DNA triple helix formation at target sites containing several pyrimidine interruptions: stabilization by protonated cytosine or 5-(1-propargylamino)dU.

DNase I footprinting has been used to study the formation of parallel triplexes at oligopurine target sequences which are interrupted by pyrimidines at regular intervals. TA interruptions are targeted with third strand oligonucleotides containing guanine, generating G x TA triplets, while CG base pairs are targeted with thymine, forming T x CG triplets. We have attempted to optimize the stability of these complexes by varying the base composition and sequence arrangement of the target sites, and by replacing the third strand thymines with the positively charged analogue 5-(1-propargylamino)dU (U(P)). For the target sequence (AAAT)(5)AA, in which pyrimidines are positioned at every fourth residue, triplex formation with TG-containing oligonucleotides is only detected in the presence of a triplex-binding ligand, though stable triplexes were detected at the target site (AAAAAT)(3)AAAA. Triplex stability at targets containing pyrimidines at every fourth residue is increased by introducing guanines into the duplex repeat unit using the targets (AGAT)(5)AA and (ATGA)(5)AA. In contrast, placing C(+) x GC triplets on the 5'-side of G x TA, using the target (AGTA)(5)TT, produces complexes of lower stability. We have attempted further to increase the stability of these complexes by using the positively charged thymine base analogue U(P), and have shown that (TU(P)TG)(5)TT forms a more stable complex with target (AAAT)(5)AA than the unmodified third strand, generating a footprint in the absence of a triplex-binding ligand. Triplex formation at (AGTA)(5)AA is improved by using the modified oligonucleotide (TCGU(P))(5)TT, generating a complex in which the charged triplets C(+) x GC and U(P) x AT alternate with uncharged triplets. In contrast, placing U(P) x AT triplets adjacent to C(+) x GC, using the third strand oligonucleotide (U(P)CGT)(5)TT, reduces triplex formation, while the third strand with both substitutions, (U(P)CGU(P))(5)TT, produces a complex with intermediate stability. It appears that, although adjacent U(P) x AT triplets form stable triplexes, placing U(P) x AT adjacent to C(+) x GC is unfavorable. Similar results were obtained with fragments containing CG inversions within the oligopurine tract, though triplexes at (AAAAAC)(3)AA were only detected in the presence of a triplex-binding ligand. Placing C(+) x GC on the 5'-side of T x CG triplets also reduces triplex formation, while a 3'-C(+) x GC produces complexes with increased stability.     

DNA triple helix formation at oligopurine sites containing multiple contiguous pyrimidines.

We have used DNase I footprinting to assess the formation of triple helices at 15mer oligopurine target sites which are interrupted by several (up to four) adjacent central pyrimidine residues. Third strand oligonucleotides were designed to generate complexes containing central (X.TA)nor (X.CG)n triplets (X = each base in turn) surrounded by C+.GC and T.AT triplets. It has previously been shown that G.TA and T.CG are the most stable triplets for recognition of single TA and CG interruptions. We show that these triplets are the most useful for recognizing consecutive pyrimidine interruptions and find that addition of each pyrimidine residue leads to a 30-fold decrease in third strand affinity. The addition of 10 microM naphthylquinoline triplex-binding ligand stabilizes each complex so that all the oligonucleotides produce footprints at similar concentrations (0.3 microM). Targets containing two pyrimidines are only bound by oligonucleotides generating (G.TA)2 and (T.CG)2 with a further 30-fold decrease in affinity. (G.TA)2 is slightly more stable than (T.CG)2. In the presence of the triplex-binding ligand the order of stability is (G.TA)2 > (C.TA)2 > (T.TA)2 > (A.TA)2 and (T.CG)2 > (C.CG)2 > (G.CG)2 = (A.CG)2. No oligonucleotide footprints are generated at target sites containing three consecutive pyrimidines, though addition of 10 microM triplex-binding ligand produces stable complexes with oligonucleotides generating (G.TA)3, (T.CG)3 and (C.CG)3, with a further 30-fold reduction in affinity. No footprints are generated at targets containing four Ts, though the ligand induces a weak interaction with the oligonucleotide generating (T.CG)4.     

Comparison of antiparallel A.AT and T.AT triplets within an alternate strand DNA triple helix.

We have examined the formation of alternate strand triple-helices at the target sequence A11(TC)6.(GA)6T11 using the oligonucleotides T11(AG)6 and T11(TG)6, by DNase I footprinting. These third strands were designed so as to form parallel T.AT triplets together with antiparallel G.GC and A.AT or T.AT triplets. We find that, although both oligonucleotides yield clear footprints at similar concentrations (0.3 microM) in the presence of manganese, only T11(TG)6 forms a stable complex in magnesium-containing buffers, albeit at a higher concentration (10-30 microM). Examination of the interaction of (AG)6 and (TG)6 with half the target site confirmed that the complex containing A.AT triplets was only stable in the presence of manganese. In contrast no binding of (TG)6 was detected in the presence of either metal ion, suggesting that the reverse-Hoogsteen T.AT triplet is less stable that G.GC. We suggest that, within the context of G.GC triplets, the rank order of antiparallel triplet stability is A.AT (Mn2+) > T.AT (Mn2+) > T.AT (Mg2+) > A.AT (Mg2+). Third strands containing a single base substitution in the centre of either the parallel or antiparallel portion showed a (10-fold) weaker interaction in manganese-containing buffers, and no interaction in the presence of magnesium.     

Base triplet nonisomorphism strongly influences DNA triplex conformation: effect of nonisomorphic G* GC and A* AT triplets and bending of DNA triplexes

Structural understanding of DNA triplexes is grossly inadequate despite their efficacy as therapeutic agents. Lack of structural similarity (isomorphism) of base triplets that figure in different DNA triplexes brings in an added complexity. Recently, we have shown that the residual twist (Deltat degrees ) and the radial difference (Deltar A) adequately define base triplet nonisomorphism in structural terms and allow assessment of their role in conferring stability as well as sequence-dependent structural variations in DNA triplexes. To further corroborate these, molecular dynamics (MD) simulations are carried out on DNA triplexes comprising nonisomorphic G* GC and A* AT base triplets under different sequential contexts. Base triplet nonisomorphism between G* GC and A* AT triplets is dominated by Deltat degrees (9.8 degrees ), in view of small Deltar (0.2 A), and is in contrast to G* GC and T* AT triplets where both Deltat degrees (10.6 degrees ) and Deltar (1.1A) are prominent. Results show that Deltat degrees alone enforces mechanistic influence on the triplex-forming purine strand so as to favor a zigzag conformation with alternating conformational features that include high (40 degrees ) and low (20 degrees ) helical twists, and high anti(G) and anti(A) glycosyl conformation. Higher thermal stability of this triplex compared to that formed with G* GC and T* AT triplets can be traced to enhanced base-stacking and counterion interactions. Surprisingly, it is found for the first time that the presence of a nonisomorphic G* GC or A* AT base triplet interrupting an otherwise mini A* AT or G* GC isomorphic triplex can induce a bend/curvature in a DNA triplex. These observations should prove useful in the design of triplex-forming oligonucleotides and in the understanding the binding affinities of this triplex with proteins.     

Relative stability of triplexes containing different numbers of T.AT and C+.GC triplets.

We have used DNase I footprinting to compare the stability of parallel triple helices containing different numbers of T.AT and C+. GC triplets. We have targeted a fragment containing the 17mer sequence 5'-AGGAAGAGAAAAAAGAA with the 9mer oligonucleotides 5'-TCCTTCTCT, 5'-TTCTCTTTT and 5'-TTTTTTCTT, which form triplexes at the 5'-end, centre and 3'-end of the target site respectively. Quantitative DNase I footprinting has shown that at pH 5.0 the dissociation constants of these oligonucleotides are 0.13, 4.7 and >30 microM respectively, revealing that increasing the proportion of C+.GC triplets increases triplex stability. The results suggest that the positive charge on the protonated cytosine contributes to triplex stability, either by a favourable interaction with the stacked pisystem or by screening the charge on the phosphate groups. In the presence of a naphthylquinoline triplex binding ligand all three oligonucleotides bind with similar affinities. At pH 6.0 these triplexes only form in the presence of the triplex binding ligand, while at pH 7.5 footprints are only seen with the oligonucleotide which generates the fewest number of C+.GC triplets (TTTTTTCTT) in the presence of the ligand.     

Recognition of base pair inversions in duplex by chimeric (alpha,beta) triplex-forming oligonucleotides

DNA recognition by triplex-forming oligonucleotides (TFOs) is usually limited by homopurine-homopyrimidine sequence in duplexes. Modifications of the third strand may overcome this limitation. Chimeric alpha-beta TFOs are expected to form triplex DNA upon binding to non-regular sequence duplexes. In the present study we describe binding properties of chimeric alpha-beta oligodeoxynucleotides in the respect to short DNA duplexes with one, three, and five base pair inversions. Non-natural chimeric TFO's contained alpha-thymidine residues inside (GT) or (GA) core sequences. Modified residues were addressed to AT/TA inversions in duplexes. It was found in the non-denaturing gel-electrophoresis experiments that single or five adjacent base pair inversions in duplexes may be recognized by chimeric alpha-beta TFO's at 10 degrees C and pH 7.8. Three dispersed base pair inversions in the double stranded DNA prevented triplex formation by either (GT) or (GA) chimeras. Estimation of thermal stability of chimeric alpha-beta triplexes showed decrease in T(m) values as compared with unmodified complexes.     

Effects of an abasic site on triple helix formation characterized by affinity cleaving.

The stability of triple helical complexes of pyrimidine oligodeoxyribonucleotides containing one abasic 1,2-dideoxy-D-ribose (phi) residue was examined by affinity cleaving. Within a pyrimidine third strand, the triplets phi.AT, phi.GC, phi.TA and phi.CG are significantly less stable than the triplets, T.AT, C+GC and G.TA. The decrease in binding produced by an abasic residue is similar to that observed with imperfectly matched natural base triplets, with phi.AT and phi.GC being less stable than phi.TA and phi.CG triplets for the sequences studied.     

NMR structural studies of intramolecular (Y+)n.(R+)n(Y-)nDNA triplexes in solution: imino and amino proton and nitrogen markers of G.TA base triple formation.

We reported previously on NMR studies of (Y+)n.(R+)n(Y-)n DNA triple helices containing one oligopurine strand (R)n and two oligopyrimidine strands (Y)n stabilized by T.AT and C+.GC base triples [de los Santos, C., Rosen, M., & Patel, D. J. (1989) Biochemistry 28, 7282-7289]. Recently, it has been established that guanosine can recognize a thymidine.adenosine base pair to form a G.TA triple in an otherwise (Y+)n.(R+)n(Y-)n triple-helix motif. [Griffin, L. C., & Dervan, P. B. (1989) Science 245, 967-971]. The present study extends the NMR research to the characterization of structural features of a 31-mer deoxyoligonucleotide that folds intramolecularly into a 7-mer (Y+)n.(R+)n(Y-)n triplex with the strands linked through two T5 loops and that contains a central G.TA triple flanked by T.AT triples. The G.TA triplex exhibits an unusually well resolved and narrow imino and amino exchangeable proton and nonexchangeable proton spectrum in H2O solution, pH 4.85, at 5 degrees C. We have assigned the imino protons of thymidine and amino protons of adenosine involved in Watson-Crick and Hoogsteen pairing in T.AT triples, as well as the guanosine imino and cytidine amino protons involved in Watson-Crick pairing and the protonated cytidine imino and amino protons involved in Hoogsteen pairing in C+.GC triples in the NOESY spectrum of the G.TA triplex. The NMR data are consistent with the proposed pairing alignment for the G.TA triple where the guanosine in an anti orientation pairs through a single hydrogen bond from one of its 2-amino protons to the 4-carbonyl group of thymidine in the Watson-Crick TA pair.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flanking sequence effects within the pyrimidine triple-helix motif characterized by affinity cleaving.

Nearest neighbor interactions affect the stabilities of triple-helical complexes. Within a pyrimidine triple-helical motif, the relative stabilities of natural base triplets T.AT, C + GC, and G.TA, as well as triplets, D3.TA and D3.CG, containing the nonnatural deoxyribonucleoside 1-(2-deoxy-beta-D-ribofuranosyl)-4-(3-benzamido)phenylimidazole (D3) were characterized by the affinity cleaving method in the context of different flanking triplets (T.AT, T.AT: T.AT, C + GC: C + GC, T.AT: G + GC, C + GC). The to be insensitive to substitutions in either the 3' or 5' directions, while the relative stabilities of triple helices containing C + GC triplets decreased as the number of adjacent C + GC triplets increased. Triple helices incorporating a G.TA interaction were most stable when this triplet was flanked by two T.AT triplets and were adversely affected when a C + GC triplet was placed in the adjacent 5' direction. Similarly, complexes containing D3.TA or D3.CG triplets were most stable when the triplet was flanked by two T.AT triplets but were destabilized when the adjacent 3' neighbor position was occupied with a C + GC triplet. This information regarding sequence composition effects in triple-helix formation establishes a set of guidelines for targeting sequences of double-helical DNA by the pyrimidine triple-helix motif.     

Alternate strand recognition of double-helical DNA by (T,G)-containing oligonucleotides in the presence of a triple helix-specific ligand.

Triple helix formation requires a polypurine- polypyrimidine sequence in the target DNA. Recent works have shown that this constraint can be circumvented by using alternate strand triplex-forming oligonucleotides. We have previously demonstrated that (T,G)-containing triplex- forming oligonucleotides may adopt a parallel or an antiparallel orientation with respect to an oligopurine target, depending upon the sequence and, in particular, upon the number of 5'-GpT-3' and 5'-TpG-3' steps [Sun et al. (1991) C.R. Acad. Sci. Paris Ser III, 313, 585-590]. A single (T,G)-containing oligonucleotide can therefore interact with two oligopurine stretches which alternate on the two strands of the target DNA. The (T,G) switch oligonucleotide contains a 5'-part targeted to one of the oligopurine sequences in a parallel orientation followed by a 3'-part that adopts an antiparallel orientation with respect to the second oligopurine sequence. We show that a limitation to the stability of such a triplex may arise from the instability of the antiparallel part, composed of reverse-Hoogsteen C.GxG and T.AxT base triplets. Using DNase I footprinting and ultraviolet absorption experiments, we report that a benzo[e]pyridoindole derivative [(3-methoxy- 7H-8-methyl-11-[(3'-amino-propyl) amino] benzo[e]pyrido [4,3-b]indole (BePI)], a drug interacting more tightly with a triplex than with a duplex DNA, strongly stabilizes triplexes with reverse-Hoogsteen C.GxG and T.AxT triplets thus allowing a stabilization of the triplex-forming switch (T,G) oligonucleotide on alternating oligopurine- oligopyrimidine 5'-(Pu)14(Py)14-3' duplex sequences. These results lead to an extension of the range of oligonucleotide sequences for alternate strand recognition of duplex DNA.     

Thermodynamic and kinetic stability of intermolecular triple helices containing different proportions of C+*GC and T*AT triplets

We have used oligonucleotides containing appropriately placed fluorophores and quenchers to measure the stability of 15mer intermolecular triplexes with third strands consisting of repeats of TTT, TTC, TCC and TCTC. In the presence of 200 mM sodium (pH 5.0) triplexes that contain only T·AT triplets are unstable and melt below 30°C. In contrast, triplets with repeats of TTC, TCC and CTCT melt at 67, 72 and 76°C, respectively. The most stable complex is generated by the sequence containing alternating C⁺·GC and T·AT triplets. All four triplexes are stabilised by increasing the ionic strength or by the addition of magnesium, although triplexes with a higher proportion of C⁺·GC triplets are much less sensitive to changes in the ionic conditions. The enthalpies of formation of these triplexes were estimated by examining the concentration dependence of the melting profiles and show that, in the presence of 200 mM sodium at pH 5.0, each C⁺·GC triplet contributes about 30 kJ mol^–1, while each T·AT contributes only 11 kJ mol^–1. Kinetic experiments with these oligonucleotides show that in 200 mM sodium (pH 5.0) repeats of TCC and TTC have half-lives of ~20 min, while the triplex with alternating C⁺·GC and T·AT triplets has a half-life of ~3 days. In contrast, the dissociation kinetics of the triplex containing only T·AT are too fast to measure.     

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.     

Analysis of GAA/TTC DNA triplexes using nuclear magnetic resonance and electrospray ionization mass spectrometry

The formation of a GAA/TTC DNA triplex has been implicated in Friedreich's ataxia. The destabilization of GAA/TTC DNA triplexes either by pH or by binding to appropriate ligands was analyzed by nuclear magnetic resonance (NMR) and positive-ion electrospray mass spectrometry. The triplexes and duplexes were identified by changes in the NMR chemical shifts of H8, H1, H4, 15N7, and 15N4. The lowest pH at which the duplex is detectable depends upon the overall stability and the relative number of Hoogsteen C composite function G to T composite function A basepairs. A melting pH (pHm) of 7.6 was observed for the destabilization of the (GAA)2T4(TTC)2T4(CTT)2 triplex to the corresponding Watson-Crick duplex and the T4(CTT)2 overhang. The mass spectrometric analyses of (TTC)6.(GAA)6 composite function(TTC)6 triplex detected ions due to both triplex and single-stranded oligonucleotides under acidic conditions. The triplex ions disappeared completely at alkaline pH. Duplex and single strands were detectable only at neutral and alkaline pH values. Mass spectrometric analyses also showed that minor groove-binding ligands berenil, netropsin, and distamycin and the intercalating ligand acridine orange destabilize the (TTC)6.(GAA)6 composite function (TTC)6 triplex. These NMR and mass spectrometric methods may function as screening assays for the discovery of agents that destabilize GAA/TTC triplexes and as general methods for the characterization of structure, dynamics, and stability of DNA and DNA-ligand complexes.     

Coralyne has a preference for intercalation between TA.T triples in intramolecular DNA triple helices.

Intercalating ligands may improve both the stability and sequence specificity of triple helices. Numerous intercalating drugs have been described, including coralyne, which preferentially binds triple helices, though its sequence specificity has been reported to be low [Lee,J.S., Latimer,L.J.P. and Hampel,K.J. (1993) Biochemistry , 32, 5591-5597]. In order to analyse the sequence preferences of coralyne we have used a combination of DNase I footprinting, UV melting, UV-visible spectrophotometry, circular dichroism and NMR spectroscopy to examine defined intermolecular triplexes and intramolecular triplexes linked either by hexaethylene glycol chains or by octandiol chains. DNase I footprinting demonstrated that coralyne has a moderate preference for triplexes over duplexes, but a substantial preference for TA.T triplets compared with CG. C+triplets. The drug was found to have essentially no effect on the melting temperatures of duplexes of the kind d(A)n.d(T)n or d(GA)n.d(TC)n. In contrast, it increased the T m for triplexes of the kind d(T)nd(A)n.dTn, but had little effect on the stability of d(TC)nd(GA).d(CT)n at either low or high pH. On binding to DNA triplexes, there is a large change in the absorption spectrum of coralyne and also a substantial fluorescence quenching that can be attributed to intercalation. The changes in the optical spectra have been used for direct titration with DNA. For triplexes d(T)6d(A)6.d(T)6, the Kd at 298 K was 0.5-0.8 microM. In contrast, the affinity for d(TC) nd(GA)n.d(CT)n triplexes was 6- to 10-fold lower and was characterized by smaller changes in the absorption and CD spectra. This indicates a preference for intercalation between TAT triples over CG.C+/TA.T triples. NMR studies confirmed interaction by intercalation. However, a single, secondary binding was observed at high concentrations of ligand to the triplex d(AGAAGA-L-TCTTCT-L-TCTTCT), presumably owing to the relatively low difference in affinity between the TA.T site and the competing, neighbouring sites.     

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.     

Site-resolved energetics in DNA triple helices containing G*TA and T*CG triads

Recognition of specific sites in double-helical DNA by triplex-forming oligonucleotides has been limited until recently to sites containing homopurine-homopyrimidine sequences. G*TA and T*CG triads, in which TA and CG base pairs are specifically recognized by guanine or by thymine, have now extended this recognition code to DNA target sites of mixed base sequences. In the present work, we have obtained a characterization of the stabilities of G*TA and T*CG triads, and of the effects of these triads upon canonical triads, in triple-helical DNA. The three DNA triplexes investigated are formed by the folding of the 31-mers d(GAAXAGGT(5)CCTYTTCT(5)CTTZTCC) with X = G, T, or C, Y = C, A, or G, and Z = C, G, or T. We have measured the exchange rates of imino protons in each triad of the three triplexes using nuclear magnetic resonance spectroscopy. The exchange rates are used to map the local free energy of structural stabilization in each triplex. The results indicate that the stability of Watson-Crick base pairs in the G*TA and T*CG triads is comparable to that of Watson-Crick base pairs in canonical triads. The presence of G*TA and T*CG triads, however, destabilizes neighboring canonical triads, two or three positions removed from the G*TA/T*CG site. Moreover, the long-range destabilizing effects induced by the T*CG triad are larger than those induced by the G*TA triad. These findings reveal the molecular basis for the lower overall stability of G*TA- and T*CG-containing triplexes.     

Footprinting studies of DNA-sequence recognition by nogalamycin.

We have studied the DNA sequence binding preference of the antitumour antibiotic nogalamycin by DNase-I footprinting using a variety of DNA fragments. The DNA fragments were obtained by cloning synthetic oligonucleotides into longer DNA fragments and were designed to contain isolated ligand-binding sites surrounded by repetitive sequences such as (A)n.(T)n and (AT)n. Within regions of (A)n.(T)n, clear footprints are observed with low concentrations of nogalamycin (< 5 microM), with apparent binding affinities for tetranucleotide sequences which decrease in the order TGCA > AGCT = ACGT > TCGA. In contrast, within regions of (AT)n, the ligand binds best to AGCT; binding to TCGA and TGCA is no stronger than to alternating AT. Within (ATT)n, the preference is for ACGT > TCGA. Although each of these binding sites contains all four base pairs, there is no apparent consensus sequence, suggesting that the selectivity is affected by local DNA dynamic and structural effects. At higher drug concentrations (> 25 microM), nogalamycin prevents DNAse-I cleavage of (AT)n but shows no interaction with regions of (AC)n.(GT)n. Regions of (A)n.(T)n, which are poorly cut by DNase I, show enhanced rates of cleavage in the presence of low concentrations of nogalamycin, but are protected from cleavage at higher concentrations. We suggest that this arises because drug binding to adjacent regions distorts the DNA to a structure which is more readily cut by the enzyme and which is better able to bind further ligand molecules.     

DNA sequence specificity of a naphthylquinoline triple helix-binding ligand.

We have examined the effect of a naphthylquinoline triplex-binding ligand on the formation of intermolecular triplexes on DNA fragments containing the target sites A6G6xC6T6 and G6A6xT6C6. The ligand enhances the binding of T6C2, but not T2C6, to A6G6xC6T6 suggesting that it has a greater effect on TxAT than C+xGC triplets. The complex with T6C2 is only stable below pH 6.0, confirming the requirement for protonation of the third strand cytosines. Antiparallel triplexes with GT-containing oligonucleotides are also stabilised by the ligand. The complex between G5T5 and A6G6xC6T6 is stabilised by lower ligand concentrations than that between T5G5 and G6A6xC6T6. The ligand does not promote the interaction with GT-containing oligonucleotides which have been designed to bind in a parallel orientation. Although the formation of antiparallel triplexes is pH independent, we find that the ligand has a greater stabilising effect at lower pH, suggesting that the active species is protonated. The ligand does not promote the binding of antiparallel GA-containing oligonucleotides at pH 7.5 but induces the interaction between A5G5 and G6A6xT6C6 at pH 5.5. Ethidium bromide does not promote the formation of any of these triplexes and destabilises the interaction of acridine-linked pyrimidine-containing third strands with these target sites.     

Intramolecular triple-helix formation at (PunPyn).(PunPyn) tracts: recognition of alternate strands via Pu.PuPy and Py.PuPy base triplets.

Triple-helical DNA shows increasing potential for applications in the control of gene expression (including therapeutics) and the development of sequence-specific DNA-cleaving agents. The major limitation in this technology has been the requirement of homopurine sequences for triplex formation. We describe a simple approach that relaxes this requirement, by utilizing both Pu.PuPy and Py.PuPy base triplets to form a continuous DNA triple helix at tandem oligopurine and oligopyrimidine tracts. [Triplex formation at such a sequence has been previously demonstrated only with the use of a special 3'-3' linkage in the third strand [Horne, D. A., & Dervan, P. B. (1990) J. Am. Chem. Soc. 112, 2435-2437].] Supporting evidence is from chemical probing experiments performed on several oligonucleotides designed to form 3-stranded fold-back structures. The third strand, consisting of both purine and pyrimidine blocks, pairs with purines in the Watson-Crick duplex, switching strands at the junction between the oligopurine and oligopyrimidine blocks but maintaining the required strand polarity without any special linkage. Although Mg2+ ions are not required for the formation of Pu.PuPy base triplets, they show enhanced stability in the presence of Mg2+. In the sequences observed. A.AT triplets appear to be more stable than G.GC triplets. As expected, triplex formation is largely independent of pH unless C+.GC base triplets are required.     

Novel Hoogsteen-like Bases for Recognition of the C-G Base Pair by DNA Triplex Formation

Effective sequence-specific recognition of duplex DNA is possible by triplex formation with natural oligonucleotides via Hoogsteen H-bonding. However, triplex formation is in practice limited to pyrimidine oligonucleotides that bind duplex A-T or G-C base pair DNA sequences specifically at homopurine sites in the major groove as T·A-T and C⁺ ·G-C triplets. Here we report the successful modelling of novel unnatural nucleosides that recognize the C-G DNA base pair by Hoogsteen-like major groove interaction. These novel Hoogsteen nucleotides are examined within model A-type and B-type conformation triplex structures since the DNA triplex can be considered to incorporate A-type and/or B-type configurational properties. Using the same deoxyribose-phosphodiester and base-deoxyribose dihedral angle configuration, a triplet comprised of a C-G base pair and the novel Hoogsteen nucleotide, Y2, replaces the central T·A-T triplet in the triplex. The presence of any structural or energetic perturbations due to the central triplet in the energy-minimized triplex is assessed with respect to the unmodified energy minimized (T·A-T)₁₁ starting structures. Incorporation of this novel triplet into both A-type and B-type natural triplex structures provokes minimal change in the configuration of the central and adjacent triplets.