Oligonucleotide-directed triple helix formation at adjacent oligopurine and oligopyrimidine DNA tracts by alternate strand recognition.

A significant limitation to the practical application of triplex DNA is its requirement for oligopurine tracts in target DNA sequences. The repertoire of triplex-forming sequences can potentially be expanded to adjacent blocks of purines and pyrimidines by allowing the third strand to pair with purines on alternate strands, while maintaining the required strand polarities by combining the two major classes of base triplets, Py.PuPy and Pu.PuPy. The formation of triplex DNA in this fashion requires no unusual bases or backbone linkages on the third strand. This approach has previously been demonstrated for target sequences of the type 5'-(Pu)n(Py)n-3' in intramolecular complexes. Using affinity cleaving and DNase I footprinting, we show here that intermolecular triplexes can also be formed at both 5'-(Pu)n(Py)n-3' and 5'-(Py)n(Pu)n-3' target sequences. However, triplex formation at a 5'-(Py)n(Pu)n-3' sequence occurs with lower yield. Triplex formation is disfavored, even at acid pH, when a number of contiguous C+.GC base triplets are required. These results suggest that triplex formation via alternate strand recognition at sequences made up of blocks of purines and pyrimidines may be generally feasible.     

RecQ and RecG helicases have distinct roles in maintaining the stability of polypurine.polypyrimidine sequences

DNA triplex structures can block the replication fork and result in double-stranded DNA breaks (DSBs). RecQ and RecG helicases may be important for replication of such sequences as RecQ resolves synthetic triplex DNA structures and RecG mediates replication restart by fork regression. Primer extension on an 88bp triplex-forming polypurine.polypyrimidine (Pu.Py) tract from the PKD1 gene demonstrated that RecQ, but not RecG, facilitated primer extension by T7 DNA polymerase. A high-throughput, dual plasmid screening system using isogenic bacterial lines deficient in RecG, RecQ, or both, revealed that RecQ deficiency increased mutation to sequence flanking this 88bp tract by eight to ten-fold. Although RecG facilitated small deletions in an 88bp mirror repeat-containing sequence, it was absolutely required to maintain a 2.5kb Pu.Py tract containing multiple mirror repeats. These results support a two-tiered model where RecQ facilitates fork progression through triplex-forming tracts and, failing processivity, RecG is critical for replication fork restart.     

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.     

Suicidal nucleotide sequences for DNA polymerization.

Studying the activity of T7 DNA polymerase (Sequenase) on open circular DNAs, we observed virtually complete termination within potential triplex-forming sequences. Mutations destroying the triplex potential of the sequences prevented termination, while compensatory mutations restoring triplex potential restored it. We hypothesize that strand displacement during DNA polymerization of double-helical templates brings three DNA strands (duplex DNA downstream of the polymerase plus a displaced overhang) into close proximity, provoking triplex formation, which in turn prevents further DNA synthesis. Supporting this idea, we found that Sequenase is unable to propagate through short triple-helical stretches within single-stranded DNA templates. Thus, DNA polymerase, by inducing triplex formation at specific sequences in front of the replication fork, causes self-termination. Possible biological implications of such 'conformational suicide' are discussed. Our data also provide a novel way to target DNA polymerases at specific sequences using triplex-forming oligonucleotides.     

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.     

Presence of divalent cation is not mandatory for the formation of intramolecular purine-motif triplex containing human c-jun protooncogene target

Modulation of endogenous gene function, through sequence-specific recognition of double helical DNA via oligonucleotide-directed triplex formation, is a promising approach. Compared to the formation of pyrimidine motif triplexes, which require relatively low pH, purine motif appears to be the most gifted for their stability under physiological conditions. Our previous work has demonstrated formation of magnesium-ion dependent highly stable intermolecular triplexes using a purine third strand of varied lengths, at the purine?pyrimidine (Pu?Py) targets of SIV/HIV-2 (vpx) genes (Svinarchuk, F., Monnot, M., Merle, A., Malvy, C., and Fermandjian, S. (1995) Nucleic Acids Res. 23, 3831-3836). Herein, we show that a designed intramolecular version of the 11-bp core sequence of the said targets, which also constitutes an integral, short, and symmetrical segment (G(2)AG(5)AG(2))?(C(2)TC(5)TC(2)) of human c-jun protooncogene forms a stable triplex, even in the absence of magnesium. The sequence d-C(2)TC(5)TC(2)T(5)G(2)AG(5)AG(2)T(5)G(2)AG(5)AG(2) (I-Pu) folds back twice onto itself to form an intramolecular triple helix via a double hairpin formation. The design ensures that the orientation of the intact third strand is antiparallel with respect to the oligopurine strand of the duplex. The triple helix formation has been revealed by non-denaturating gel assays, UV-thermal denaturation, and circular dichroism (CD) spectroscopy. The monophasic melting curve, recorded in the presence of sodium, represented the dissociation of intramolecular triplex to single strand in one step; however, the addition of magnesium bestowed thermal stability to the triplex. Formation of intramolecular triple helix at neutral pH in sodium, with or without magnesium cations, was also confirmed by gel electrophoresis. The triplex, mediated by sodium alone, destabilizes in the presence of 5'-C(2)TC(5)TC(2)-3', an oligonucleotide complementary to the 3'-oligopurine segments of I-Pu, whereas in the presence of magnesium the triplex remained impervious. CD spectra showed the signatures of triplex structure with A-like DNA conformation. We suggest that the possible formation of pH and magnesium-independent purine-motif triplexes at genomic Pu?Py sequences may be pertinent to gene regulation.     

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.     

Triplex formation by oligonucleotides containing novel deoxycytidine derivatives.

Homopurine sequences of duplex DNA are binding sites for triplex-forming oligodeoxyribopyrimidines. The interactions of synthetic duplex DNA targets with an oligodeoxyribopyrimidine containing N4-(6-amino-2-pyridinyl)deoxycytidine (1), a nucleoside designed to interact with a single C-G base pair interruption of the purine target tract, was studied by UV melting, circular dichroism spectroscopy and dimethylsulfate alkylation experiments. Nucleoside 1 supports stable triplex formation at pH 7.0 with formation of a 1-Y-Z triad, where Y-Z is a base pair in the homopurine tract of the target. Selective interaction was observed when Y-Z was C-G, although A-T and, to a lesser extent, T-A and G-C base pairs were also recognized. The circular dichroism spectra of the triplex having a 1-C-G triad were similar to those of a triplex having a C(+)-G-C triad, suggesting that the overall structures of the two triplexes are quite similar. Removal of the 6-amino group from 1 essentially eliminated triplex formation. Reaction of a triplex having the 1-C-G triad with dimethylsulfate resulted in a 50% reduction of methylation of the G residue of this triad. In contrast, the G of a similar triplex containing a U-C-G triad was not protected from methylation by dimethylsulfate. These results are consistent with a binding mode in which the 6-amino-2-pyridinyl group of 1 spans the major groove of the target duplex at the 1-C-G binding site and forms a hydrogen bond with the O6 of G. An additional stabilizing hydrogen bond could form between the N4 of the imino tautomer of 1 and the N4 amino group of C.     

In vivo persistence of DNA triple helices containing psoralen-conjugated oligodeoxyribonucleotides.

Triple helices represent an attractive method for modulating specific gene expression. In particular, cross-linking between a triplex-forming oligonucleotide (TFO) and its duplex DNA target, typically through the formation of psoralen photoadducts, allows efficient blocking of elongation by RNA polymerases in vitro. However, in vivo, this approach is limited by DNA repair of the photoadduct. Here we describe the use of an oligodeoxyribonucleotide 19mer psoralen-modified TFO to form covalent linkages between an oligonucleotide and both strands of the targeted duplex DNA, thereby efficiently blocking expression of a luciferase reporter gene. Most importantly, we demonstrate that both the psoralen cross-link and the purine-motif triplex remained intact for at least 72 h post-transfection, indicating that such species can persist for an extended period of time in vivo. These findings support the feasibility of an antigene approach for the therapeutic regulation of specific gene expression.     

Secondary binding sites for heavily modified triplex forming oligonucleotides

In order to enhance DNA triple helix stability synthetic oligonucleotides have been developed that bear amino groups on the sugar or base. One of the most effective of these is bis-amino-U (B), which possesses 5-propargylamino and 2′-aminoethoxy modifications. Inclusion of this modified nucleotide not only greatly enhances triplex stability, but also increases the affinity for related sequences. We have used a restriction enzyme protection, selection and amplification assay (REPSA) to isolate sequences that are bound by the heavily modified 9-mer triplex-forming oligonucleotide B₆CBT. The isolated sequences contain A_n tracts (n = 6), suggesting that the 5′-end of this TFO was responsible for successful triplex formation. DNase I footprinting with these sequences confirmed triple helix formation at these secondary targets and demonstrated no interaction with similar oligonucleotides containing T or 5-propargylamino-dU.     

Structural polymorphism exhibited by a homopurine.homopyrimidine sequence found at the right end of human c-jun protooncogene

Homopurine·homopyrimidine (Pu·Py) tracts are likely to play important biological role in eukaryotes. Using circular dichroism, UV-thermal denaturation and gel electrophoresis, we have analyzed the structural polymorphism of a 21-bp Pu·Py DNA segment within human c-jun protooncogene 3′-region, a potential target for triplex formation. Results show that below physiological pH and in the presence of Na⁺/K⁺ with Mg²⁺ the duplex is destabilized/disproportionated, resulting in strand mediated structural transitions to the self-associated structures of G- and C-rich strands separately, identified as G-quadruplex and i-motif species. A significant differential behavior of the monovalent cations was observed, accordingly the presence of Na⁺ in acidic as well as neutral pH facilitated the duplex formation, while K⁺ favored the formation of self-associated structures. In Na⁺ and Mg²⁺, under acidic and neutral pH conditions, the duplex displayed triphasic and biphasic melting profiles, respectively. This self-association property of oligonucleotides might limit their use as duplex targets in triplex formation. Study is also relevant for understanding structural and biological properties of DNA sequence containing homopurine tracts.     

A-tract DNA disfavours triplex formation.

Optimisation of DNA triplex stability is of fundamental importance in the anti-gene strategy. In the present work, thermal denaturation studies by UV-spectrophotometry and structural and dynamical characterizations by NMR spectroscopy have been used systematically to investigate the effects on triplex stability of isolated insertions of different base triplets into an otherwise homogeneous 15-mer dT x dA-dT oligo-triplex. It is found that insertion of a single central C(+) x G-C or T x D-T triplet (D=2,6-diaminopurine) leads to a pronounced stabilization (up to 20 deg. C if the cytosine base is C5 methylated) at acidic as well as neutral pH. To a smaller degree, this is the case also for a C(+) x I-C triplet insertion.Using imino proton exchange measurements, it is shown that insertion of a DT base-pair in the underlying duplex perturbs the intrinsic A-tract structure in the same way as has been shown for a GC insert. We propose that the intrinsic properties of A-tract duplex DNA (e. g. high propeller twist and rigidity) are unfavourable for triplex formation and that GC- or DT-inserts stabilize the triplex by interfering with the A-tract features of the underlying duplex. The C(+) x I-C triplet without the N2 amino group in the minor groove is readily accommodated within the typical, highly propeller-twisted A-tract structure. This might be related to its smaller effect on the stability of the corresponding triplex.These results may be valuable for understanding DNA triplex formation in vivo as well as for the design of efficient triplex-forming oligonucleotides and in choosing suitable target sequences in the anti-gene strategy.     

Factors influencing the extent and selectivity of alkylation within triplexes by reactive G/A motif oligonucleotides.

G/A motif triplex-forming oligonucleotides (TFOs) complementary to a 21 base pair homopurine/homopyrimidine run were conjugated at one or both ends to chlorambucil. These TFOs were incubated with several synthetic duplexes containing the targeted homopurine run flanked by different sequences. The extent of mono and interstrand cross-linking was compared with the level of binding at equilibrium. Covalent modification took place within a triple-stranded complex and usually occurred at guanine residues in the flanking double-stranded DNA. The efficiency of alkylation was dependent upon the sequence of the flanking duplex, the solution conditions, and the rate of triplex formation relative to the rate of chlorambucil reaction. Self-association of the TFOs as parallel duplexes was demonstrated and this did not interfere with triple strand formation. With an optimal target, cross-linking of the triplex was very efficient when incubation was carried in a physiological buffer supplemented with the triplex selective intercalator coralyne.     

6-Oxocytidine a novel protonated C-base analogue for stable triple helix formation.

2'-O-Methyl-3'-O-phosphoramidite building blocks of 6-oxocytidine 6 and its 5-methyl derivative 7, respectively, were synthesized and incorporated via phosphoramidite chemistry in 15 mer oligodeoxynucleotides [d(T72T7), S2; d(T73T7), S3] to obtain potential Py.Pu.Py triplex forming homopyrimidine strands. UV thermal denaturation studies and CD spectroscopy of 1:1 mixtures of these oligomers and a 21 mer target duplex [d(C3A7GA7C3)-d(G3T7CT7G3), D1] with a complementary purine tract showed a nearly pH-independent (6.0-8.0) triple helix formation with melting temperatures of 21-19 degrees C and 18.5-17.5 degrees C, respectively (buffer system: 50 mM sodium cacodylate, 100 mM NaCl, 20 mM MgCl2). In contrast, with the corresponding 15mer deoxy-C-containing oligonucleotide [d(T(7)1T7), S1] triplex formation was observed only below pH 6.6. Specificity for the recognition of Watson-Crick GC-base pairs was observed by pairing the modified C-bases of the 15mers with all other possible Watson-Crick-base compositions in the target duplex [d(C3A7XA7C3)-d(G3T7YT7G3), X = A,C,T; Y = T,G,A, D2-4]. Additionally, the Watson-Crick-pairing of the modified oligomers S2 and S3 was studied.     

A stable complex between homopyrimidine oligomers and the homologous regions of duplex DNAs

When plasmid DNA duplexes carrying the regular homopurine-homopyrimidine inserts (dGA)n, (dTC)n and (dG)n, (dC)n are preincubated with homologous labeled oligo(dPy) ((dTC)n and (dC)n respectively) at acid pH, the label co-electrophoreses with the duplex DNA. Thus, a very strong complex is formed. Complementary oligo(dPu) does not form a complex under these conditions. No binding is observed for oligo(dPy) with non-homologous inserts as well as with vector plasmids without inserts. The complex is formed equally well with linear, nicked or superhelical DNA. The complex is not detected at pH greater than 6. Complex formation leads to very little, if any, unwinding of the duplex. The observed complex appears to be the Py.Pu.Py triplex consisting of TAT and CGC base-triads with protonated cytosines. Two-dimensional gel electrophoresis patterns show that the presence of homologous oligo(dPy) destabilizes the formation of the H form.     

A psoralen-conjugated triplex-forming oligodeoxyribonucleotide containing alternating methylphosphonate-phosphodiester linkages: synthesis and interactions with DNA.

A psoralen-conjugated oligodeoxyribopyrimidine (1443), PS-pTTTTCTTTTCTTCTT, where PS is trimethylpsoralen and C is 5-methyl-2'-deoxycytidine, that contains alternating methylphosphonate-phosphodiester internucleotide linkages was synthesized. The ability of 1443 to form triple-stranded complexes with a purine tract in a synthetic DNA duplex was studied. Irradiation of solutions containing the DNA target and 10 microM 1443 or 0.25 microM of a similar psoralen-conjugated oligodeoxyribonucleotide that contained all phosphodiester linkages, (1193), with long-wavelength UV light resulted in approximately 80% formation of interstrand cross-links at pH 7.0, 37 degrees C, in the presence of 20 mM magnesium chloride. The extent of triplex formation as monitored by photo-cross-linking decreased over the pH range 5.5-8.0, and the apparent pK of the 5-methylcytosines (C) in 1443 was approximately one-half of a pH unit less than that of the 5-methylcytosines in 1193. Oligomer 1443 formed triplexes in the absence of magnesium, and maximum triplex formation was observed in solutions containing 2.5 mM magnesium, whereas maximal triplex formation by the fully charged 1193 was not observed until the magnesium concentration was 10 mM or higher. Unlike the all-phosphodiester backbone of 1193, the alternating methylphosphonate-phosphodiester backbone of 1193 is resistant to hydrolysis by exonucleases in fetal calf serum. The nuclease resistance of 1443 and its ability to form triplexes at very low magnesium concentrations suggests that triplex-forming oligomers with alternating methylphosphonate-phosphodiester backbones may be good candidates for use as antigene reagents in cell culture.     

Intermolecular triplex formation distorts the DNA duplex in the regulatory region of human papillomavirus type-11.

A conformational distortion in the DNA duplex at the regulatory region of human papillomavirus type-11 next to an intermolecular triplex, formed with a synthetic oligonucleotide, was investigated with several chemical probes. The sequence targeted for triplex formation borders on the binding sites for the regulatory proteins encoded by the viral E2 open reading frame. Dimethyl sulfate, diethyl pyrocarbonate, and OsO4 all react to a greater extent with nucleotides in the duplex that are immediately adjacent to the triplex as compared to other bases throughout the duplex. This hypermodification was observed on both the polypurine and polypyrimidine strands of the duplex DNA. Similar hyperreactivity of bases flanking a triplex also was seen when the contiguous target polypurine tract was effectively extended by mutating interrupting pyrimidines in the human papillomavirus type-11 sequence to purines. We propose that this hyperreactivity is due to a structural distortion caused by the junction between the triplex and the duplex tracts.