(Received for publication, October 12, 1994; and in revised form, December 21, 1994)
From the
We have targeted the
d(GA
G
)
d(C
T
C
)
duplex for triplex formation with
d(G
T
G
) in the presence of
MgCl
. The resulting triple helix,
d(G
T
G
)*d(G
A
G
)
d(C
T
C
),
is considerably weaker than the related triplex,
d(G
A
G
)*d(G
A
G
)
d(C
T
C
),
and melts in a biphasic manner, with the third strand dissociating at
temperatures about 20-30 °C below that of the remaining
duplex. This is in distinct contrast to the
d(G
A
G
)*d(G
A
G
)
d(C
T
C
)
triplex, which melts in essentially a single transition. Gel
electrophoresis under non-denaturing conditions shows the presence of
the
d(G
T
G
)*d(G
A
G
)
d(C
T
C
)
triplex as a band of low mobility compared to the duplex or the single
strand bands. Binding of the d(G
T
G
)
third strand and the purine strand of the duplex can be monitored by
imino proton NMR spectra. While these spectra are typically very broad
for intermolecular triplexes, the line widths can be dramatically
narrowed by the addition of two thymines to both ends of the pyrimidine
strand. Thermodynamic analysis of UV melting curves shows that this
triplex is considerably less stable than related triplexes formed with
the same duplex. The orientation of the third strand was addressed by a
combination of fluorescence energy transfer and UV melting experiments.
Results from these experiments suggest that, in the unlabeled triplex,
the preferred orientation of the third strand is parallel to the purine
strand of the duplex.
DNA triple helix formation has been the focus of much attention recently in terms of its potential use as a method for selective regulation of gene expression(1) . Oligonucleotides can inhibit protein synthesis in several different ways. In the antisense strategy, an oligonucleotide binds to a targeted mRNA molecule in a sequence-specific manner to prevent subsequent translation of the message into protein. Alternatively, binding of an oligonucleotide directly to a gene or gene promoter, via triplex formation, can arrest or block transcription. The process of down-regulating gene expression through triple helix formation is referred to as the antigene strategy. Triple helix formation has also been utilized in the development of artificial nucleases, created by tethering a cleaving agent to a triplex-forming oligonucleotide(2, 3, 4) . This leads to breaks in double-stranded DNA at very specific sites. Several studies have demonstrated the feasibility of using oligonucleotides in gene regulation(5, 6, 7) . Several properties need to be better understood in order to design optimally effective therapeutic agents based on triplex formation. These include sequence specificity of triplex formation, stability of the complex formed, delivery of the oligonucleotide into cells, and resistance of the oligonucleotides to endogenous nucleases.
The primary requirement
for triple helix formation is a homopurine-homopyrimidine sequence in
the target duplex. Typically, the third strand sequence is also
homopurine or homopyrimidine. Depending on the nature of the third
strand, there are two main categories of triple helices: pyrimidine*
purine*pyrimidine (pyr*purpyr) or purine*purine
pyrimidine
(pur*pur
pyr), where * represents Hoogsteen base pair formation
between the third strand and the purine strand of the duplex and
denotes the Watson-Crick base pair of the duplex. Most studies to date
have focused on pyr*pur
pyr triplexes. This type of triplex is
more stable at low pH because the cytosines on the third strand require
protonation in order to form Hoogsteen hydrogen bonds with the purine
strand of the duplex. In contrast, pur*pur
pyr triplexes are
stable at neutral pH. Thermal denaturation studies on pur*pur
pyr
triplexes often reveal a single transition, suggesting the simultaneous
dissociation of all three strands(8) . In most examples of
pyr*pur
pyr triplexes, a biphasic transition is observed, with the
third strand dissociating at a lower temperature than the
duplex(9, 10, 11, 12) . This
suggests that a pyrimidine third strand may be less stable, in general,
than a purine third strand.
Unlike the Watson-Crick base-paired
double helix, in which the two strands are bound antiparallel to each
other, the third strand of a triple helix can be either parallel or
antiparallel with respect to the purine strand to which it is
hydrogen-bonded. The polarity of the third strand in the triplex is
dependent on the sequence as well as the base
composition(13, 14) . A homopyrimidine third strand
containing cytosine and/or thymine binds to the purine strand in a
parallel orientation. The binding of a homopurine oligonucleotide to
the purine strand depends on the sequence. Experimental evidence has
been reported for both parallel (15) and antiparallel (16) orientations of the third strand in triplexes composed
solely of G*GC triplets. But if the third strand contains both Gs
and As, the polarity of the third strand will be antiparallel with
respect to the purine strand(8, 17) , since As with
anti-glycosidic bond conformations can form only reverse-Hoogsteen
hydrogen bonds (13) and hence direct the oligonucleotide in an
antiparallel orientation.
Hogan and co-workers (18, 19) have reported antigene activity of
oligonucleotides composed of G and T bases. However, there have been
only a limited number of physical studies on triplexes formed by such
sequences. Helene and co-workers (13, 20) have
demonstrated that the orientation of a G, T third strand depends on its
sequence. Calculations indicate that, for a 10-mer triplex whose third
strand is composed of equal number of G and T bases (with corresponding
G and A bases in the purine strand of the target duplex), the third
strand will be antiparallel to the duplex purine strand if there are
three or more GpT or TpG steps; otherwise it will be parallel.
According to the calculations of Sun and Helene(13) , this can
be explained in terms of a balance between the preference for the
parallel orientation (Hoogsteen hydrogen bonding) in an all G third
strand and the better tolerance for the lack of isomorphism between
G*GC and T*A
T triplets in the antiparallel orientation
(reverse Hoogsteen hydrogen bonding). Radhakrishnan et al.(21) carried out NMR studies on an intramolecular triplex
composed of a third strand containing Gs and one T. These studies
provided clear evidence for the distortion induced by insertion of a
T*A
T triplet within a stack of G*G
C triplets.
In earlier
studies, we have targeted the
d(GA
G
)
d(C
T
C
) for triple helix formation
with either d(C
T
C
), to make a
pyr*pur
pyr triplex(9) , or with
d(G
A
G
), to make a pur*pur
pyr
triplex(8) . We have recently described the Fourier transform
infrared spectrum of the pyr/pur*pur
pyr intermolecular triplex
d(G
T
G
)*d(G
A
G
)
d(C
T
C
) (22) . Here we employ UV, CD, gel electrophoresis, and NMR to
further characterize this triplex. While results from fluorescence
energy transfer experiments are inconclusive regarding the orientation
of the third strand relative to the purine strand of the duplex, UV
melting analysis of fluorescently labeled triplexes suggests that the
preferred orientation of the third strand is parallel to the purine
strand of the duplex in the unlabeled triplex.
Oligodeoxynucleotides were synthesized using standard
phosphoramidite chemistry on an automated DNA synthesizer as described
earlier(8) . The deprotected oligonucleotides were extensively
dialyzed against 1 mM Tris-HCl with several changes of buffer
over a period of 2-3 days. The purity of the resulting
oligonucleotides was checked by NMR and gel electrophoresis and found
to be greater than 95%. Molar extinction coefficients of various
oligonucleotides in 1 mM Tris-HCl were determined by phosphate
analysis(23) , and the concentration of the stock solutions was
determined using the following values:
d(GA
G
):
= 11500 cm
(mol of
base)
L; d(C
T
C
):
= 8300 cm
(mol of
base)
L; d(G
T
G
):
= 9900 cm
(mol of
base)
L;
d(T
C
T
C
T
):
= 8500 cm
(mol of
base)
L. All experiments were carried out in buffer
containing 10 mM Tris-HCl with 50 mM MgCl
at pH 7.4. Duplex and triplex samples were prepared by mixing the
oligonucleotides at appropriate ratios in the desired buffer, followed
by heating at 80 °C for about 5 min and cooling slowly to room
temperature. The samples were equilibrated at 5 °C overnight before
use.
Labeled oligonucleotides were synthesized using phosphoramidite
chemistry on ABI 394 DNA synthesizers. The 3`-labels were coupled to
oligonucleotides prepared using the 3`-DMT-C6 amine-ONTM controlled
pore glass (Clontech). The 5`-carboxytetramethlyrhodamine was added to
a 5`-aminohexyl-derivatized oligonucleotide (Glen Research). All amino
oligonucleotides were ion-exchanged to their lithium salts by
precipitation from ethanol-acetone as described
previously(24) . The crude, derivatized oligonucleotides were
dissolved in sodium carbonate buffer (0.25 ml, 0.1 M, pH 9.0)
and treated with
5-carboxytetramethylrhodamine-N-hydroxysuccinimidyl ester
(Applied Biosystems, 3 µl in MeSO) or Malachite Green
isothiocyanate (1 mg in 50 µl of dry N,N-dimethylformamide, Molecular Probes). After
coupling overnight at room temperature, the oligonucleotides were again
precipitated as their lithium salts from ethanol-acetone. The
oligonucleotides were resuspended in triethylammonium acetate buffer
(0.1 M, pH 7.0, buffer A), filtered, and purified by
reverse-phase HPLC (
)on a Waters 996 Diode Array Detector
system with a Hamilton PRP-1 column (300
7 mm) using a gradient
of 0-40% acetonitrile in buffer A at a flow rate of 2 ml/min.
The 5`-Malachite Green oligonucleotides were made directly on the
DNA synthesizer using a leuco-Malachite Green phosphoramidite, details
of which will be published elsewhere. ()The oligonucleotides
were coupled to the Malachite Green amidite using normal activation and
oxidation steps. On removal from the instrument, the synthesis columns
were treated with a solution of freshly prepared iodosobenzene in
dichloromethane (0.01 M, 30 s) and washed with more solvent
and air-dried. The derivatized controlled pure glass was treated with
ammonia for 4 h at 55 °C, then the supernatants were passed through
NAP-10 columns (Pharmacia LKB Biotechnol), and the desired
oligonucleotides were purified by reverse phase-HPLC as described
above.
The UV absorbance and melting studies were carried out on a Gilford 2600 UV/Vis spectrophotometer equipped with a Gilford 2527 thermoprogrammer or on a Cary 3 spectrophotometer. UV melts were done with a heating rate of 0.25 or 0.3 deg/min. CD spectra were recorded on a Jasco J500A spectropolarimeter using cells of 0.1-, 1.0-, or 10-mm optical pathlength, with temperature controlled by an external circulating water bath. CD spectra reported are the average of eight scans. CD melting experiments were done by manually changing the temperature of the bath and letting it equilibrate at the desired temperature for 5 min. The cell temperature was monitored by attachment of a microprobe directly onto the sample cell. The ellipticity data were collected at various wavelengths, and each point on the melting curve represents the average of 100 readings taken over a period of 100 s. Mixing curves were constructed from data obtained from the CD spectra of samples containing varying mole ratios of duplex and the third strand, with the total concentration of the duplex plus the third strand held constant.
Thermodynamic parameters for the formation of
the triplex were estimated from the concentration dependence of the
thermal melting temperature of the triplex in the concentration range
10 to 850 µM. Since the two transitions were well
separated from each other over a wide concentration range, we were able
to follow the concentration dependence of each transition separately.
Assuming a two-state model for each transition, we analyzed the
biphasic melting curves according to the procedure described by Pilch et al.(9) using the equation, 1/T = (R/
H°)lnC +
(
S° - 0.188R)/
H° for each
individual transition.
H° and
S°
are the enthalpy and entropy changes, respectively, associated with the
respective structural transitions, T
is the
temperature corresponding to the maximum of the dA/d(1/T) plot derived from the melting
curves, where A is the absorbance, C is the total
concentration of the d(G
T
G
) strand
(which is equal to the total concentration of each of the other two
strands), and R is the gas constant.
H° and
S° were calculated from the slope and intercept of
the straight lines obtained by fitting the data points on
1/T
versus lnC plots by linear
regression. The UV melting curves used for the thermodynamic analysis
were obtained at a heating rate of 0.25°/min; a window of ±
2 °C was used for calculating the derivative. The reversibility of
the transitions was checked by heating the sample at 1 °C/min up to
90 °C followed by cooling at the same rate. No hysterisis was
observed in the melting curves showing that the equilibrium is
maintained at each point on the melting curve.
Gel electrophoresis
was carried out under non-denaturing conditions in 15% polyacrylamide
gels containing acrylamide and bis(acrylamide) in a 29:1 ratio, cast in
90 mM Tris-borate and 50 mM MgCl. Samples
for electrophoresis were prepared in 90 mM Tris-borate and 50
mM MgCl
and were diluted into loading buffer
containing 90 mM Tris-borate, 50 mM MgCl
,
and 5% Ficoll, so that the final concentration ranged from 50 to 60
µM in single strand, duplex, or triplex. Then, 40 µl
of each sample were loaded into the wells, and electrophoresis was
carried out in buffer containing 90 mM Tris-borate and 50
mM MgCl
, in the cold room (4 °C) or at room
temperature (25 °C) at about 7 V/cm for 24 h. Gels were then
visualized by UV shadowing on a fluorescent background and
photographed.
Proton NMR spectra of the samples were recorded at 500
MHz on a General Electric spectrometer (GN-500) equipped with an Oxford
Instruments magnet and a Nicolet 1280 computer. Spectra of the samples
were taken in aqueous solutions containing 10 mM Tris-HCl,
0-50 mM MgCl, and 85% H
O, 15%
D
O, at 5 or 20 °C. MgCl
titrations were
carried out by adding aliquots of MgCl
into samples
containing the duplex and the third strand in equimolar ratios (1
mM each) dissolved in the buffer. After each addition of
MgCl
, the samples were heated at 80 °C for 5 min and
cooled. The spectra were acquired using a T331 pulse sequence for
solvent suppression, with a pulse repetition time of 5 s and interval
delay of 109 µs.
We have shown previously that the oligonucleotide
d(GT
G
), used here as the third
strand for triplex formation, can, in the presence of monovalent
cations such as Na
or K
,
self-associate to form a quadruplex structure(25) . This
quadruplex structure can be eliminated by dialysis against 1 mM Tris-HCl buffer. Tris, a bulky cation, does not promote quadruplex
formation of d(G
T
G
), nor do the
concentrations of Mg
used in this study to stabilize
the triplex (data not shown).
Figure 1:
CD spectra of
d(GT
G
)*d(G
A
G
)
d(C
T
C
)
triplex
(-),d(G
A
G
)
d(C
T
C
)
duplex (-
-), d(G
T
G
)
(
) and mathematical sum of the spectra of
d(G
A
G
)
d(C
T
C
)
and d(G
T
G
) (- - -), all at 0.5
mM in structure (single strand, duplex, or triplex) in buffer
containing 10 mM Tris-HCl (pH = 7.5) and 50 mM MgCl
, recorded at 5 °C in cells of 0.1-mm
pathlength.
Thermal
denaturation of the
d(GT
G
)*d(G
A
G
)
d(C
T
C
) triplex was studied by
temperature induced changes in the CD spectrum at the same high
oligonucleotide concentrations. Fig. 2a shows a typical
thermal denaturation profile of the triplex monitored by CD changes at
240 and 277 nm. As the temperature increases, the ellipticity values at
the two wavelengths also increase, except for a small decrease at high
temperature for the 277 nm band. The change in ellipticity with
temperature is biphasic and cooperative for both wavelengths, with the
two transitions centered around 38 and 60 °C. It is evident from Fig. 2a that the low temperature transition is most
readily followed at 277 nm while the high temperature transition is
best monitored at 240 nm. Fig. 2b shows the CD melting
profile of the duplex at the same two wavelengths. The duplex shows
only one transition around 60 °C. The ellipticity at 277 nm does
not show any cooperative change in the lower temperature region, where
the triplex shows a large change. As in the case of the triplex,
however, the temperature profile shows a relatively small but
cooperative decrease in ellipticity around 60 °C. The 240-nm
melting curve is also monophasic, with a transition at the same
temperature. Hence the transition that occurs around 38 °C for the
triplex is due to the dissociation of the third strand from the triplex
while that at 60 °C is due to the dissociation of the duplex into
single strands.
Figure 2:
CD melting curves of the triplex,
d(GA
G
)*
d(G
A
G
)
d(C
T
C
) (a) and the duplex,
d(G
A
G
)
d(C
T
C
) (b) in 10 mM Tris-HCl and 50 mM MgCl
, monitored by the change in ellipticity at 277 nm (filled circle) or 240 nm (open circle) bands of the
CD spectrum. Sample conditions are the same as in Fig. 1.
The differences in the CD spectra elicited by the
binding of d(GT
G
) to the duplex
were utilized to construct a mixing curve that provides further
evidence for complex formation between the duplex and the third strand
and also determines the stoichiometry of the complex formed. Fig. 3presents the mixing curve for the binding of
d(G
T
G
) to the duplex monitored by
the CD changes associated with complex formation for the 277 nm band.
The titration of the third strand into the duplex was carried out by
varying the mole ratio of the duplex and the third strand, keeping the
total concentration, duplex plus third strand, constant. The
discontinuous change in slope in the mixing curve observed when the
mixture contains equal amounts of the duplex and the third strand
demonstrates that the stoichiometry of the complex formed is 1 duplex/1
strand of d(G
T
G
).
Figure 3:
CD mixing curve for the binding of
d(GT
G
) to the
d(G
A
G
)
d(C
T
C
)
duplex monitored by the ellipticity changes for the CD band at 277 nm.
Sample conditions are the same as in Fig. 1.
Figure 4:
Concentration variation of
T, determined from UV melting curves, for both the low
temperature (open circle) and high temperature (closed
circle) melting transition for the triplex
d(G
T
G
)*d(G
A
G
)
d(C
T
C
)
in the same buffer as in Fig. 1.
Figure 5:
Polyacrylamide gel electrophoresis pattern
of various complexes of d(GT
G
),
d(G
A
G
), and
d(C
T
C
) under non-denaturing
conditions in the presence of 40 mM MgCl
at 4
°C (A) or at 25 °C (B). Lane 1,
d(G
A
G
); lane 2,
d(C
T
C
); lane 3,
d(G
T
G
); lane 4,
d(G
A
G
) +
d(C
T
C
); and lane 5,
d(G
A
G
) +
d(C
T
C
) +
d(G
T
G
).
Figure 6:
Imino proton region of the NMR spectrum
of
d(GA
G
)
d(C
T
C
)
duplex + d(G
T
G
) in 85%
H
O, 15% D
O containing 10 mM tris/HCl
in the absence of any MgCl
(A) or in the presence
of 50 mM MgCl
(B), at 20 °C. Sample
concentration is 1 mM in each
strand.
In an effort to sharpen
the NMR lines, we designed another triplex in which the pyrimidine
strand has two extra thymines at each end, the other two strands being
same as in the original triplex. With this ``overhang''
triplex,
d(GT
G
)*d(G
A
G
)
d(TTC
T
C
TT), we were able to reduce
the temperature and maintain reasonably narrow line widths for the
imino proton peaks. Fig. 7provides a comparison of the spectra
obtained for this triplex with that of the ``blunt end''
triplex, d(G
T
G
)*
d(G
A
G
)
d(C
T
C
),
at 5 °C. The blunt end spectrum is very broad and featureless
whereas the overhang spectrum has fairly well resolved peaks. In this
case, the additional hydrogen bonds due to Hoogsteen base pairing of
the third strand to the duplex are clearly seen. The improvement in the
triplex spectrum due to the presence of the overhang thymines is quite
remarkable.
Figure 7:
Imino proton spectrum of the
d(GT
G
)*d(G
A
G
)
d(T
C
T
C
T
)
overhang triplex (A) and the blunt end triplex,
d(G
T
G
)*d(G
A
G
)
d(C
T
C
) (B) in the presence of 50 mM MgCl
, at 5
°C. Other conditions are the same as in Fig. 5.
The possible hydrogen bonding schemes for the
interaction of the third strand with the purine strand of the duplex
are depicted in Fig. 8. In either the Hoogsteen or reverse
Hoogsteen modes, there are six guanine imino protons and four thymine
imino protons involved in base pair formation involving the third
strand. This results in a maximum of 20 imino protons potentially
detectable by NMR. In the spectrum in Fig. 7there are only
approximately 16 observable protons, most likely due to fraying of the
triplex ends. While we have not made assignments for these imino
protons, evidence for formation of G*GC triplets can be found in
both CD spectra (see above) as well as Fourier transform infrared
results(22) , while the latter also provides evidence for
formation of T*A
T triplets.
Figure 8:
Hoogsteen (left) and reverse
Hoogsteen (right) base pairing patterns for T*AT and
G*G
C triplets. - - - -, Watson-Crick hydrogen bonding
interactions; , Hoogsteen or reverse Hoogsteen
interactions.
We have attempted
to gather information about the orientation of the third strand in the
triplex
d(GT
G
)*d(G
A
G
)
d(C
T
C
) via energy transfer
measurements. Oligonucleotides were labeled with TAMRA (donor) or
Malachite Green (acceptor) at the 3` or 5` end (see Fig. 9).
TAMRA, a rhodamine derivative, has an absorbance maximum at 560 nm and
strong fluorescence emission in the 570-650 nm range. Malachite
Green, the acceptor, has an absorbance in the 550-700 nm region
and is non-fluorescent. This choice of donor-acceptor pair simplifies
the analysis, since only the donor is fluorescent. Hence, the
efficiency of energy transfer can be calculated directly from the
decrease in donor fluorescence in the presence of the acceptor without
interference from acceptor fluorescence. Triplex samples using labeled
oligonucleotides were made using the same conditions as that used for
the unlabeled samples and at the same high DNA concentrations.
Figure 9: Structure of oligonucleotide conjugates for studies on labeled triplexes.
Somewhat unexpectedly, the fluorescence was efficiently quenched
(88% energy transfer efficiency) when TAMRA was at either the 5` or
3` end of the third strand, with Malachite Green fixed at the 3` end of
the purine strand of the duplex. Control experiments carried out on
duplex samples revealed an efficiency of transfer of 56% with both
labels at the 3` end and 87% with one label at the 3` end, the other at
the 5` end. These results, expected for antiparallel duplexes, are
similar to those reported on other duplexes(29) . Thus the most
likely interpretation of our energy transfer results on the triplex
samples is that the donor and the acceptor are positioned at the same
end of the molecule in both doubly labeled samples. This implies that
the orientation of the third strand is determined by the favorable
interaction between donor and acceptor molecules tethered to the
oligonucleotides. Thus, any inherent free energy difference between the
two orientations of the third strand in the absence of labels must be
small compared to the magnitude of the donor-acceptor interaction.
If there is an inherent preference for one orientation of the third strand over the other, one may expect some difference in the thermal stability of the two doubly labeled triplexes. Fig. 10shows the first derivative UV melting curves for the completely unlabeled triplex (sample A), the doubly labeled triplex with the third strand labeled either at the 5` end (sample B), or at the 3` end (sample C). As mentioned above, the unlabeled triplex possesses two transitions; as the concentrations used for the UV melts are significantly lower than those used for the CD melts, these transitions occur at somewhat lower temperatures. The first one, centered at about 18 °C, arises from the dissociation of the third strand while the second one, around 55 °C, is due to the melting of the duplex. While samples B and C also melt in a biphasic manner, there is a significant difference in transition temperatures. The first transition of each of these samples is shifted to higher temperature than in the unlabeled sample. In sample B, this transition occurs at 42 °C, while for sample C, it is further shifted to 57 °C. The second transition occurs at 62 °C in both samples, which is somewhat higher than the second transition in the unlabeled triplex.
Figure 10:
First derivative plots of the UV melting
curves of the unlabeled triplex,
d(GT
G
)*d(G
A
G
)
d(C
T
C
)
(
) and fluorescently labeled triplexes with the
acceptor at the 3` end of the purine strand of the duplex and the donor
at the 5` end (-
-
-) or at the 3` end (-) of the third
strand. All samples contained 0.033 mM triplex.
The remarkable increase in stability of the third strand observed in
samples B and C must arise from the interaction between the two dye
molecules in close proximity to each other. This large effect supports
the hypothesis that the orientation of the third strand is determined
by the favorable donor-acceptor interaction in these samples. As a
result of this interaction, the donor and acceptor molecules are
positioned at the same end of the triplex in both samples B and C. In
order for this to occur, the third strand in sample B will have to bind
to the duplex in an antiparallel orientation with respect to the purine
strand whereas for sample C, the binding has to be parallel to the
purine strand. Thermal denaturation of the two triplexes, B and C,
shows that sample C forms a much more stable triplex compared to sample
B, implying that the triplex with the third strand parallel to the
purine strand of the duplex is more stable than the one with
antiparallel orientation. If we assume that the stabilization due to
the dye-dye interaction is similar in both cases, we may infer that, in
the absence of this interaction, the preferred orientation of the third
strand in the triplex,
d(GT
G
)*d(G
A
G
)
d(C
T
C
),
is parallel to the purine strand of the underlying duplex. This agrees
with the predictions based on theoretical calculations that for a
triplex forming oligonucleotide with 50% each of Gs and Ts, the
preferred orientation will be parallel to the purine strand if the
number of GpT or TpG steps is 3 or smaller(13) .
The data presented above provide several lines of evidence
for triple helix formation by the binding of a mixed purine/pyrimidine
oligonucleotide, d(GT
G
), to a
homopurine-homopyrimidine duplex,
d(G
A
G
)
d(C
T
C
).
It was clear from the beginning of these studies that this triplex,
d(G
T
G
)*
d(G
A
G
)
d(C
T
C
),
was strikingly less stable than the closely related pur*pur
pyr
triplex,
d(G
A
G
)*d(G
A
G
)
d(C
T
C
), because its formation
required substantially higher oligonucleotide concentrations. Unlike
the latter triplex, the G/T third strand triplex exhibits a biphasic
melting profile as measured both by CD and UV absorbance.
Circular
dichroism, which is inherently more sensitive to conformational changes
than absorbance, proved to be a useful tool in studying triplex
formation for the G/T third strand considered here. The large negative
bands in the triplex CD spectrum, with minima at 277 and 210 nm, are
not present in either the duplex or the
d(GT
G
) spectra and hence result
from the binding of the third strand to form the triplex. The negative
ellipticity near 277 nm has been described before in
poly(dG)*poly(dG)
poly(dC) triplexes(30, 31) .
Any changes in the third strand binding state should, then, primarily
affect these bands. The CD melting curve recorded at 277 nm has a large
cooperative increase in ellipticity at 38 °C, indicating the
dissociation of the third strand in this temperature region. This
melting curve also shows a small cooperative decrease in ellipticity in
the temperature range corresponding to dissociation of the duplex. In
comparison, the melting curve of the duplex shows a small but
non-cooperative increase in ellipticity at 277 nm at low temperatures,
followed by a larger, cooperative decrease in ellipticity at 60 °C
as it denatures. These observations indicate that the 277 nm band
primarily tracks the binding of the third strand.
The CD melting
curves presented in this study show some similarities with melting
curves reported for the premelting transitions of a 45-base pair long
duplex composed of blocks of d(A)d(T)
distributed between random GC base pairs(32) . For this
duplex, the ellipticity at the longer wavelength band of the CD
spectrum gradually increased with increasing temperature until the
dissociation of the duplex. This premelting transition was attributed
to temperature-dependent structural changes in the A tract of the B
form double helix. At low temperature the double helix is bent due to
the presence of the A tract. As the temperature is increased, the helix
straightens out. The ellipticity change at 270 nm due to premelting is
several times higher than that associated with the subsequent duplex
dissociation. But in the case of
d(G
A
G
)
d(C
T
C
),
the total ellipticity change for the 277 nm band in the temperature
region below denaturation of the duplex is actually smaller than that
due to the duplex dissociation. In contrast, the triplex melting curve
for
d(G
T
G
)*d(G
A
G
)
d(C
T
C
),
recorded at 277 nm, exhibits a much larger change in the low
temperature region compared to that corresponding to the duplex
dissociation. Unlike the low temperature region of the duplex melting
curve, however, this transition is highly cooperative. It is very
unlikely that this transition is due to the premelting changes in the A
tract of the triple helix. The binding of the third strand in the major
groove of the duplex is expected to make the helix more rigid compared
to the duplex and hence less bending, if any, may be expected in the
triplex.
The thermodynamic stability of the
d(GT
G
)*d(G
A
G
)
d(C
T
C
) triplex is considerably
lower than that of related triplexes with the same underlying duplex
but all purine or all pyrimidine third strands. Thus, at 25 °C,
G° for binding of the third strand to the underlying
duplex is -5.8 kcal/mol, corresponding to a binding constant of
1.8
10
. Both
H° and
S° are less negative for this triplex than for the
other triplexes based on this duplex (8, 9) with the
balance overall leading to a lower stability in terms of
G°. This decrease in stability arises, at least
partially, from the presence of GpT and TpG steps which is
destabilizing due to the non-isomorphic nature of the G*G
C and
T*A
T triplets(13) .
Gel retardation experiments
carried out at two different temperatures were undertaken to
demonstrate formation of the triple helix and also to confirm that the
low temperature transition observed in the CD melting studies is indeed
due to the dissociation of the third strand from the duplex. The
experiments were carried out at two temperatures, one well below the
midpoint of the first transition and the second at a temperature above
the first transition midpoint but below the starting point of the
second transition. The presence of a slowly migrating species at low
temperature demonstrates formation of the triplex. A similar gel
retardation pattern was reported for the
d(GA
G
)*d(G
A
G
)
d(C
T
C
)
triplex(8) . The smearing of this band may be due to end-to-end
association of the triplex molecules. At the higher temperature, the
slowly migrating band, due to the triplex, is not present while the
band due to the duplex remains unaffected, indicating dissociation of
the third strand from the triplex at this higher temperature. These
experiments clearly show that the triplex formed at low temperature
dissociates into duplex and single strand in the temperature region
well below the dissociation of the duplex, confirming the conclusions
derived from the CD studies on the biphasic melting of this triplex.
Additional evidence for triplex formation is obtained from the NMR
spectrum of the mixture of the duplex and the third strand in the
presence of MgCl. The exchangeable proton region of the NMR
spectrum of the mixture in the absence of MgCl
shows peaks
from imino protons that are in slow exchange with solvent due to the
duplex formation through Watson-Crick hydrogen bonding. Appearance of
new imino protons in the presence of MgCl
results from the
formation of hydrogen bonds between the third strand and the duplex.
These new resonances arise from the imino protons on the third strand
guanines and thymines interacting with the duplex purine strand, as
illustrated in Fig. 8, thereby confirming the formation
G*G
C and T*A
T base triplets.
Intermolecular triplexes typically exhibit very broad NMR lines (33, 34, 35) , compared to those of duplexes, despite the fact that their overall dimensions, and hence correlation time, differ only slightly from that of duplexes. In earlier studies involving DNA duplexes, we observed evidence of end-to-end stacking, based on concentration-dependent nuclear Overhauser effect between terminal deoxynucleosides(36) . Because of the larger surface area for stacking in the case of a terminal triplet compared to a terminal base pair, this effect should be enhanced for triplexes. Thus, minimization or elimination of this stacking between the molecules should improve the spectrum. In an effort to diminish the efficiency of intermolecular stacking, we designed a triplex with one strand possessing extra thymines on each end. These extra terminal residues are expected to disrupt the end-to-end stacking between triplex molecules, thereby minimizing line broadening. Spectra shown in the Fig. 7clearly demonstrate the effectiveness of this design. The intermolecular stacking interaction may also be responsible for some effects seen in optical and electrophoretic studies, such as the relatively large slope observed for the low temperature base lines in the UV melting profiles of triplexes and also characteristic smearing of triplex bands in gel electrophoresis experiments(8) .
The
orientation of the third strand with respect to the underlying duplex
is a critical element in designing triplex-forming oligonucleotides. A
homopyrimidine third strand binds to the duplex with an orientation
parallel to the purine strand. There is some experimental evidence for
the antiparallel orientation in triplexes composed solely of A*AT
triplets(37) . Also, there have been reports of both parallel (15) and antiparallel (16, 38) third strand
orientations in triplexes composed solely of G*G
C triplets.
Theoretical calculations, however, indicate greater stability for the
parallel orientation in triplexes containing only G*G
C
triplets(15, 20) . Oligonucleotides composed of Gs and
As have been shown to bind antiparallel to the purine strand of the
duplex(8, 17) . Theoretical studies have suggested
that a third strand containing both Gs and Ts can bind either parallel
or antiparallel to the purine strand of the
duplex(13, 20) . A parallel orientation is preferred
if the number of TpG or GpT steps is less than three for a sequence
containing an equal number of Gs and Ts. An increase in the number of
purine-pyrimidine steps will result in an antiparallel orientation for
the third strand.
We have addressed this question using fluorescence resonance energy transfer experiments. Our results showed extensive quenching of the donor fluorescence regardless of which end of the third strand was labeled. The most likely explanation of this observation is that, in the doubly labeled triplexes, the orientation of the labeled third strand is determined by the favorable interaction between the donor and acceptor labels rather than by the inherent preference for orientation of the third strand in unlabeled triplexes. Thus, we significantly perturbed the triplex by adding the fluorescence labels and were prevented from ascertaining the desired information. UV melting studies, however, did provide suggestive evidence by showing that the triplex with the third strand labeled at the same end as the purine strand was substantially more stable, in agreement with the predictions of Helene and co-workers(13, 20) .
Other reports have appeared concerning the strand polarity in triplexes containing third strands composed of Gs and Ts. Beal and Dervan (17) have studied the binding of a triplex-forming oligonucleotide containing Gs and Ts. This oligonucleotide, containing over 70% Gs, was observed to bind antiparallel to the purine strand of the duplex target. Based on the number of TpG or GpT steps, this is consistent with earlier predictions(13, 20) . However, a recent report on the oligonucleotide d(GGGTTGG), conjugated to an intercalating agent, states that this oligonucleotide, which also has about 70% Gs but only two GpT or TpG steps, binds to the purine strand in an antiparallel orientation(39) . This is unexpected based on our experimental results reported here as well as the calculations of Sun et al. (13, 20). One possible explanation for this discrepancy is the presence of the tethered intercalator, which may have influenced the orientation of the third strand.
In summary, we
have demonstrated the formation of the
d(GT
G
)*d(G
A
G
)
d(C
T
C
)
triplex in the presence of MgCl
by various
spectrophotometric measurements, imino proton NMR and gel
electrophoresis. While both UV and CD melting curves are biphasic, the
latter show more pronounced changes for dissociation of the triplex.
The third strand of this triplex binds less tightly than that of
d(G
A
G
)*d(G
A
G
)
d(C
T
C
) and appears to have the
opposite orientation. This preference in strand orientation is not very
strong, as the presence of a donor-acceptor pair of fluorescence labels
can alter the orientation of the third strand.