A physico-chemical study of triple helix formation by an oligodeoxythymidylate with N3' -> P5' phosphoramidate linkages
A physico-chemical study of triple helix formation by an oligodeoxythymidylate with N3 ' -> P5 ' phosphoramidate linkagesBei-wen Zhou-Sun, Jian-sheng Sun1, Sergei M. Gryaznov2, Jean Liquier, Thérèse Garestier1, Claude Hélène1 and Eliane Taillandier*
Laboratoire de Spectroscopie Biomoléculaire, CNRS URA 1430, UFR Santé Médecine Biologie Humaine, Université Paris-Nord, 74, rue Marcel Cachin, 93017 Bobigny, France, 1Laboratoire de Biophysique, Muséum National d'Histoire Naturelle, INSERM U201, CNRS URA481, 43, rue Cuvier, 75231 Paris Cedex 05, France and 2Lynx therapeutics, Inc., 3832 Bay Center Place, Hayward, CA 94545, USA
Received December 31, 1996;Revised and Accepted March 19, 1997
ABSTRACT
Non-denaturing gel retardation assay, DNA melting experiments and FTIR spectroscopy were used to characterize the triple helix formed by a 15mer 2'-deoxythymidylate with N3' -> P5' phosphoramidate linkages with its target sequence. The results indicate that: (i) the pentadecadeoxythymidylate with phosphoramidate linkages [dT15(np)] is highly potent to form a triple helix with a dT15-dA15target duplex through Hoogsteenbase-pairing; (ii) it forms a dT15(np)-dA15xdT15(np) triplex with the single-stranded oligo-2'-deoxyadenylate (dA15) without detectable double-helical intermediate; (iii) it does not only form a triple helix on the dT15-dA15target duplex, but also partially displaces the dT15 strand from the dT15-dA15 duplex to form a dT15(np)-dA15xdT15(np) complex.
INTRODUCTION
Over the past few years, oligonucleotides have been receiving increased attention as a potential new class of pharmacologically active compounds. They can be used to interfere with gene expression in a sequence-specific manner by targeting either mRNA via double helix formation (antisense oligonucleotides or ribozymes) or chromosomal DNA via triple helix formation (antigene oligonucleotides) or proteins (sense oligonucleotides and aptamers) (1 -5 ). These possible applications have raised a great deal of interest in the development of oligonucleotide analogs with increased resistance to nucleases and/or enhanced binding to nucleic acid targets (6 ). The so-called antigene strategy is based on local triple helix formation via the binding of an oligonucleotide to the major groove of double-helical DNA (7 ,8 ). It was demonstrated that local triple helix formation could inhibit transcription in vitro/ex vivo by competing with the binding of transcriptional factors to the promotor, or blocking the initiation or the elongation step of transcription (for review see 3 ). Up to now there is no convincing evidence of an endogenous transcriptional inhibition in vivo by triple helix formation. Nevertheless recent studies which show that the endogenous targets are accessible to antigene oligonucleotides within the chromosomal environment (9 ) are encouraging for further antigene oligonucleotide developments.
The synthesis and some hybridization properties of a new family of oligonucleotide analogs containing N3' -> P5' phosphoramidate linkages have been recently described (10 ). An oligonucleotide phosphoramidate containing thymines and cytosines and/or guanines binds very strongly to double-helical DNA at the targeted oligopurine-oligopyrimidine segment and inhibits in vitro the transcription of the nef gene of the human immunodeficiency virus (HIV) at submicromolar concentration (11 ).
In an attempt to understand the strong binding of antigene oligonucleotides with N3' -> P5' phosphoramidate linkages to the targeted oligopurine-oligopyrimidine DNA sequences, we report here a study of the triple helix formed by a 15mer 2'-deoxythymidylate with phosphoramidate linkages with its target sequence by gel retardation assay, DNA melting experiments and FTIR spectroscopy. Gel retardation assay and DNA melting experiments were carried out in order to determine the thermal stability, the nature and the stoichiometry of the formed complexes. The study by FTIR spectroscopy of an oligonucleotide with N3' -> P5' phosphoramidate linkages is aimed at characterizing the interactions involved upon duplex and triplex formation by such an oligonucleotide. In the present manuscript, the double and triple helices are designated as Y-R and Y-RxH. The letters R and Y or H stand for purine and pyrimidine strands, the symbols - and x indicate Watson-Crick and Hoogsteen hydrogen bonds, respectively.
MATERIALS AND METHODS
Oligonucleotides and polynucleotides
Two 2'-deoxyoligonucleotides (dT15, dA15) and one modified 2'-deoxyoligonucleotide (7c-dA15) containing four dispersed 7-deaza-2'-deoxyadenines (Table 1 ) with phosphodiester linkages (OliGold grade) were purchased from Eurogentech (Belgium). They were ethanol precipitated. The 2'-deoxyoligonucleotide dT15(np) with N3' -> P5' phosphoramidate linkages was synthesized as described earlier (10 ). All these oligonucleotides were purified using a Ultrafree-MC filter (Millipore). Double-stranded polynucleotide poly(dA)-poly(dT) was purchased from Pharmacia and used without further purification. The concentrations were determined by the measurement at 25oC of their absorbance at 260 nm with a Kontron Uvikon 941 spectrophotometer using an extinction coefficient calculated according to a nearest-neighbor model (12 ) for oligonucleotides, and using an extinction coefficient of 6000/M/cm per nucleotide for polynucleotides.
PO and PN are abbreviations for the phosphodiester and the N3' -> P5' phosphoramidate linkage, respectively. A stands for 7-deaza-2'-deoxyadenine.
Gel retardation assay
A non-denaturing 10% acrylamide gel (40:1 acrylamide/bis-acrylamide) was prepared with 50 mM HEPES buffer, pH 7.0, without or in presence of 10 mM MgCl2. One strand was labelled at the 5'-end by T4 polynucleotide kinase using [[gamma]-32P]ATP. The labelled strand (10 nM) was completed with its unlabelled analog to 2 [mu]M. Samples prepared with different stoichiometries of strands were incubated at 4oC or room temperature overnight in a 50 mM HEPES buffer, pH 7.0, containing 10% sucrose without or in presence of 10 mM MgCl2. Electrophoresis was performed for 3-4 h with 2 W power at 4oC. Gels were dried, autoradiographed and analyzed on a Molecular Dynamics Phosphorimager.
DNA melting experiments
Double and triple helix stabilities were measured by DNA melting experiments which were carried out on an Uvikon 941 spectrophotometer using quartz cuvettes of 1 cm optical pathlength. The temperature of the cell holder was varied by circulating liquid using a Huber waterbath controlled by aHuber PD415 programmer at a rate of 0.1oC/min. Temperature monitoring was achieved by using a thermocouple in a control cuvette. Samples were dissolved at 1 [mu]M strand concentration in 10 mM sodium cacodylate buffer at pH 7.0 containing 0.1 or 0.4 M sodium chloride. All melting curves of the samples obtained upon cooling or heating were superimposable indicating that the equilibrium was achieved. The melting temperature (Tm) was evaluated as the temperature of half-association (or half-dissociation) of the formed complexes determined by the first derivative of the melting curve.
FTIR spectroscopy experiments
The samples were studied in the presence of 0.5 Na+ per nucleotide at pH 7.0. They were prepared in H2O or D2O solutions in ZnSe cells at 25 mM strand concentration. Deuteration experiments were performed by drying the samples under nitrogen and redissolving in D2O (>99.8% purity, purchased from Euriso-Top CEA). FTIR spectra were recorded using a Perkin Elmer 2000 spectrophotometer monitored by the Galaxy GRAMS386 program (1 cm-1 resolution). Spectra were generally obtained by accumulation of 25 scans. Temperature was controlled by a Specac temperature controller.
RESULTS AND DISCUSSION
Gel retardation assay
The experiments presented in Figure 1 A show the formation of the dT15(np)-dA15xdT15(np) and dT15-dA15xdT15(np) triple helices in presence of 10 mM MgCl2. A 7c-dA15 labelled strand containing 7-deaza-2'deoxyadenine residues has been used in lanes 1-5 instead of a dA15 labelled strand in lanes 6-11.
DNA melting experiments
Table 2 summarizes the melting temperatures of the samples containing different combinations of two oligomers. The dA15+dT15 sample with 1:1 stoichiometry (duplex) presents a melting temperature of 35.5oC in 100 mM NaCl. The complex with 1:2 stoichiometry obtained by addition of a second dT15 strand has also a monophasic melting profile in 100 mM NaCl and the measured Tm is similar. Therefore, the dT15-dA15xdT15 triplex is not formed under low salt condition in agreement with earlier studies (13 ,14 ). In the presence of 400 mM NaCl, a biphasic melting profile is observed (Fig. 2 , inset). It is noticed that the increase of the optical density (transition amplitude) upon temperature increase in 100 mM NaCl, which corresponds to the melting of the dA15-dT15 duplex is the same for the 1:1 and the 1:2 stoichiometry solutions. However the hyperchromicity is different in both cases, as for the 1:2 sample the absorption contains a contribution of the non base-paired dT15 strand at all temperatures.
Characteristics of the melting curves of different samples in a buffer containing 10 mM sodium cacodylate, pH 7.0, 0.1 M sodium chloride
Strands
A15+dT15
7c-dA15+dT15
7c-dA15+dT15(np)
dA15+dT15(np)
Stoichiometry
1:1
1:2
1:1
1:1
1:1
1:2
Tm (+-0.5oC)
35.5
36.5
29.0
32.0
45.0
45.5
[Delta]A (260 nm)
0.06
0.06
0.03
0.03
0.06
0.12
Hypochromism [Delta]A/A (260 nm)
23%
17%
13%
13%
20%
30%
The strand concentration of one equimolar of oligomer is 1 [mu]M. [Delta]A(260 nm) represents the amplitude of the melting transition as measured by the difference of absorbance at 260 nm after and before the transition.
FTIR spectroscopy: triple helix formation
Triple helix formation can be characterized by the FTIR spectra recorded in D2O solutions in the spectral domain between 1750 and 1550 cm-1 presented in Figure 3 . We show here the spectra ofdA15+dT15(np) samples with 1:1 and 1:2 stoichiometry (Fig. 3 c and d) and of the complex between poly(dT)-poly(dA) and dT15(np) prepared so as to contain a 1:1:1 stoichiometry (Fig. 3 e). Their spectral profiles are similar to that of the canonical triple helix dT15-dA15xdT15 (Fig. 3 b) which is easily stabilized at 0.5 Na+ per nucleotide (15 ,16 ).
CONCLUSION
In summary, three techniques (gel retardation, DNA melting and FTIR spectroscopy) were used to investigate triple helix formation by a 15mer deoxythymidylate with N3' -> P5' phosphoramidate linkages on its target sequence. Together, they have consistently determined the nature and the stoichiometry of the formed complexes, as well as the interactions involved in each complex. The results clearly indicate that: (i) the pentadecadeoxythymidylate with phosphoramidate linkages is highly potent to form a triple helix with its target sequence through Hoogsteen base pairing; (ii) it forms a triplex with the single-stranded pentadecadeoxyadenylate without any detectable double-helical intermediate; (iii) it does not only form a triple helix on the dT15-dA15 target duplex, but also displaces, at least partially, the dT15 strand from dT15-dA15 to form a new complex, the dT15(np)-dA15x dT15(np) triplex.
REFERENCES
1 Hélène,C. and Toulmé,J.J. (1990) Biochem. Biophys. Acta., 1049, 99-125.
