exerted by the junctions
and flanking segments upon the A-tracts cannot be directly elucidated
from the thermodynamic parameters reported here, some clues can,
however, be derived. Interactions of the TFO with A-tract targets
containing two flanking ends that are either CG- or AT-rich (20-2(CGC)FE and 20-2(TAT)FE, respectively) exhibit very similar Ka values. Yet, the H and
S values of these two processes differ (Table II).
Specifically, the formation of dT18*20-2(CGC)FE is
associated with a less favorable enthalpy change and a less unfavorable
entropy change than those that accompany the formation of
dT18*20-2(TAT)FE.
We propose that the apparent enthalpy-entropy compensation that leads
to similar Ka values for the two processes implies a
different mode of contextual effects. The relatively rigid CG-rich
flanking ends enforce a particular conformation upon the junctions,
which is propagated along the A-tract. Presumably, this perturbation
affects the structural features of the major groove of the adenine run
in such a way that renders the configuration along this groove less
favorable for the formation of hydrogen bonds between the TFO and the
target duplex. The structural perturbation exerted upon the A-tract by
the flexible AT-rich flanking ends is relatively smaller, allowing for
a more optimal interaction of the A-tract with the TFO. This difference
should be, and indeed is, reflected by a less favorable H
value for dT18*20-2(CGC)FE formation than that
accompanying the formation of dT18*20-2(TAT)FE. The
relatively weak interaction between the TFO and the duplex 20-2(CGC)FE
results in a smaller attenuation of the conformational freedom of the
interacting species, which is, in turn, associated with a less
unfavorable entropy change. Notably, such a mode of enthalpy-entropy
compensation, where the experimental conditions are fixed while the
structure of the interacting molecules varies (congener series), has
been previously discussed and demonstrated (33, 34). The particularly
large unfavorable entropy revealed by the formation of
dT18*20-2(TAT)FE may be interpreted in terms of previous
findings, according to which triplex formation results in an enhanced
base pair stacking at the 5' junction between the triplex and the
duplex (35). If such a pronounced stacking is propagated, it would
influence a flexible AT-rich flanking sequence to a higher extent than
it would affect the robust GC-rich end, leading to an attenuated
conformational freedom within the AT-rich segments. We thus propose
that in double-stranded targets flanked by CG-rich segments, the
dominating contextual effects are those exerted upon the A-tract by the
junctions between the A-tract and the flanking segments. In contrast,
contextual effects in duplexes flanked by AT-rich segments are mainly
exerted upon these flanking sequences by the junctions between the
triplex and duplex motifs, with a minimal structural perturbation
within the target A-tract. This interpretation is clearly consistent
with the relatively high melting temperature of
dT18*20-2(TAT)FE (Table III).
The open-ended A-tract target and the A-tract targets flanked by
one or two CG-rich segments provide an example of a partial enthalpy-entropy compensation within a congener series. As a general trend, going from the 18-0FE through A-tracts that contain a single flanking end to the target with two junction sites is associated with a significant decrease of the negative H
values that is only partially compensated by a decrease of unfavorable
S contributions. The thermodynamic parameters associated
with the binding of a TFO to adenine runs flanked by one junction at
either the 5' or 3' end of the A-tract are intriguing. Although the
H revealed by the interaction between dT18
and 20-1FE/5' is more favorable than that characterizing the binding
of dT18 to 20-1FE/3', the association constant of the
former process is slightly smaller. The difference in the
Ka values derives from an unfavorable entropic
contribution that is more pronounced in dT18*20-1FE/5'. We
propose that the interpretation suggested for the difference between
the parameters revealed by the formation of
dT18*20-2(CGC)FE and dT18*20-2(TAT)FE also
applies to the interaction of the TFO with 20-1FE/5' and 20-1FE/3'.
Specifically, the junction between the A-tract and the 5' flanking
sequence is influenced by the 5' triplex-duplex junction to a larger
extent than the 3' junction, because of the enhanced base pair stacking
at the 5' end of the triplex-duplex motif. This influence and the
resulting perturbation at the 5' flanking sequence results in a larger
negative entropy contribution, leading to a slight destabilization of
20-1FE/5' relative to 20-1FE/3'.
In this study we show that even a particularly inflexible DNA
motif such as A-tracts may be structurally perturbed by neighboring sequences in such a way that will affect the propensity of this motif
to interact with other molecules. The transmitted equilibrium structural effects are finely tuned by the composition of the flanking
sequences as well as by their location relative to the A-tract. Because
the unique properties of A-tracts derive from the intrinsic rigidity
and invariance of this motif, even subtle structural perturbations
sustained by the A-tracts are likely to be consequential, as indeed is
demonstrated here. Moreover, the particularly strong stacking
interactions between the base pairs along the A-tract allow the
structural perturbation to be propagated over this motif. The
contextual effects reported here were studied through the
interaction of adenine runs with a single-stranded DNA ligand, thus
providing potentially important insights into a more refined choice of
TFOs applied for the antigene strategy. Such contextual effects are,
however, likely to apply to the interaction of A-tracts with other
DNA-binding species such as drugs and proteins. As such, the current
results highlight the structural complexity of DNA molecules and
provide a new dimension to the notion that significant information
might be encoded by DNA structural parameters.
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FOOTNOTES |
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* This work was supported by the Israel Science Foundation founded by the Academy of Sciences and Humanities and by the Minerva Foundation, Germany.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. E-mail:
avi.minsky@weizmann.ac.il.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M100619200
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ABBREVIATIONS |
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The abbreviations used are: ITC, isothermal titration calorimetry; HPLC, high pressure liquid chromatography; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); Tm, melting temperature; TFO, triplex-forming oligonucleotide.
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REFERENCES |
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