©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
An Unusually Stable Purine(Purine-Pyrimidine) Short Triplex
THE THIRD STRAND STABILIZES DOUBLE-STRANDED DNA (*)

Fedor Svinarchuk (1) (2)(§), Jacques Paoletti (1), Claude Malvy (1)

From the (1) Laboratoire de Biochimie-Enzymologie, CNRS URA 147, Institute Gustave Roussy, rue Camille Desmoulins, 94805 Villejuif Cedex, France and (2) Department of Biochemistry, Novosibirsk Institute of Bioorganic Chemistry, 8 Prospect Lavrenteva, Novosibirsk 630090, Russia.

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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, 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.


INTRODUCTION

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)() 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.




MATERIALS AND METHODS

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.


RESULTS

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).


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.


DISCUSSION

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(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.


FOOTNOTES

*
This work was supported by the ``Association pour la Recherche sur le Cancer'' research fellowship (to F. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Laboratoire de Biochimie-Enzymologie, CNRS URA 147, Institute Gustave Roussy, rue Camille Desmoulins, 94805 Villejuif Cedex, France. Tel.: 33-1-45-59-70-45; Fax: 33-1-46-78-41-20.

The abbreviations used are: DMS, dimethyl sulfate; MOPS, 4-morpholinepropanesulfonic acid.


ACKNOWLEDGEMENTS

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.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.