Synthesis and properties of triple helix-forming oligodeoxyribonucleotides containing 7-chloro-7-deaza-2'-deoxyguanosine.

Multiple incorporations of 7-chloro-7-deaza-2'-deoxyguanosine in place of 2'-deoxyguanosine have been performed into a triple helix-forming oligodeoxyribonucleotide involving a run of six contiguous guanines designed to bind in a parallel orientation relative to the purine strand of the DNA target. The ability of these modified oligodeoxyribonucleotides to form triple helices in a buffer containing monovalent cations was studied by UV--melting curves analysis, gel shift assay and restriction enzyme protection assay. In the presence of Na(+), the incorporation of two, three or five modified nucleosides in the third strand has improved the efficacy of formation of the triplex as compared to that formed with the unmodified oligonucleotide. The stabilities of the three modified triplexes were similar. The coupling of 6-chloro-2-methoxy-9-(omega-hexylamino)-acridine to the 5'-end of the oligonucleotides containing modified nucleosides led to an increase in triplex stability similar to that observed when the acridine was added to the 5'-end of the unmodified oligonucleotide. In the presence of K(+), only the oligodeoxyribonucleotides containing modified G retained the ability to form triple helices with the same efficiency. The incorporation of the modified nucleoside has two effects: (i) it decreases TFO self-association, and (ii) it slightly increases triplex stability. The enhanced ability of the modified oligonucleotides containing 7-chloro-7-deaza-2'-deoxyguanosine over the parent oligomer to form triple helices was confirmed by inhibition of restriction enzyme cleavage using a circular plasmid containing the target sequence.     

Parallel and antiparallel G*G.C base triplets in pur*pur.pyr triple helices formed with (GA) third strands.

Triple helices with G*G.C and A*A.T base triplets with third GA strands either parallel or antiparallel with respect to the homologous duplex strand have been formed in presence of Na (+) or Mg(2+) counterions. Antiparallel triplexes are more stable and can be obtained even in presence of only monovalent Na(+) counterions. A biphasic melting has been observed, reflecting third strand separation around 20 degrees C followed by the duplex -> coil transition around 63 degrees C. Parallel triplexes are far less stable than the antiparallel ones. Their formation requires divalent ions and is observed at low temperature and in high concentration conditions. Different FTIR signatures of G*G.C triplets in parallel and antiparallel triple helices with GA rich third strands have been obtained allowing the identification of such base triplets in triplexes formed by nucleic acids with heterogeneous compositions. Only S-type sugars are found in the antiparallel triplex while some N-type sugar conformation is detected in the parallel triplex.     

Physico-chemical studies on DNA triplexes containing an alternate third strand with a non-nucleotide linker

Differential scanning calorimetric (DSC), circular dichroism (CD) and molecular mechanics studies have been performed on two triple helices of DNA. The target duplex consists of 16 base pairs in alternate sequence of the type 5′-(purine)_m(pyrimidine)_m-3′. In both the triplexes, the third oligopyrimidine strand crosses the major groove at the purine–pyrimidine junction, with a simultaneous binding of the adjacent purine tracts on alternate strands of the Watson–Crick duplex. The switch is ensured by a non-nucleotide linker, the 1,2,3 propanetriol residue, that joins two 3′–3′ phosphodiester ends. The third strands differ from each other for a nucleotide in the junction region. The resulting triple helices were termed 14-mer-PXP and 15-mer-PXP (where P=phosphate and X=1,2,3-propanetriol residue) according to the number of nucleotides that compose the third strand. DSC data show two independent processes: the first corresponding to the dissociation of the third strand from the target duplex, the second to the dissociation of the double helix in two single strands. The two triple helices show the same stability at pH 6.6. At pH 6.0, the 15-mer-PXP triplex is thermodynamically more stable than the 14-mer-PXP triplex. Thermodynamic data are discussed in relation to structural models. The results are useful when considering the design of oligonucleotides that can bind in an antigene approach to the DNA for therapeutic purposes.     

Chemical modification of the third strand: differential effects on purine and pyrimidine triple helix formation.

DNA triple helices offer exciting perspectives toward oligonucleotide-directed control of gene expression. Oligonucleotide analogues are routinely used with modifications in either the backbone or the bases to form more stable triple-helical structures or to prevent their degradation in cells. In this article, different chemical modifications are tested in a model system, which sets up a competition between the purine and pyrimidine motifs. For most modifications, the DeltaH degrees of purine triplex formation is close to zero, implying a nearly temperature-independent affinity constant. In contrast, the pyrimidine triplex is strongly favored at lower temperatures. The stabilization induced by modifications previously known to be favorable to the pyrimidine motif was quantified. Interestingly, modifications favorable to the GT motif (propynyl-U and dU replacing T) were also discovered. In a system where two third strands compete for triplex formation, replacement of the GA or GT strand by a pyrimidine strand may be observed at neutral pH upon lowering the temperature. This purine-to-pyrimidine triplex conversion depends on the chemical nature of the triplex-forming strands and the stability of the corresponding triplexes.     

Synthesis and binding properties of oligo-2'-deoxyribonucleotides conjugated with triple-helix-specific intercalators: benzo[e] and benzo[g] pyridoindoles

DNA binding compounds, such as benzo[e] (BePI) and benzo[g] pyridoindole (BgPI) derivatives, exhibit preferential stabilization of triple helices. We report here the synthesis of a series of pyrimidine triple-helix-forming oligo-2'-deoxyribonucleotides conjugated with these molecules. BePI was coupled to the 5-position of 2'-deoxyuridine via two linkers of different sizes attached to its 11-position and placed at either the 5'-end, inside the sequence, or at both the 5'-end and the internal positions using periodate oxidation of a diol-containing oligonucleotide followed by reductive coupling with amino-linked BePI. The same BePI derivatives were also linked to the oligonucleotide chain via internucleotidic phosphorothiolate or phosphoramidate linkages. A mixture of diastereoisomers was prepared as well as separate pure Rp and Sp isomers. A BePI derivative, with two different linkers attached to its 3-position, and BgPI derivatives were also linked to the 5-position of a 2'-deoxyuridine located at either the 5'-end or inside the sequence, as well as to the beta- anomeric position of an additional 2'- deoxyribose placed inside the sequence. The binding properties of these oligonucleotide-benzopyridoindoles conjugates with their double-stranded DNA target was studied by absorption spectroscopy.     

Recognition of nucleic acid double helices by homopyrimidine 2', 5'-linked RNA.

We have studied the effect of a 2',5'-RNA third strand backbone on the stability of triple helices with a 'pyrimidine motif' targeting the polypurine strand of duplex DNA, duplex RNA and DNA/RNA hybrids. Comparative experiments were run in parallel with DNA and the regioisomeric RNA as third strands adopting the experimental design of Roberts and Crothers. The results reveal that 2',5'-RNA is indeed able to recognize double helical DNA (DD) and DNA (purine):RNA (pyrimidine) hybrids (DR). However, when the duplex purine strand is RNA and the duplex pyrimidine strand is DNA or RNA (i.e. RD or RR), triplex formation is not observed. These results exactly parallel what is observed for DNA third strands. Based on T m data, the affinities of 2',5'-RNA and DNA third strands towards DD and DR duplexes were similar. The RNA third strand formed triplexes with all four hairpins, as previously demonstrated. In analogy to the arabinose and 2'-deoxyribose third strands, the possible C2'- endo pucker of 2',5'-linked riboses together with the lack of an alpha-2'-OH group are believed to be responsible for the selective binding of 2',5'-RNA to DD and DR duplexes, over RR and RD duplexes. These studies indicate that the use of other oligonucleotide analogues will prove extremely useful in dissecting the contributions of backbone and/or sugar puckering to the recognition of nucleic acid duplexes.     

Spectroscopic studies on the formation and thermal stability of DNA triplexes with a benzoannulated delta-carboline-oligonucleotide conjugate

A benzoannulated delta-carboline with a phenyl substituent has been covalently tethered to the 3'-end of a triplex-forming oligonucleotide and its ability to bind and stabilize DNA triple helices has been examined by various spectroscopic methods. UV thermal melting experiments were conducted with different hairpin duplexes and with a complementary single-stranded oligonucleotide as targets for the conjugate. The delta-carboline ligand preferentially binds triplexes over duplexes and leads to a temperature increase of the triplex-to-duplex transition by up to 23 degrees C. The results obtained from UV, CD and fluorescence measurements suggest that the delta-carboline ligand exhibits specific interactions with a triplex and favors binding by intercalation at the triplex-duplex junction.     

Stable DNA triple helix formation using oligonucleotides containing 2'-aminoethoxy,5-propargylamino-U

We have prepared oligonucleotides containing the novel base analogue 2'-aminoethoxy,5-propargylamino-U in place of thymidine and examined their ability to form intermolecular and intramolecular triple helices by DNase I footprinting and thermal melting studies. The results were compared with those for oligonucleotides containing 5-propargylamino-dU and 2'-aminoethoxy-T. We find that the bis-substituted derivative produces a large increase in triplex stability, much greater than that produced by either of the monosubstituted analogues, which are roughly equipotent with each other. Intermolecular triplexes with 9-mer oligonucleotides containing three or four base modifications generate footprints at submicromolar concentrations even at pH 7.5, in contrast to the unmodified oligonucleotide, which failed to produce a footprint at pH 5.0, even at 30 microM. UV- and fluorescence melting studies with intramolecular triplexes confirmed that the bis-modified base produces a much greater increase in T(m) than either modification alone.     

DNA triplex stabilization by a delta-carboline derivative tethered to third strand oligonucleotides

A delta-carboline derivative was covalently coupled to a 7 mer oligonucleotide at its 5'- or 3'-end. The stability of triplexes formed from the conjugates and a double-helical target was studied by UV melting experiments. Compared to the unmodified control triple helices, triplexes with the conjugate exhibit a significantly higher stability. However, the degree of stabilization depends on the particular triplex structure formed.     

Oligonucleotide N3'-->P5' phosphoramidates as potential therapeutic agents

Uniformly modified nucleic acids analogues, oligonucleotide N3'-->P5' phosphoramidates, containing 3'-amino instead of 3'-hydroxyl nucleosides, were synthesized and studied. These compounds form very stable duplexes with complementary native phosphodiester DNA and exceptionally stable duplexes with RNA strands. Increases in duplex melting temperature, deltaTm, relatively to their phosphodiester counterparts, reaches 2.9-3.5 degrees C per modified nucleoside. Moreover, the phosphoramidate compounds form extremely stable triple stranded complexes with single or double stranded DNA oligomers under near physiological salt and pH conditions. Melting temperatures of these triplexes usually exceed that of the isosequential phosphodiester counterparts by up to 35 degrees C. For 11-15-mers 2'-deoxyphosphoramidates are structurally and functionally similar to the native RNA molecules and thus can be used as RNA decoys. They are resistant to enzymatic digestion by nucleases both in vitro and in vivo. Oligonucleotide phosphoramidates apparently are cell permeable, and they have a good bioavailability and biodistribution, while being non-toxic in mice at therapeutically relevant doses. Duplexes of the several studied phosphoramidates with complementary RNA strands apparently are not substrates for RNase H in vitro. Despite that, these compounds exerted high sequence-specific antisense activity in various cell lines and in SCID mice. The observed in vitro lack of RNase H recognition of the RNA:phosphoramidate duplexes may result in better specificity in biological activity of these compounds relative to RNase H inducing oligonucleotides. Experimental results also indicate that oligonucleotide phosphoramidates can be used as efficient and specific modulators of gene expression by an antigene mechanism of action. Finally, the oligo-2'-deoxyphosphoramidate double stranded complexes can structurally mimic native RNA complexes, which could be efficiently and specifically recognized by the RNA binding proteins, such as HIV-1 Rev and Tat.     

Triple helix formation with Drosophila satellite repeats. Unexpected stabilization by copper ions

The Drosophila melanogaster (AAGAGAG)(n) satellite repeat represents up to 1.5% of the entire fly genome and may adopt non-B DNA structures such as pyrimidine triple helices. UV melting and electrophoretic mobility shift assay experiments were used to monitor the stability of intermolecular triple helices as a function of size, pH, and backbone or base modification. Three to four repeats of the heptanucleotide motif were sufficient to allow the formation of a stable complex, especially when modified TFOs were used. Unexpectedly, low concentrations (40-100 microM) of Cu(2+) were found to favor strongly pyrimidine triplex formation under near-physiological conditions. In contrast, a much higher magnesium concentration was required to stabilize these triplexes significantly, suggesting that copper may be an essential stabilizing factor for pyrimidine triplexes.     

Solvent effects on model d(CG·G)7 and d(TA·T)7 DNA triple helices

Using free energy molecular mechanics, we find that the molecular effects of solvent are critical in determining relative stabilities in DNA triple helices or triplexes. The continuum solvent model is unable to differentiate the thermodynamics reflecting the basic solvation differences around the occupied major groove in triplexes. In order to avoid the local minimum problem, which is a major limitation of any modeling study, we started our computations with multiple structures rather than relying on the optimization of a single reference structure. By constructing triplex models with different initial helical twists, helical rises, and sugar-pucker permutations, we explore the potential surface and the structural preference with respect to these variations. We find that in order to accommodate a third strand in triplex formation, the backbone geometry of the B-DNA duplex target has to be adjusted into A-DNA-like form with a deep major groove. This is achieved by concerted adjustment in torsions β, ε, and ζ around the phosphate groups. However, the sugar pucker displays a more rich variation, resulting in conformations not usually associated with the canonical duplex structures. © 1995 John Wiley & Sons, Inc.     

Extensive sugar modification improves triple helix forming oligonucleotide activity in vitro but reduces activity in vivo

We are developing triple helix forming oligonucleotides (TFOs) for gene targeting. Previously, we synthesized bioactive TFOs containing 2'-O-methylribose (2'-OMe) and 2'-O-aminoethylribose (2'-AE) residues. Active TFOs contained four contiguous 2'-AE residues and formed triplexes with high thermal stability and rapid association kinetics. In an effort to further improve bioactivity, we synthesized three series of TFOs containing the 2'-AE patch and additional ribose modifications distributed throughout the remainder of the oligonucleotide. These were either additional 2'-AE residues, the conformationally locked BNA/LNA ribose with a 2'-O,4'-C-methylene bridge, or the 2'-O,4'-C-ethylene analogue (ENA). The additionally modified TFOs formed triplexes with greater thermal stability than the reference TFO, and some had improved association kinetics. However, the most active TFOs in the biochemical and biophysical assays were the least active in the bioassay. We measured the thermal stability of triplexes formed by the TFOs in each series on duplex targets containing a change in sequence at a single position. The Tm value of the variant sequence triplexes increased as the number of all additional modifications increased. A simple explanation for the failure of the improved TFOs in the bioassay was that the increased affinity for nonspecific targets lowered the effective nuclear concentration. Enhancement of TFO bioactivity will require chemical modifications that improve interaction with the specific targets while retaining selectivity against mismatched sequences.     

Oligonucleotides containing a lysine residue as 3'-3' junction for alternate strand triple helix formation

Oligonucleotides with a 3'-3' inversion of polarity site assured by one lysine residue have been synthesized, characterized and used as third strands in alternate strand triple helix formation. UV melting studies and molecular mechanics calculations have been carried out to investigate the stability and the geometry of these new triplexes.     

Evidence for a DNA triplex in a recombination-like motif: I. Recognition of Watson-Crick base pairs by natural bases in a high-stability triplex

Data are presented on a triplex type with two parallel homologous strands for which triplex formation is almost as strong as duplex formation at least for some sequences and even at pH 7 and 0.2 M NaCl. The evidence mainly rests upon comparing thermodynamic properties of similar systems. A paperclip oligonucleotide d(A12C4T12C4A12) with two linkers C4 obviously can form a triplex with parallel back-folded adenine strand regions, because the single melting transition of this complex splits in two transitions by introducing mismatches only in the third strand region. Respectively, a hairpin duplex d(A12C4T12) and a single strand d(A12) form a triplex as a 1:1 complex in which the second adenine strand is parallel oriented to the homologous one in the Watson-Crick paired duplex. In this system the melting temperature T(m) of the triplex is practically the same as that of the duplex d(A12)-d(T12), at least within a complex concentration range of 0.2-4.0 microM. The melting behaviour of complexes between triplex stabilizing ligand BePI and the system hairpin duplex plus single strand supports the triplex model. Non-denaturing gel electrophoresis suggests the existence of a triplex for a system in which five of the twelve A-T*A base triads are substituted by C-G*C base triads. The recognition between any substituted Watson-Crick base pair (X-Y) in the hairpin duplex d(A4XA7C4T7YT4) and the correspondingly replaced base (Z) in the third strand d(A4ZA7) is mutually selective. All triplexes with matching base substitutions (Z = X) have nearly the same stability (T(m) values from 29 to 33.5 degrees C), whereas triplexes with non-matching substitutions (Z not equal X) show a clearly reduced stability (T(m) values from 15 to 22 degrees C) at 2microM equimolar oligonucleotide concentration. Most nucleic acid triple helices hitherto known are limited to homopurine-homopyrimidine sequences in the target duplex. A stable triplex formation is demonstrated for inhomogeneous sequences tolerating at least 50% pyrimidine content in the homologous strands. On the basis of the surprisingly similar thermodynamic parameters for duplex and triplex, and of the fact that this triplex type seems to be more stable than many other natural DNA triplexes known, and on the basis of semiempirical and molecule mechanical calculations, we postulate bridging interactions of the third strand with the two other strands in the triplex according to the recombination motif. This triplex, denoted by us 'recombination-like form', tolerates heterogeneous base sequences.     

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.     

Spectroscopic studies of chimeric DNA-RNA and RNA 29-base intramolecular triple helices.

Fourier transform infrared (FTIR), UV absorption and exchangeable proton NMR spectroscopies have been used to study the formation and stability of two intramolecular pH-dependent triple helices composed by a chimeric 29mer DNA-RNA (DNA double strand and RNA third strand) or by the analogous 29mer RNA. In both cases decrease of pH induces formation of a triple helical structure containing either rU*dA.dT and rC+*dG.dC or rU*rA.rU and rC+*rG.rC triplets. FTIR spectroscopy shows that exclusively N-type sugars are present in the triple helix formed by the 29mer RNA while both N- and S-type sugars are detected in the case of the chimeric 29mer DNA-RNA triple helix. Triple helix formation with the third strand RNA and the duplex as DNA appears to be associated with the conversion of the duplex part from a B-form secondary structure to one which contains partly A-form sugars. Thermal denaturation experiments followed by UV spectroscopy show that a major stabilization occurs upon formation of the triple helices. Monophasic melting curves indicate a simultaneous disruption of the Hoogsteen and Watson-Crick hydrogen bonds in the intramolecular triplexes when the temperature is increased. This is in agreement with imino proton NMR spectra recorded as a function of temperature. Comparison with experiments concerning intermolecular triplexes of identical base and sugar composition shows the important role played by the two tetrameric loops in the stabilization of the intramolecular triple helices studied.     

Chemical modification of pyrimidine TFOs: effect on i-motif and triple helix formation

In order to form more stable triple helical structures or to prevent their degradation in cells, oligonucleotide analogs are routinely used, either in the backbone or among the bases. The target sequence chosen for this study is a 16-base-long oligopurine-oligopyrimidine region present in the human neurotrophin 4/5 gene. Seven different chemical modifications were tested for their effect on (i) triple helix formation and (ii) i-DNA stability. i-DNA is a tetrameric structure involving hemiprotonated C x C+ base pairs, which may act as a competing structure for triplex formation, especially in the case of a cytosine-rich third strand. At acid pH, oligophosphoramidates formed the most stable triple helix, whereas oligonucleotides including 5-propynyl-dU formed a stable i-motif which precluded triplex formation. Only two candidates stabilized triple helices at neutral pH: oligonucleotides with phosphoramidate linkage and phosphodiester oligonucleotides containing 5-methyl-dC and 5-propynyl-dU.