Positively charged oligonucleotides overcome potassium-mediated inhibition of triplex DNA formation.

The formation of triplex DNA using unmodified, purine-rich oligonucleotides (ODNs) is inhibited by physiologic levels of potassium. Changing negative phosphodiester bonds in a triplex forming oligonucleotide (TFO) to neutral linkages causes a small increase in triplex formation. When phosphodiester bonds in a TFO are converted to positively-charged linkages the formation of triplex DNA increases dramatically. In the absence of KCl, a 17mer TFO containing 11 positively-charged linkages at a concentration of 0.2 microM converts essentially all of a 30 bp target duplex to a triplex. Less than 15% of the target duplex is shifted by 2 microMolar of the unmodified TFO. In 130 mM KCl, triplex formation is undetectable using the unmodified TFO, while triplex formation is nearly complete with 2 microM positively-charged TFO. With increasing potassium, TFOs containing a higher proportion of modified linkages show enhanced triplex formation compared with those less modified. In contrast with unmodified TFOs, triplex formation with more heavily modified TFOs can occur in the absence of divalent cations. We conclude that replacement of phosphodiester bonds with positively-charged phosphoramidate linkages results in more efficient triplex formation, suggesting that these compounds may prove useful for in vivo applications.     

Triplex targeting of a native gene in permeabilized intact cells: covalent modification of the gene for the chemokine receptor CCR5.

A 12 nucleotide oligodeoxyribopurine tract in the gene for the chemokine receptor CCR5 has been targeted and covalently modified in intact cells by a 12mer triplex forming oligonucleotide (TFO) bearing a reactive group. A nitrogen mustard placed on the 5'-end of the purine motif TFO modified a guanine on the DNA target with high efficiency and selectivity. A new use of a guanine analog in these TFOs significantly enhanced triplex formation and efficiency of modification, as did the use of the triplex-stabilizing intercalator coralyne. This site-directed modification of a native chromosomal gene in intact human cells under conditions where many limitations of triplex formation have been partially addressed underscores the potential of this approach for gene control via site-directed mutagenesis.     

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 molecular beacon strategy for real-time monitoring of triplex DNA formation kinetics

We used a molecular beacon (MB) containing a 15-mer triplex-forming oligonucleotide (TFO) to probe in real-time the kinetics of triplex DNA formation in the left side of the TCl tract (502-516) of the c-src proto-oncogene in vitro. The metal ions Na+, K+, and Mg2+ stabilized triplex DNA at this site. The pseudo-first-order rate constant (kpsi) and the second-order association rate constant (k1) for the binding of the MB to the target duplex in 10 mM sodium phosphate buffer, pH 7.3, increased from 3.2 +/- 0.9 to 15 +/- 2.8 x 10(-3) s(-1) and 6.4 +/- 1.8 to 30 +/- 5.6 x 102 M(-1) s(-1), respectively, on increasing the MgCl2 concentration from 1 to 2.5 mM. Similar values were obtained for the triplex DNA stabilized by NaCl (100-250 mM). Surprisingly, the values were around 2 times higher in the presence of KCl. The AG of triplex formation in the presence of 1 mM MgCl2, 150 mM NaCl, and 150 mM KCl were -7.8 +/- 0.3, -8.2 +/- 0.3 and -8.7 +/- 0.7 kcal/mol respectively, despite significant differences in the values of deltaH and deltaS, suggesting enthalpy-entropy compensation in the stabilization of the triplex DNA by these metal ions. These results show the utility of MBs ih probing triplex DNA formation and in evaluating kinetic and thermodynamic parameters important for the design and development of TFOs as triplex DNA-based therapeutic agents.     

Sulfur signaling: is the agent sulfide or sulfane?

Triplex-forming oligonucleotides (TFOs) are sequence-dependent DNA binders that may be useful for DNA targeting and detection. A sensitive and convenient method to monitor triplex formation by a TFO and its target DNA duplex is required for the application of TFO probes. Here we describe a novel design by which triplex formation can be monitored homogeneously without prelabeling the target duplex. The design uses a TFO probe tagged with a fluorophore that undergoes fluorescence resonance energy transfer with fluorescent dyes that intercalate into the target duplex. Through color compensation analysis, the specific emission of the TFO probe reveals the status of the triple helices. We used this method to show that triple helix formation with TFOs is magnesium dependent. We also demonstrated that the TFO probe can be used for detection of sequence variation in melting analysis and for DNA quantitation in real-time polymerase chain reaction.     

Improved synthesis of daunomycin conjugates with triplex-forming oligonucleotides. The polypurine tract of HIV-1 as a target

Triple helix-forming oligonucleotides (TFOs) are promising agents for the control of gene expression, as they can selectively bind to a chosen oligopyrimidine.oligopurine region of a gene of interest thus interfering with its expression. The stability of the triplex formed by the TFO and the duplex is often too poor for successful applications of TFOs in vivo and the conjugation of a DNA intercalating moiety to the TFO is a common way to enhance the TFO affinity for its target. In a previous work, we investigated the properties of daunomycin conjugated TFO (dauno-TFO) and found that this class of compounds showed a higher degree of affinity than native oligonucleotides for an oligopyrimidine.oligopurine duplex target and that the presence of the amino sugar increases such stability. Here, we report a significantly improved synthetic procedure for the preparation of the conjugates, based on the protection of the daunosamine moiety by N-trifluoroacetylation. This protecting group is removed as a final step from the conjugation product by mild basic hydrolysis to give the desired dauno-TFO. Compared to the previous synthetic procedure, the improvement is important. The synthesis is now more reproducible and no side products are formed. Moreover, the thus protected daunomycin derivative is very stable, up to at least one year. Two dauno-TFOs, differing by the length of the oligonucleotide moiety, were prepared to target the polypurine tract (PPT) of HIV-1. Triplex formation by these compounds with model duplexes was studied by UV spectroscopy, thermal gradient gel electrophoresis (TGGE) and gel electrophoretic mobility shift. The experimental results demonstrate that dauno-TFOs bind to the PPT of HIV-1 more strongly than the unconjugated TFOs.     

DNA triplex structures are stabilized by the incorporation of 3'-endo blocked pyrimidine nucleosides in the Hoogsteen strand

A short route to pyrimidine locked nucleosides has been developed for their incorporation in triplex forming oligonucleotides (TFO). Compared to oligonucleotides built with standard nucleosides, the modified TFOs containing 3'-endo blocked residues formed, with their corresponding DNA duplexes, more stable triple helix systems, an effect which might be ascribed to the 3'-endo pucker of the modified nucleoside residues.     

Triplex staples: DNA double-strand cross-linking at internal and terminal sites using psoralen-containing triplex-forming oligonucleotides

A method has been developed to attach 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen to the 5 position of thymine bases during solid-phase oligonucleotide synthesis. UV irradiation of triplex-forming oligonucleotides (TFOs) containing internally attached psoralens produces photoadducts at TpA steps within target duplexes, thus relaxing the constraints on selection of psoralen target sequences. Photoreaction of TFOs containing two psoralens, located at the 5'- and 3'-ends, has been used to create double-strand cross-links (triplex staples) at both termini of the TFO. Such complexes have no free single-stranded ends. TFOs containing 4'-(hydroxymethyl)-4,5',8-trimethylpsoralen, 3-methyl-2-aminopyridine, and 5-(3-aminoprop-2-ynyl)deoxyuridine formed photoadducts with target duplexes under near-physiological conditions.     

Stem-loop oligonucleotides as tools for labelling double-stranded DNA

We report on a sequence-specific double-stranded DNA labelling strategy in which a stem-loop triplex forming oligonucleotide (TFO) is able to encircle its DNA target. Ligation of this TFO to either a short hairpin oligonucleotide or a long double-stranded DNA fragment leads to the formation of a topological complex. This process requires the hybridization of both extremities of the TFO to each other on a few base pairs. The effects of different factors on the formation of these complexes have been investigated. Efficient complex formation was observed using both GT or TC TFOs. The stem-loop structure enhances the specificity of the complex. The topologically linked TFO remains associated with its target even under conditions that do not favour triple-helix formation. This approach is sufficiently sensitive for detection of a 20-bp target sequence at the subfemtomolar level. This study provides new insights into the mechanics and properties of stem-loop TFOs and their complexes with double-stranded DNA targets. It emphasizes the interest of such molecules in the development of new tools for the specific labelling of short DNA sequences.     

Synthesis of twisted intercalating nucleic acids possessing acridine derivatives. Thermal stability studies

Twisted intercalating nucleic acids (TINA) possessing acridine derivatives have been synthesized via the postsynthetic modifications of oligonucleotides possessing insertions of (R)-1-O-(4-iodobenzyl)glycerol (8) or (R)-1-O-(4-ethynylbenzyl)glycerol (9) at the 5'-end or in the middle as a bulge. In the first postsynthetic step, oligonucleotides 8 and 9 on the CPG support were treated with a Sonogashira coupling reaction mixture containing 9-chloro-2-ethynylacridine or 9-chloro-2-iodoacridine, respectively. After the postsynthetic step, treatment of the oligonucleotides with 32% aq ammonia or 50% ethanolic solution of tris(2-aminoethyl)amine led to the substitution of chloride on acridine concurrent with deprotection of the bases and cleavage of the oligonucleotides from CPG. Molecular modeling of the parallel triplex with a bulged insertion of the monomer (R)-3-O-[4-(9-aminoacridin-2-ylethynyl)benzyl]glycerol in the triplex-forming oligonucleotide (TFO) showed that the acridine moiety was stacking between the bases of the duplex, while phenyl was placed between the bases of the TFO. Thermal denaturation studies and fluorescence properties of TINA-acridine oligonucleotide duplexes and triplexes are discussed.     

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.     

133 Designed DNA crystals with a triple-helix veneer

DNA has been used as a tool for the self-assembly of nano-sized objects and arrays in two and three-dimensions. Triplex-forming oligonucleotides (TFOs) can be exploited to recognize and introduce functionality at precise duplex regions within these DNA nanostructures (Rusling et al., 2012). Here we have examined the feasibility of using TFOs to bind to specific locations within a 3-turn DNA tensegrity triangle motif. The tensegrity triangle is a rigid DNA motif with three-fold rotational symmetry, consisting of three helices directed along three linearly independent directions (Liu et al., 2004). The triangles form a three-dimensional crystalline lattice stabilized via sticky-end cohesion (Zheng et al., 2009). The TFO 5′-TTCTTTCTTCTCT was used to target the tensegrity motif containing an appropriately embedded oligopurine–oligopyrimidine binding site. Formation of DNA triplex in the motif was characterized by an electrophoretic mobility shift assay (EMSA), UV melting studies and FRET analysis. Non-denaturing gel analysis of annealed DNA motifs showed a band with slower mobility only in the presence of TFO and only when the DNA motif contained the triplex binding site. Experiments were undertaken at pH 5.0, since the formation of a triplex with cytidine-containing TFOs requires slightly acidic conditions (pH<?6.0). TFOs with modified C-analogs and T-analogs having a higher pK _a worked at a more neutral pH, also evidenced by EMSA. UV melting studies revealed that the melting point of the 3-turn triangle was 64?°C and the TFO binding increased the melting point to 80?°C. FRET analysis was done by labeling the triangle with fluorescein and the TFO with a cyanine dye (Cy5). The FRET melting curve revealed that a signal was observed only when the TFO was bound to the DNA motif and the results were consistent with UV melting studies. These results indicate that a TFO can be specifically targeted to the tensegrity triangle motif.     

Investigation of the formation and intracellular stability of purine.(purine/pyrimidine) triplexes.

In a previous work we showed that a short triple helix-forming oligonucleotide (TFO) targeted to the murine c-pim-1 proto-oncogene promoter gives a very stable triple helix under physiological conditions in vitro . Moreover, this triplex was stable inside cells when preformed in vitro . However, we failed to detect triplex formation for this sequence inside cells in DMS footprinting studies. In the present work, in order to determine whether our previous in vivo results are limited to this particular short triplex or can be generalized to other purine.(purine/pyrimidine) triplexes, we have tested three other DNA targets already described in the literature. All these purine.(purine/pyrimidine) triplexes are specific and stable at high temperature in vitro . In vivo studies have shown that the preformed triplexes are stable inside cells for at least 3 days. This clearly demonstrates that intracellular conditions are favourable for the existence of purine. (purine/pyrimidine) triplexes. The triplexes can also be formed in nuclei. However, for all the sequences tested, we were unable to detect any triple helix formation in vivo in intact cells by DMS footprinting. Our results show that neither (i) chromatinization of the DNA target, (ii) intracellular K+concentration nor (iii) cytoplasmic versus nuclear separation of the TFO and DNA target are responsible for the intracellular arrest of triplex formation. We suggest the existence of a cellular mechanism, based on a compartmentalization of TFOs and/or TFO trapping, which separates oligonucleotides from the DNA target. Further work is needed to find oligonucleotide derivatives and means for their delivery to overcome the problem of triplex formation inside cells.     

Stability and kinetics of nucleic acid triplexes with chimaeric DNA/RNA third strands

A series of chimaeric DNA/RNA triplex-forming oligonucleotides (TFOs) with identical base sequence but varying sequential composition of the sugar residues were prepared. The structural, kinetic, and thermodynamic properties of triplex formation with their corresponding double-helical DNA target were investigated by spectroscopic methods. Kinetic and thermodynamic data were obtained from analysis of nonequilibrium UV-melting and annealing curves in the range of pH 5.1-6.7 in a 10 mM citrate/phosphate buffer containing 0.1 M NaCl and 1 mM EDTA. It was found that already single substitutions of ribo- for deoxyribonucleotides in the TFOs greatly affect stability and kinetics of triplex formation in a strongly sequence dependent manner. Within the sequence context investigated, triplex stability was found to increase when deoxyribonucleotides were present at the 5'-side and ribonucleotides in the center of the TFO. Especially the substitution of thymidines for uridines in the TFO was found to accelerate both the association and dissociation process in a strongly position-dependent way. Differential structural information on triplexes and TFO single-strands was obtained from CD-spectroscopy and gel mobility experiments. Only minor changes were observed in the CD spectra of the triplexes at all pH values investigated, and the electrophoretic mobility was nearly identical in all cases, indicating a high degree of structural similarity. In contrast, the single-stranded TFOs showed high structural variability, as determined in the same way. The results are discussed in the context of the design of TFOs for therapeutic or biochemical applications.     

Binding of triple helix forming oligonucleotides to sites in gene promoters

A class of triplex-forming oligodeoxyribonucleotides (TFOs) is described that can bind to naturally occurring sites in duplex DNA at physiological pH in the presence of magnesium. The data are consistent with a structure in which the TFO binds in the major groove of double-stranded DNA to form a three-stranded complex that is superficially similar to previously described triplexes. The distinguishing features of this class of triplex are that TFO binding apparently involves the formation of hydrogen-bonded G.GC and T.AT triplets and the TFO is bound antiparallel with respect to the more purine-rich strand of the underlying duplex. Triplex formation is described for targets in the promoter regions of three different genes: the human c-myc and epidermal growth factor receptor genes and the mouse insulin receptor gene. All three sites are relatively GC rich and have a high percentage of purine residues on one strand. DNase I footprinting shows that individual TFOs bind selectively to their target sites at pH 7.4-7.8 in the presence of millimolar concentrations of magnesium. Electrophoretic analysis of triplex formation indicates that specific TFOs bind to their target sites with apparent dissociation constants in the 10(-7)-10(-9) M range. Strand orientation of the bound TFOs was confirmed by attaching eosin or an iron-chelating group to one end of the TFO and monitoring the pattern of damage to the bound duplex DNA. Possible hydrogen-bonding patterns and triplex structures are discussed.     

Suppression of c-myc oncogene expression by a polyamine-complexed triplex forming oligonucleotide in MCF-7 breast cancer cells.

Polyamines are excellent stabilizers of triplex DNA. Recent studies in our laboratory revealed a remarkable structural specificity of polyamines in the induction and stabilization of triplex DNA. 1,3-Diaminopropane (DAP) showed optimum efficacy amongst a series of synthetic diamines in stabilizing triplex DNA. To utilize the potential of this finding in developing an anti-gene strategy for breast cancer, we treated MCF-7 cells with a 37mer oligonucleotide to form triplex DNA in the up-stream regulatory region of the c-myc oncogene in the presence of DAP. As individual agents, the oligonucleotide and DAP did not downregulate c-myc mRNA in the presence of estradiol. Complexation of the oligonucleotide with 2 mM DAP reduced c-myc mRNA signal by 65% at 10 microM oligonucleotide concentration. In contrast, a control oligonucleotide had no significant effect on c-myc mRNA. The expression of c-fos oncogene was not significantly altered by the triplex forming oligonucleotide (TFO). DAP was internalized within 1 h of treatment; however, it had no significant effect on the level of natural polyamines. These data indicate that selective utilization of synthetic polyamines and TFOs might be an important strategy to develop anti-gene-based therapeutic modalities for breast cancer.     

Triplex formation on DNA targets: how to choose the oligonucleotide

Triplex-forming oligonucleotides (TFOs) are sequence-specific DNA binders. TFOs provide a tool for controlling gene expression or, when attached to an appropriate chemical reagent, for directing DNA damage. Here, we report a set of rules for predicting the best out of five different triple-helical binding motifs (TM, UM, GA, GT, and GU, where M is 5-methyldeoxycytidine and U is deoxyuridine) by taking into consideration the sequence composition of the underlying duplex target. We tested 11 different triplex targets present in genes having an oncogenic role. The rules have predictive power and are very useful in the design of TFOs for antigene applications. Briefly, we retained motifs GU and TM, and when they do form a triplex, TFOs containing G and U are preferred over those containing T and M. In the case of the G-rich TFOs, triplex formation is principally dependent on the percentage of G and the length of the TFO. In the case of the pyrimidine motif, replacement of T with U is destabilizing; triplex formation is dependent on the percentage of T and destabilized by the presence of several contiguous M residues. An equation to choose between a GU and TM motif is given.     

Triplex formation by morpholino oligodeoxyribonucleotides in the HER-2/neu promoter requires the pyrimidine motif

Triplex-forming oligonucleotides (TFOs) are good candidates to be used as site-specific DNA-binding agents. Two obstacles encountered with TFOs are susceptibility to nuclease activity and a requirement for magnesium for triplex formation. Morpholino oligonucleotides were shown in one study to form triplexes in the absence of magnesium. In the current study, we have compared phosphodiester and morpholino oligonucleotides targeting a homopurine–homopyrimidine region in the human HER2/neu promoter. Using gel mobility shift analysis, our data demonstrate that triplex formation by phosphodiester oligonucleotides at the HER-2/neu promoter target is possible with pyrimidine-parallel, purine-antiparallel and mixed sequence (GT)-antiparallel motifs. Only the pyrimidine-parallel motif morpholino TFO was capable of efficient triple helix formation, which required low pH. Triplex formation with the morpholino TFO was efficient in low or no magnesium. The pyrimidine motif TFOs with either a phosphodiester or morpholino backbone were able to form triple helices in the presence of potassium ions, but required low pH. We have rationalized the experimental observations with detailed molecular modeling studies. These data demonstrate the potential for the development of TFOs based on the morpholino backbone modification and demonstrate that the pyrimidine motif is the preferred motif for triple helix formation by morpholino oligonucleotides.     

Poly(L-lysine)-graft-dextran copolymer: amazing effects on triplex stabilization under physiological pH and ionic conditions (in vitro).

Triplex DNA formation involving unmodified triplex-forming oligonucleotides (TFOs) is very unstable under physiological conditions. Here, we report a novel strategy to stabilize both purine and pyrimidine motif triplex DNA within the rat alpha1 (I) collagen gene promoter under physiologically relevant conditions by a poly(L-lysine)- graft -dextran copolymer. Using an in vitro electrophoretic mobility shift assay, we show that the copolymer almost completely abrogates the inhibitory effects of physiological concentrations of monovalent cations, particularly potassium ion (K+), on purine motif triplex formation involving very low concentrations of an unmodified guanine-rich TFO. Of importance, pH dependency in pyrimidine motif triplex formation involving an unmodified cytosine-rich TFO is also significantly overcome by the copolymer. Finally, the triplex-stabilizing efficiency of the copolymer is remarkably higher than that of other oligocations, like spermine and spermidine. We suggest that the ability of the graft copolymer to stabilize triplex DNA under physiologically relevant pH and salt concentrations will be a cue for further progress in the antigene strategy.