From the
Classical models for DNA triple helix formation assume the
stabilization of these structures through the formation of Hoogsteen
hydrogen bonds. This assumes that G-rich duplex DNA is more stable than
triplex DNA. We report the results of co-migration assay, dimethyl
sulfate footprint, and UV spectroscopic melting studies that reveal
that at least in some cases of short (13-mer) purine(purine-pyrimidine)
triplex the stability of double-stranded DNA is increased by the
binding of the third strand. Under conditions which are usually
considered as physiological (10 mM MgCl
Homopurine-homopyrimidine regions in DNA have attracted a great
deal of attention in connection with their possible role in gene
regulation in eukaryotes
(1, 2) . These regions also
raise the possibility of manipulating gene expression through
artificial triple helix formation
(3, 4) . Displacement
of DNA-bound regulatory proteins from their recognition sites or
polymerase blockage by triplex-forming oligonucleotides might provide a
general strategy for the alteration of sequence-specific function in
eukaryotes
(5, 6) . While the kinetic parameters of
formation of an oligodeoxyribonucleotide-directed triple helix as well
as its stability were carefully investigated for the
pyrimidine(purine-pyrimidine) structural
motif
(7, 8, 9, 10) , little is known
about purine(purine-pyrimidine) triple helical structural
motifs
(11, 12, 13) that may also be used for
sequence-specific DNA recognition.
Purine oligodeoxynucleotides bind
specifically to purine sequences in double-helical DNA to form a local
triple-helical structure
(12, 14) . These
oligonucleotides bind in the major groove antiparallel to the
Watson-Crick purine strand through reverse Hoogsteen base pairing. For
each of the triplets A-AT, G-GC the third strand base is presumed to
form two hydrogen bonds to the purine of the Watson-Crick base pair
(Fig. 1). One would expect that the stability of the triplex
would be less than the corresponding G-rich duplex structure. In the
previous work we have demonstrated the formation of a very stable
triplex with a polypurine oligodeoxynucleotide 5`-GGGGAGGGGGAGG-3`
targeted to the promoter region of c-pim-1 gene
(15) .
This triplex was stable up to 65 °C in a co-migration assay. Even
the symmetrical part of this oligonucleotide has a triplex melting
temperature of 56 °C. To further clarify the mechanisms governing
triplex formation we have extended this work to another
oligodeoxynucleotide sequence 5`-GGAGGGGGAGGGG-3`. The targets for this
sequence can be found in many mammalian genes, for instance in the
promoter sequences of bovine cytokeratin
(16) , human major
histocompatibility class III HLA factor B
(17) , and in the 3`
end of the human c-jun protooncogene
(18) . Here we
report the results of co-migration assay, dimethyl sulfate
(DMS)
At first the stability and precision of the interaction was
investigated by a co-migration assay in a non-denaturing polyacrylamide
gel electrophoresis at 65 °C (Fig. 2). This experiment
clearly indicates that under our experimental condition the interaction
with the oligonucleotide occurs only with DNA containing the target
sequence (BglI-XbaI fragment), and there is no
interaction with the plasmid pBluescript SK used as a control. The
temperature condition of the co-migration assay implies an interaction
of high stability. Addition of 0.1% SDS did not change the pattern of
the co-migration assay (data not shown). To further clarify the nature
of the interaction, we used DMS footprinting. DMS modifies the N-7
position of guanines leading to chain cleavage after treatment with
piperidine. This chemical will not react with the
purine(purine-pyrimidine) triplex because the N-7 position of purines
in double-stranded DNA is protected by Hoogsteen base
pairing
(20) . As shown in Fig. 3A (lane
2), the guanines located within the target site for the
``antiparallel'' oligonucleotide 5`-GGAGGGGGAGGGG-3` are less
reactive with DMS than guanines external to the third strand binding
site, indicating triplex formation under experimental conditions. This
protection is weaker with ``parallel'' oligonucleotide
5`-GGGGAGGGGGAGG-3`, and the first two guanines from the 5` end of the
target sequence are not protected at all (Fig. 3A,
lane 1). This experiment shows that in fact both
oligonucleotides bind the purine tract of the double helical DNA in an
antiparallel orientation to the duplex purine strand (Fig. 3,
A and B).
Our results show that factors other than the hydrogen bonds
take part in the stabilization of the triplex. It is not a hydrophobic
interaction since addition of 0.1% SDS did not change the pattern of
the co-migration assay. These factors are strongly sequence-dependent.
For example, the melting temperature of the triplex with the
oligonucleotide 5`-GGGGGGAAAAAGA-3` (the target sequence
5`-AGAAAAAGGGGGG-3`) is only 26 °C
(22) , and there was no
triplex formation in the case of oligonucleotide 5`-AGGAGGAGGAGA-3`
(target sequence, 5`-AGAGGAGGAGGA-3`) in the presence of
Mg
We thank Dr. E. Lescot for oligonucleotides synthesis;
Dr. A. Deroussent for mass spectroscopy analysis; and Drs. M. Lee, M.
Grigoriev, and J.-R. Bertrand for the helpful discussion.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, 150
mM Na
or K
) and with a rate
of heating/cooling of 1 °C/min, there is a good reversibility of
the melting profiles which is consistent with a high rate of triplex
formation. Other factors than Hoogsteen hydrogen bonds should therefore
be involved in triplex stabilization. We suggest that oligonucleotides
with similar properties could be efficient agents for artificial gene
regulation.
(
)
footprint and UV spectroscopic melting
studies that reveal that the third strand can increase the stability of
the double-stranded DNA used in this report. This is new when one
refers to the other melting studies performed with oligonucleotides
forming triplexes
(5, 8, 10, 19) .
Figure 1:
The
widespread models for A-AT and G-GC triplets within a triple helix
motif where the third strand is antiparallel to the purine Watson-Crick
strand and bases are in the
anticonformation.
Oligonucleotides
Oligonucleotides were
synthesized using the Applied Biosystems 391A DNA synthesizer and
precipitated twice with 10 volumes of 3% solution of LiClO in acetone. The pellet was washed with acetone, dried, and
dissolved in water. Mass spectroscopy analyses have shown more than 95%
purity of the oligonucleotides. For the co-migration assay
oligonucleotides were purified by electrophoresis in 20% polyacrylamide
denaturing gel. The 5` end of the oligonucleotide was radiolabeled with
[
-
P]ATP (Amersham Corp.) and T4
polynucleotide kinase (New England Biolabs) according to the
manufacturer's instructions. Oligonucleotides were labeled at a
specific activity of 200 Ci/mmol.
Plasmid Construction
The target sequence
5`-cGGGGAGGGGGAGGa-3`/3`-catggCCCCTCCCCCTCCttcga-5` was cloned in
plasmid pBluescript II in the KpnI and HindIII sites
(the length of resulting plasmid pST2 is 2947 base pairs).
Co-migration Assay
For the co-migration assay
XbaI-BglI fragment (242 base pairs) of the pST2 (1.2
pM) was mixed with nonspecific control DNA (parent plasmid
pBluescript II (1.5 pM)) and incubated with 1.0 pM
5-P-labeled oligonucleotide 5`-GGAGGGGGAGGGG-3` in 10
µl of buffered solution containing 10 mM MgCl
,
50 mM sodium acetate, and 20 mM Tris acetate, pH 7.5.
After 10 min of incubation of the mixture at 55 °C, the triple
helix formation was monitored by 5% polyacrylamide gel electrophoresis
in the same buffer. The co-migration assay at high temperature was
performed by immersion of the mini protein II apparatus (Bio-Rad) into
a water thermostat. The temperature was monitored by the thermometer
immersed into the upper chamber of electrophoresis apparatus.
Probing with DMS
To prepare a DNA fragment for
modification by DMS, the pST2 plasmid was cut with XbaI
restriction enzyme, 3`-labeled with Klenow fragment of DNA polymerase
I, and digested with BglI restriction enzyme. A smaller
labeled fragment (about 0.3 pM) was dissolved in 20 µl of
the buffer, 50 mM MOPS, pH 7.2, 50 mM NaCl, and 10
mM MgCl. Then 15 pM oligonucleotide
5`-GGAGGGGGAGGGG-3` or 5`-GGGGAGGGGGAGG-3` was added. The mixture was
incubated 2 h at 37 °C. Then 2 µl of 5% DMS were added, and the
reaction was performed for 3 min at 25 °C. The reaction was stopped
by the addition of a 5-µl solution containing 10% mercaptoethanol,
1 mM EDTA, and 0.1 M sodium acetate. After double
precipitation in ethanol, the samples were treated with 50 µl of
10% piperidine at 95 °C for 20 min, and the products of cleavage
were separated in 6% polyacrylamide denaturing gel.
UV Spectroscopic Temperature-dependent Melting
Studies
Absorbance of the oligonucleotide mixtures was measured
at 258 nm as a function of temperature with an Uvicon 941
spectrophotometer equipped with a Huber cryothermostat and Huber PD410
temperature programmer through software developed for
T recording. The rate of temperature
increase is specified in the figure legends. The buffer contains 10
mM MgCl
, 20 mM Tris acetate, pH 7.5, 50
mM sodium acetate. Concentration of each oligonucleotide is
1.15 µM. Before melting studies, all samples were heated
to 80 °C for 15 min and then allowed to return slowly to room
temperature.
Figure 2:
A, autoradiogram of a 5% nondenaturing
polyacrylamide gel showing the results of the co-migration assay at 65
°C. Lane 1, co-migration assay with oligonucleotide
5`-GGAGGGGGAGGGG-3`; lane 2, migration of the DNA without the
oligonucleotide; lane 3, 5-P-labeled
oligonucleotide 5`-GGAGGGGGAGGGG-3` alone. B, the same gel as
A after staining with ethidium bromide. M, markers of
length (R DNA of
174-HaeIII digest, New
England Biolabs); P, plasmid pBluescript SK (control
nonspecific DNA); F, XbaI-BglI restriction
fragment of the plasmid pST2; O, oligonucleotide
5`-GGAGGGGGAGGGG-3`.
Figure 3:
Autoradiogram of a 6% polyacrylamide
sequencing gel showing the results of DMS footprinting experiments.
Reactions were performed on the 3` end-labeled
XbaI-BglI restriction fragment of the plasmid pST2.
A, lane 1, DMS treatment in the presence of a 50-fold
excess of the parallel oligonucleotide 5`-GGGGAGGGGGAGG-3`; lane
2, DMS treatment in the presence of 50-fold excess of the
antiparallel oligonucleotide 5`-GGAGGGGGAGGGG-3`; lane 3, DMS
treatment without oligonucleotide; lane 4, A+G reaction.
B, scheme illustrating the most probable explanation of the
experimental results.
Since the temperature condition of the
co-migration assay is very close to the predicted melting temperature
of the targeted duplex, we monitored the stability of the targeted
duplex and of the resulting triplex by UV spectroscopic temperature
dependent melting studies (Fig. 4). As shown in this figure in
both cases, duplex or triplex, there is only one transition. There was
no transition in the melting curves for any of the purine stands or for
both of them mixed together in the temperature range from 30 to 80
°C. The transition corresponding to the triplex presents a melting
temperature which is 5 °C higher than that corresponding to the
duplex. By increasing the ratio of third strand oligonucleotide over
double-stranded DNA, we can observe that for the ratio between 1.0 and
1.2 we no longer detect the transition corresponding to double-stranded
DNA (Fig. 5). This stoichiometry suggests the generation of
triple helix structure. Furthermore, by comparing the melting curve
obtained with either increasing or decreasing the temperature (rate of
heating/cooling of 1 °C/min), we observe a very good reversibility
of the melting profiles. This indicates a higher rate of triplex
formation when compared to the triplexes investigated by other authors
(7, 8). For example, Rougee et al.(8) describe melting
curves with a hysteresis profile using a rate of heating/cooling of
0.12 °C/min.
Figure 4:
Melting temperature curves (A)
and their derivatives (B) of the equimolar mixture of the
oligonucleotides 5`-cGGGGAGGGGGAGGa-3` and
5`-agcttCCTCCCCCTCCCCggtac-3` (duplex) and 5`-cGGGGAGGGGGAGGa-3`,
3`-catggCCCCTCCCCCTCCttcga-5` and 5`-GGAGGGGGAGGGG-3`
(Triplex).
Figure 5:
Dependence of the T from
the ratio duplex/third strand. Curve numbers 1, 2,
3, 4, 5, 6, 7, and 8 correspond to the ratio 1/0, 1/0.2, 1/0.4, 1/0.6, 1/1, 1/1.2,
1/1.4, and 1/1.6 of duplex to the third strand. Conditions are the same
as described under ``Materials and Methods'' except the rate
of temperature increase was 1 °C/min.
The difference between the melting temperature of
duplex and triplex decreases with increasing NaCl concentration.
However, at 150 mM NaCl the triplex is still more stable than
the duplex (T= 70.5 and 68.1
°C, respectively). Addition of EDTA completely abolished the
difference between melting curves of duplex and triplex. Magnesium is
the only divalent ion in the solution. This therefore confirms that
triplex formation does not take place without magnesium
(15) ,
which was also shown by co-migration assay (data not shown). This
behavior agrees with the known rules for purine(purine-pyrimidine)
triplex formation
(12, 13, 21) . It is worth
mentioning that the stabilizing effect of the same oligonucleotide
without monovalent cations has been shown with 37-base pair duplex DNA
(5`-CGGGGAGGGGGAGGTAAAATTGTGGAAAAGGAAGGGA-3`/5`-agctTCCCTTCCTTTTCCA-CAATTTTACCTCCCCCTCCCCGgtac-3`).
The corresponding melting points are 78.7 °C (triplex) and 76.3
°C (duplex). We have not found any differences in the melting
profiles for triplex formation when K
was used instead
of Na
.
(21) . We have shown that superhelicity can
also influence the formation of triplex
(15) , therefore it can
be assumed that the conformation of DNA might be one of the
contributing factors. On the other hand, the presence of the divalent
cations is a requirement for the formation of a
purine(purine-pyrimidine) triplex (12, 21). We suggest that a
sequence-dependent conformation of the triplex may favor immobilization
of divalent cations through ionic bonds with the phosphates of the DNA
backbone (possibly between the phosphates in the purine chain of the
duplex and the phosphates of the third strand) and further interaction
presumably involving the imidazole nitrogen and the oxygen 6 of the
third strand and so stabilizing the whole structure. Another
possibility is that the magnesium changes the whole structure of
double-stranded DNA, making it more favorable for the third strand
binding, and that further stabilization takes place due to the stacking
interaction between the bases of the third strand. We hope this work
will stimulate modeling studies of triplex structure and search for
other oligonucleotide sequences with similar properties which could be
good targets for gene-targeted therapy.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.