Different Effects of Nonintercalative Antitumor Drugs on DNA Triple Helix Stability: SN-18071 Promotes Triple Helix Formation

Abstract

The interaction of the nonintercalating bisquaternary ammonium heterocyclic drugs SN- 18071 and SN-6999 with a DNA triple helix has been studied using thermal denaturation and CD spectroscopy. Our data show, that both minor groove binders can bind to the triple helix of poly(dA)-2poly(dT) under comparable ionic conditions, but they influence the stability of the triplex relative to the duplex structure of poly(dA)-poly(dT) in a different manner. SN- 18071, a ligand devoid of forming hydrogen bonds, can promote triplex formation and thermally stabilizes it up to 500 mM Na+ concentration. SN-6999 destabilizes the triplex to duplex equibilirium whereas it stabilizes the duplex. The binding constant of SN-18071 is found to be greater than that to the duplex. The stabilizing effect of SN-18071 is explained by electrostatic inetractions of three ligand molecules with the three grooves of the triple stranded structure. From the experiments it is concluded that SN-6999 binds to the triplex minor groove thereby destabilizing the triplex similar as previously reported for netropsin.     

Comparative circular dichroism and fluorescence studies of oligodeoxyribonucleotide and oligodeoxyribonucleoside methylphosphonate pyrimidine strands in duplex and triplex formation

An analogue of the homopyrimidine oligodeoxyribonucleotide d(CT)8 has been synthesized. This analogue, d(CT)8 contains nonionic methylphosphonate internucleoside linkages. The pH-dependent conformational transitions of d(CT)8 have been studied and its ability to form duplexes and triplexes with the normal homopurine oligonucleotide d(AG)8 has also been investigated as a function of pH. Circular dichroism spectroscopy and ethidium bromide fluorescence enhancement have been used to monitor pH-dependent conformational transitions driven by the protonation of cytosine residues, and the different behavior of d(CT)8 and d(CT)8 has been compared. It was possible to form self-associated complexes by using either d(CT)8 or d(CT)8, and both compounds combined with d(AG)8 to form duplex or triplex DNA. At neutral pH, the CD spectrum of d(AG)8.d(CT)8 duplex was quite different from the CD spectrum of d(AG)8.d(CT)8 duplex, reflecting most likely a difference in conformation. The duplex to triplex transition characteristic of this DNA sequence occurred at a lower pH when d(CT)8 was substituted for d(CT)8; however, at pH 4.2, triplex containing d(CT)8 was similar in conformation to triplex containing d(CT)8. Several of these observations can be related to the alterations in electrostatic and steric interactions that occur when the negatively charged phosphodiester backbone of d(CT)8 is replaced with a nonionic methylphosphonate backbone.     

FTIR and UV spectroscopy studies of triplex formation between pyrimidine methoxyethylphosphoramidates alpha-oligodeoxynucleotides and ds DNA targets

The ability of non-ionic methoxyethylphosphoramidate (PNHME) alpha-oligodeoxynucleotides (ODNs), alpha dT(15) and alpha dCT dodecamer, to form triplexes with their double-stranded DNA targets was evaluated. Thermal stability of the formed complexes was studied by UV thermal denaturation and the data showed that these PNHME alpha-ODNs formed much more stable triplexes than phosphodiester (PO) beta-ODNs did (Delta Tm = + 20 degrees C for alpha dCT PNHME). In addition, FTIR spectroscopy was used to determine the base pairing and the strand orientations of the triplexes formed by alpha dT(15) PNHME compared to phosphodiester ODNs with beta or alpha anomeric configuration. While beta dT(15) PO failed to form a triplex with a long beta dA(n) x beta dT(n) duplex, the Tm of the Hoogsteen part of the triplex formed by alpha dT(15) PNHME reached 40 degrees C. Moreover alpha dT(15) PNHME displaced the beta dT(15) strand of a shorter beta dA(15) x beta dT(15) duplex. The alpha dCT PNHME and alpha dT(15) PNHME third strands were found antiparallel in contrast to alpha dT(15) PO which is parallel to the purine strand of their duplex target. The uniform preferential Hoogsteen pairing of the nucleotides alpha dT and alpha dC combining both replacements might contribute to the improve stability of the triplexes.     

Binding mode of 4',6-diamidino-2-phenylindole and ethidium to poly(dG).poly(dC).poly(dC)(+) triplex and poly(dG).poly(dC) duplex

Optical spectroscopic properties of 4',6-diamidino-2-phenylindole (DAPI) and ethidium bromide complexed with poly(dG).poly(dC).poly(dC)(+) triplex and poly(dG).poly(dC) duplex were compared in this study. When complexed with both duplex and triplex, ethidium is characterized by hypochromism and a red shift in the absorption spectrum, a complicate induced circular dichroism (CD) band in the polynucleotide absorption region, and a negative reduced linear dichroism signal in both polynucleotide and drug absorption regions. The spectral properties for both duplex- and triplex-bound ethidium are identical and both can be understood by the intercalation binding mode. In contrast, the absorption and CD spectra of DAPI complexed with triplex differ from those of the DAPI-duplex complex, although both complexes can be understood by the intercalation binding mode. Considering that the third strand runs along the major groove of the template duplex, we conclude that the DAPI molecule partially intercalates near the major groove of the duplex, where the third strand can affect its spectroscopic properties.     

5-(1-propargylamino)-2'-deoxyuridine (UP): a novel thymidine analogue for generating DNA triplexes with increased stability

We have used quantitative DNase I footprinting and UV-melting studies to examine the formation of DNA triplexes in which the third strand thymines have been replaced by 5-propargylamino-dU (UP). The intra-molecular triplex A6-L-T6-L-(UP)5T (L = two octanediol residues) shows a single UV-melting transition which is >20 degrees higher than that of the parent triplex A6-L-T6-L-T6at pH 5.5. Although a single transition is observed at all pHs, the melting temperature (Tm) of the modified oligonucleotide decreases at higher pHs, consistent with the requirement for protonation of the amino group. A similar intramolecular triplex with a longer overhanging duplex shows two melting transitions, the lower of which is stabilised by substitution of T by UP, in a pH dependent fashion. Triplex stability increases by approximately 12 K for each T to UP substitution. Quantitative footprinting studies have examined the interaction of three UP-containing 9mer oligonucleotides with the different portions of the 17mer sequence 5'-AGGAAGAGAAAAAAGAA. At pH 5.0, the UP-containing oligo-nucleotides footprint to much lower concentrations than their T-containing counterparts. In particular (UP)6CUPT binds approximately 1000-fold more tightly than the unmodified oligonucleotide T6CTT. Oligonucleotides containing fewer UP residues are stabilised to a lesser extent. The affinity of these modified third strands decreases at higher pHs. These results demonstrate that the stability of DNA triplexes can be dramatically increased by using positively charged analogues of thymine.     

Triple helix formation with purine-rich phosphorothioate-containing oligonucleotides covalently linked to an acridine derivative.

Purine-rich (GA)- and (GT)-containing oligophosphorothioates were investigated for their triplex-forming potential on a 23 bp DNA duplex target. In our system, GA-containing oligophosphorothioates (23mer GA-PS) were capable of triplex formation with binding affinities lower than (GA)-containing oligophosphodiesters (23mer GA-PO). The orientation of the third strand 23mers GA-PS and GA-PO was antiparallel to the purine strand of the duplex DNA target. In contrast, (GT)-containing oligophosphorothioates (23mer GT-PS) did not support triplex formation in either orientation, whereas the 23mer GT-PO oligophosphodiester demonstrated triplex formation in the antiparallel orientation. GA-PS oligonucleotides, in contrast to GT-PS oligonucleotides, were capable of self-association, but these self-associated structures exhibited lower stabilities than those formed with GA-PO oligonucleotides, suggesting that homoduplex formation (previously described for the 23mer GA-PO sequence by Noonberg et al.) could not fully account for the decrease in triplex stability when phosphorothioate linkages were used. The 23mer GA-PS oligonucleotide was covalently linked via its 5'-end to an acridine derivative (23mer Acr-GA-PS). In the presence of potassium cations, this conjugate demonstrated triplex formation with higher binding affinity than the unmodified 23mer GA-PS oligonucleotide and even than the 23mer GA-PO oligonucleotide. A (GA)-containing oligophosphodiester with two phosphorothioate linkages at both the 5'- and 3'-ends exhibited similar binding affinity to duplex DNA compared with the unmodified GA-PO oligophosphodiester. This capped oligonucleotide was more resistant to nucleases than the GA-PO oligomer and thus represents a good alternative for ex vivo applications of (GA)-containing, triplex-forming oligonucleotides, allowing a higher binding affinity for its duplex target without rapid cellular degradation.     

A parallel stranded linear DNA duplex incorporating dG.dC base pairs

DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA.dT base pairs. We have substituted four dA.dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1.D2) with dG.dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG.dC base pairs (ps-D5.D6) is 10-16 degrees C lower and the van't Hoff enthalpy difference delta HvH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl2) compared to that of ps-D1.D2. Based on energy minimizations of a ps-[d(T5GA5).d(A5CT5)] duplex using force field calculations we propose a model for the conformation of a trans dG.dC base pair in a ps helix.     

Parallel intramolecular DNA triple helix with G and T bases in the third strand stabilized by Zn(2+) ions

We present evidence of formation of an intramolecular parallel triple helix with T•A.T and G•G.C base triplets (where • represents the hydrogen bonding interaction between the third strand and the duplex while . represents the Watson–Crick interactions which stabilize the duplex). The third GT strand, containing seven GpT/TpG steps, targets the polypurine sequence 5′-AGG-AGG-GAG-GAG-3′. The triple helix is obtained by the folding back twice of a 36mer, formed by three dodecamers tethered by hydroxyalkyl linkers (-L-). Due to the design of the oligonucleotide, the third strand orientation is parallel with respect to the polypurine strand. Triple helical formation has been studied in concentration conditions in which native gel electrophoresis experiments showed the absence of intermolecular structures. Circular dichroism (CD) and UV spectroscopy have been used to evidence the triplex structure. A CD spectrum characteristic of triple helical formation as well as biphasic UV and CD melting curves have been obtained in high ionic strength NaCl solutions in the presence of Zn²⁺ ions. Specific interactions with Zn²⁺ ions in low water activity conditions are necessary to stabilize the parallel triplex.     

NMR characterisation of a triple stranded complex formed by homo-purine and homo-pyrimidine DNA strands at 1:1 molar ratio and acidic pH.

Homo-purine (d-TGAGGAAAGAAGGT) and homo-pyrimidine (d-CTCCTTTCTTCC) oligomers have been designed such that they are complementary in parallel orientation. When mixed in a 1:1 molar ratio, the system adopts an antiparallel duplex at neutral pH with three mismatched base pairs. On lowering the pH below 5.5, a new complex is formed. The NMR results show the coexistence of a intermolecular pyrimidine.purine:pyrimidine DNA triplex and a single stranded oligopurine at this pH. The triplex is stabilized by five T.A:T, four C+.G:C and two mismatched triads, namely, C+.G-T and T.A-C. This triplex is further stabilized by a Hoogsteen C+.G base-pair on one end. Temperature dependence of the imino proton resonances reveals that the triplex dissociates directly into single strands around 55 degrees C, without duplex intermediates. Parallel duplexes are not formed under any of the conditions employed in this study.     

Proton NMR and optical spectroscopic studies on the DNA triplex formed by d-A-(G-A)7-G and d-C-(T-C)7-T.

Triplex and duplex formation of two deoxyribohexadecamers d-A-(G-A)-G (a) and d-C-(T-C)-T (b) have been studied by UV, CD, fluorescence, and proton NMR spectroscopy. Optical studies of a and b at dilute concentrations (microM range) yielded results similar to those seen for polymers of the same sequence, indicating that these hexadecamers have properties similar to the polymers in regard to triplex formation. The CD spectra of concentrated NMR samples (mM range) are similar to those observed at optical concentrations at both low and high pH, making possible a correlation between CD and NMR studies. In NMR spectra, two imido NH-N hydrogen bonded resonance envelopes at 12.6 and 13.7 ppm indicate that only the duplex conformation is present at pH greater than 7.7. Four new NH-N hydrogen-bonded resonance envelopes at 12.7, 13.5, 14.2, and 14.9 ppm are observed under acidic conditions (pH 5.6) and the two original NH-N resonances gradually disappear as the pH is lowered. Assignment of these four peaks to Watson-Crick G.C. Hoogsteen T.A Watson-Crick A.T, and Hoogsteen C+.G hydrogen-bonded imidos, respectively, confirm the formation of triple-stranded DNA NMR results also show that triplex is more stable than duplex at the same salt condition and that triplex melts to single strands directly without going through a duplex intermediate. However, in the melting studies, a structural change within the triple-stranded complex is evident at temperatures significantly below the major helix-to-coil transition. These studies demonstrate the feasibility of using NMR spectroscopy and oligonucleotide model compounds a and b for the study of DNA triplex formation.     

Solution structure of an O6-[4-oxo-4-(3-pyridyl)butyl]guanine adduct in an 11 mer DNA duplex: evidence for formation of a base triplex

The pyridyloxobutylating agents derived from metabolically activated tobacco-specific nitrosamines can covalently modify guanine bases in DNA at the O(6) position. The adduct formed, O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine ([POB]dG), results in mutations that can lead to tumor formation, posing a significant cancer risk to humans exposed to tobacco smoke. A combined NMR-molecular mechanics computational approach was used to determine the solution structure of the [POB]dG adduct within an 11mer duplex sequence d(CCATAT-[POB]G-GCCC).d(GGGCCATATGG). In agreement with the NMR results, the POB ligand is located in the major groove, centered between the flanking 5'-side dT.dA and the 3'-side dG.dC base pairs and thus in the plane of the modified [POB]dG.dC base pair, which is displaced slightly into the minor groove. The modified base pair in the structure adopts wobble base pairing (hydrogen bonds between [POB]dG(N1) and dC(NH4) amino proton and between [POB]dG(NH2) amino proton and dC(N3)). A hydrogen bond appears to occur between the POB carbonyl oxygen and the partner dC's second amino proton. The modified guanine purine base, partner cytosine pyrimidine base, and POB pyridyl ring form a triplex via this unusual hydrogen-bonding pattern. The phosphodiester backbone twists at the lesion site, accounting for the unusual phosphorus chemical shift differences relative to those for the control DNA duplex. The helical distortions and wobble base pairing induced by the covalent binding of POB to the O(6)-position of dG help explain the significant decrease of 17.6 degrees C in melting temperature of the modified duplex relative to the unmodified control.     

Triplex formation at physiological pH by 5-Me-dC-N4-(spermine) [X] oligodeoxynucleotides: non protonation of N3 in X of X*G:C triad and effect of base mismatch/ionic strength on triplex stabilities.

Oligodeoxynucleotide (ODN) directed triplex formation has therapeutic importance and depends on Hoogsteen hydrogen bonds between a duplex DNA and a third DNA strand. T*A:T triplets are formed at neutral pH and C+*G:C are favoured at acidic pH. It is demonstrated that spermine conjugation at N4 of 5-Me-dC in ODNs 1-5 (sp-ODNs) imparts zwitterionic character, thus reducing the net negative charge of ODNs 1-5. sp-ODNs form triplexes with complementary 24mer duplex 8:9 show foremost stability at neutral pH 7.3 and decrease in stability towards lower pH, unlike the normal ODNs where optimal stability is found at an acidic pH 5.5. At pH 7.3, control ODNs 6 and 7 carrying dC or 5-Me-dC, respectively, do not show any triple helix formation. The stability order of triplex containing 5-Me-dC-N4-(spermine) with normal and mismatched duplex was found to be X*G:C approximately X*A:T > X*C:G > X*T:A. The hysteresis curve of sp-ODN triplex 3*8:9 indicated a better association with complementary duplex 8:9 as compared to unmodified ODN 6 in triplex 6*8:9. pH-dependent UV difference spectra suggest that N3 protonation is not a requirement for triplex formation by sp-ODN and interstrand interaction of conjugated spermine more than compensates for loss in stability due to absence of a single Hoogsteen hydrogen bond. These results may have importance in designing oligonucleotides for antigene applications.     

Binding of netropsin to a DNA triple helix.

The interaction of netropsin, a minor groove binding drug, with T-A-T triple helix and A-T double helix was studied using circular dichroism spectroscopy and thermal denaturation. The triple helix was made by an oligonucleotide (dA)12-x-(dT)12-x-(dT)12, where x is a hexaethylene glycol chain bridged between the 3' phosphate of one strand and the 5' phosphate of the following strand. This oligonucleotide is able to fold back on itself to form a very stable triplex. Changing the conditions allows the same oligonucleotide in a duplex form with a (dT)12 dangling arm. Circular dichroism spectroscopy demonstrates that netropsin can bind to the triple helical structure. Spectral analysis shows that the bound drug exhibits a conformation and an environment similar in double-stranded and in triple-stranded structure. However, the binding constant to the triple-stranded structure is found smaller than the binding constant to the double-stranded one. Thermal denaturation experiments demonstrate that netropsin destabilizes the triplex whereas it stabilizes the duplex.     

A triple-stranded "clip" from the oligonucleotide 3'-(dA)10-pO(Ch2Ch2O)3p-(dT)10-pO(CH2CH2O)3-p-(dT)10(-5)]

The temperature dependence of the UV- and CD-spectra of the oligonucleotides 3'-d(A)10-L-(T)10-5' [anti(AT)], 3'-d(A)10-L-d(T)10-3' [par(AT)] and 3'-d(A)10-L-(dT)10-L-(dT)10-5' [tripl(ATT)] (L = -PO(CH2CH2O) 3p-) in the phosphate buffer at pH 7 under different concentrations of NaCL and in the presence or absence of 0.01 M MgCl2 was studied. All registered structural changes are the result of intramolecular processes if the concentrations of the oligonucleotides is low (about 2.2.10(-5) M). Par(AT) and anti(AT) exist in the only two forms, transforming into each other: under low temperatures they exist as hairpins with the parallel or antiparallel orientation of chains accordingly which transform into unfolded chains when the temperature increased. In contrast trip(ATT) exists in the three different forms depending on the temperature and ion conditions. They are: the three- stranded clip, the two-stranded hairpin with a single stranded "tail" and completely unfolded chain. For the first time this work presents thermodynamic parameters of the triplex formation from deoxyoligonucleotides depending on NaCl concentration. We have registered the CD spectra to one-, two-, and three-stranded forms. Ethidium bromide binding to three-stranded "clip" was investigated, and it was established that molecules of the dye may intercalate into the "clip" with formation of stable complexes (the constant of association 10(6) M-1). It is maximum three molecules of ethidium bromid which may bound to one molecule of the three-stranded clip. It has been shown that the suggested synthetic model (three oligonucleotide blocks combined by hydroxyalkyl chains) is the most convenient for physico-chemical investigations of triplexes today.     

Acceleration of DNA strand exchange by polycation comb-type copolymer.

The accelerating effect of cationic substances on DNA strand exchange reaction between 20 bp DNA duplex and its complementary single strand was studied. A comb-type polycationic copolymer which is composed of poly (L-lysine) backbone and dextran graft chain (PLL-g-Dex) and known to stabilize triplex DNA expedites the strand exchange reaction under physiological relevant conditions. Electrostatically small excess of the copolymer increased DNA strand exchange rate by 300-fold while large excess of spermine or cethyltrimethylammonium bromide, cationic detergent known to promote markedly hybridization of complementary DNA strands, showed slight effect. It should be noted that the copolymer promotes the strand exchange reaction while it stabilizes double stranded DNA.     

alpha-DNA X: alpha and beta tetrathymidilates covalently linked to oxazolopyridocarbazolium (OPC): comparative stabilization of oligo beta-[dT]:oligo beta-[dA] and oligo alpha-[dT]:oligo beta-[dA] duplexes by the intercalating agent.

The influence of the intercalating oxazolopyridocarbazolium (HOPC) on the stabilization of modified oligonucleotides: alpha-T4c5OPC or beta-T4c5OPC associated to beta-oligo (dA) was studied. It appears that the situation is different from what has been observed for the interaction of these modified oligonucleotides with poly (rA). The higher free energy of formation of the alpha-T4c5OPC :beta-oligo(dA), when compared to beta-T4c5OPC, is essentially due to the overall stability added to this system by the intercalator. This enhanced stability comes from a higher number of binding sites of HOPC for the alpha:beta duplex together with a lower van't Hoff energy of formation of the alpha:beta duplex.     

Detection and kinetic studies of triplex formation by oligodeoxynucleotides using real-time biomolecular interaction analysis (BIA).

Real-time biomolecular interaction analysis (BIA) has been applied to triplex formation between oligodeoxynucleotides. 5'-Biotinylated oligonucleotides were immobilised on the streptavidin-coated surface of a biosensor chip and subsequently hybridised to their complementary strand. Sequence-specific triplex formation was observed when a suitable third-strand oligopyrimidine was injected over the surface-bound duplex. In addition, a single-stranded oligonucleotide immobilised on the chip surface was able to capture a DNA duplex by triplex recognition. The presence of spermine increases the rate of association between the third strand and immobilised duplex, but at elevated spermine concentrations non-specific association is observed. A preliminary kinetic analysis of triplex formation at pH 5.2 by an 11mer third strand containing thymine, cytosine and uracil is reported. Values for the association and dissociation rate constants were determined to be (1.9 +/- 0.2) x 10(3) M-1 s-1 and (8.1 +/- 1.9) x 10(-5) s-1, respectively.     

Design and simple routes of synthesis of oligonucleotide conjugates for studies of DNA triple helix formation

A series of oligonucleotides conjugated to intercalators, as well as fluorescent and lipophilic substances, minor groove binders and photoactive molecules were synthesized for studies of their ability to form a stable triple helix. Purine-rich short double stranded DNA fragments from HIV-1 genome and pyrimidine 16-mer oligodeoxyribonucleotide were used as models. A conjugate of a dipyrido[3,2-a:2',3'-c]phenazine-ruthenium (II) complex and a triple helix-forming oligonucleotide was constructed. Upon sequence-specific duplex and triplex formation of the conjugate, the ruthenium complex becomes highly fluorescent. The attached ruthenium complex induces a stabilization of the DNA triple helix and a significant increase of the time of residence of the third strand on the duplex.     

DESIGN AND SIMPLE ROUTES OF SYNTHESIS OF OLIGONUCLEOTIDE CONJUGATES FOR STUDIES OF DNA TRIPLE HELIX FORMATION

A series of oligonucleotides conjugated to intercalators, as well as fluorescent and lipophilic substances, minor groove binders and photoactive molecules were synthesized for studies of their ability to form a stable triple helix. Purine-rich short double stranded DNA fragments from HIV-1 genome and pyrimidine 16-mer oligodeoxyribonucleotide were used as models. A conjugate of a dipyrido[3,2-a:2′,3′-c]phenazine-ruthenium (II) complex and a triple helix-forming oligonucleotide was constructed. Upon sequence-specific duplex and triplex formation of the conjugate, the ruthenium complex becomes highly fluorescent. The attached ruthenium complex induces a stabilization of the DNA triple helix and a significant increase of the time of residence of the third strand on the duplex.     

Circular dichroism and UV melting studies on formation of an intramolecular triplex containing parallel T*A:T and G*G:C triplets: netropsin complexation with the triplex.

We have used circular dichroism and UV absorption spectroscopy to characterize the formation and melting behaviour of an intramolecular DNA triple helix containing parallel T*A:T and G*G:C triplets. Our approach to induce and to stabilize a parallel triplex involves the oligonucleotide 5'-d(G4A4G4[T4]C4T4C4-[T4]G4T4G4) ([T4] represents a stretch of four thymine residues). In a 10 mM sodium cacodylate, 0.2 mM disodium EDTA (pH 7) buffer, we have shown the following significant results. (i) While in the absence of MgCl2 this oligonucleotide adopts an intramolecular hairpin duplex structure prolonged by the single strand extremity 5'-d([T4]G4T4G4), the presence of millimolar concentrations of MgCl2generates an intramolecular triplex (via double hairpin formation). (ii) In contrast to the antiparallel triplex formed by the oligonucleotide 5'-d(G4T4G4[T4]G4A4G4[T4]C4T4C4), the parallel triplex melts in a biphasic manner (a triplex to duplex transition followed by a duplex to coil transition) and is less stable than the antiparallel one. The enthalpy change associated with triplex formation (-37 kcal/mol) is approximately half that of duplex formation (-81 kcal/mol). (iii) The parallel triple helix is disrupted by increasing the concentration of KCl(>10 mM), whereas, under the same conditions, the antiparallel triplex remains stable. (iv) Netropsin, a natural DNA minor groove-binding ligand, binds to the central site A4/T4of the duplex or triplex in an equimolar stoichiometry. Its association constant K is smaller for the parallel triplex ( approximately 1 x 10(7) M-1) than for the antiparallel one ( approximately 1 x 10(8) M-1). In contrast to the antiparallel structure, netropsin binding has no apparent effect on thermal stability of the parallel triple helix.