Stabilisation of TG- and AG-containing antiparallel DNA triplexes by triplex-binding ligands

We have used DNase I footprinting to examine the interaction of several triplex-binding ligands with antiparallel TG- and AG-containing triplexes. We find that although a 17mer TG-containing oligonucleotide on its own fails to produce a footprint at concentrations as high as 30 µM, this interaction can be stabilised by several ligands. Within a series of disubstituted amidoanthraquinones we find that the 2,7- regioisomer affords the best stabilisation of this TG triplex, though the 1,8- isomer also stabilises this interaction to some extent. By contrast the 1,5- and 2,6- regioisomers show no interaction with TG triplexes. Similar studies with a 13mer AG-containing oligonucleotide show the opposite pattern of stabilisation: the 2,6- and 1,5- isomers stabilise this triplex, but the 2,7- and 1,8-compounds do not. The polycyclic compound BePI strongly stabilises TG- but not AG-containing triplexes, while a substituted naphthylquinoline interacts with both antiparallel triplex motifs.     

DNA sequence specificity of triplex-binding ligands.

We have examined the ability of naphthylquinoline, a 2,7-disubstituted anthraquinone and BePI, a benzo[e]pyridoindole derivative, to stabilize parallel DNA triplexes of different base composition. Fluorescence melting studies, with both inter- and intramolecular triplexes, show that all three ligands stabilize triplexes that contain blocks of TAT triplets. Naphthylquinoline has no effect on triplexes formed with third strands composed of (TC)n or (CCT)n, but stabilizes triplexes that contain (TTC)n. In contrast, BePI slightly destabilizes the triplexes that are formed at (TC)n (CCT)n and (TTC)n. 2,7-Anthraquinone stabilizes (TC)n (CCT)n and (TTC)n, although it has the greatest effect on the latter. DNase I footprinting studies confirm that triplexes formed with (CCT)n are stabilized by the 2,7-disubstituted amidoanthraquinone but not by naphthylquinoline. Both ligands stabilize the triplex formed with (CCTT)n and neither affects the complex with (CT)n. We suggest that BePI and naphthylquinoline can only bind between adjacent TAT triplets, while the anthraquinone has a broader sequence of selectivity. These differences may be attributed to the presence (naphthylquinoline and BePI) or absence (anthraquinone) of a positive charge on the aromatic portion of the ligand, which prevents intercalation adjacent to C+GC triplets. The most stable structures are formed when the stacked rings (bases or ligand) alternate between charged and uncharged species. Triplexes containing alternating C+GC and TAT triplets are not stabilized by ligands as they would interrupt the alternating pattern of charged and uncharged residues.     

Binding of a homologous series of anthraquinones to DNA

The DNA binding characteristics of a series of homologous 2,6-disubstituted anthraquinone threading intercalators bearing one to four ethylene glycol units in their side chains have been studied. Binding constants were measured via surface plasmon resonance (SPR). These compounds bind to an AT-rich hairpin with slightly higher affinity than to a GC-rich hairpin. The binding constants decrease as the length of the side chain increases.     

Tuning G-quadruplex vs double-stranded DNA recognition in regioisomeric lysyl-peptidyl-anthraquinone conjugates

Anthraquinone is a versatile scaffold to provide effective DNA binders. This planar system can be easily conjugated to protonable side chains: the nature of the lateral groups and their positions around the tricyclic moiety largely affect the DNA recognition process in terms of binding affinity and mode, as well as sequence and structure of the target nucleic acid. Starting from an anthracenedione system symmetrically functionalized with N-terminal lysyl residues, we incremented the length of side chains by introducing a Gly, Ala, or Phe spacer, characterized by different flexibility, lipophilicity, and bulkiness. Moreover, 2,6, 2,7, 1,8, and 1,5 regioisomers were examined to yield a small bis(lysyl-peptidyl) anthracenedione library. By merging spectroscopic, enzymatic, and cellular results, we showed that the proper combination of a basic aminoacid (Lys) with a more hydrophobic residue (Phe) can provide selective G-quadruplex recognition, in particular when side chains are located at positions 2,6 or 2,7. In fact, while these derivatives effectively bind G-quadruplex structures, they behave at the same time as rather poor double-stranded DNA intercalators. As a result, the Lys-Phe substituted anthraquinones are poorly cytotoxic but still able to promote a senescence mechanism in cancer cells. This combination of chemical and biological properties foresees potentially valuable applications in anticancer medicinal chemistry.     

1,8-Naphthyridin-2,7-(1,8H)-dione is an effective mimic of protonated cytosine in peptide nucleic acid triplex recognition systems

A novel bicyclic mimic of protonated cytosine [1,8-naphthyridin-2,7-(1,8H)-dione, (K)] for Hoogsteen type triplex recognition of guanine has been designed for incorporation into peptide nucleic acids. Bis-PNA clamps with the K base incorporated in the Hoogsteen strand showed a significant stabilization of the triplexes at pH 7 as compared to similar triplexes with PNA oligomers containing either cytosine (6.7 degrees C per unit) or pseudoisocytosine (1.5 degrees C per unit). Cooperative stabilization was observed when the K units were placed in adjacent positions ( approximately 3 degrees C per unit).     

Secondary binding sites for triplex-forming oligonucleotides containing bulges, loops, and mismatches in the third strand

We have used DNase I footprinting to examine the binding of five different 17-mer oligonucleotides to a 53-base oligopurine tract containing four pyrimidine interruptions. Although all the expected triplexes formed with high affinity (K(d) approximately 10-50 nM), one oligonucleotide produced a footprint at a second site with about 20-fold lower affinity. We have explored the nature of this secondary binding site and suggest that it arises when each end of the third strand forms a 7-mer triplex with adjacent regions on the duplex, generating a contiguous 14-base triplex with a bulge in the center of the third strand oligonucleotide. This unusual binding mode was examined by use of oligonucleotides that were designed with the potential to form different length third-strand loops of various base composition. We find that triplexes containing single-base bulges are generally more stable than those with dinucleotide loops, though triplexes can be formed with loops of up to nine thymines, generating complexes with submicromolar dissociation constants. These structures are much more stable than those formed by adding two separate 7-mer oligonucleotides, which do not generate DNase I footprints, though a stable complex is generated when the two halves are covalently joined by a hexa(ethylene glycol) linker. MPE produces less clear footprints, presumably because this cleavage agent binds to triplex DNA, but confirms that the oligonucleotides can bind in unexpected places. These results suggest that extra care needs to be taken when designing long triplex-forming oligonucleotides so as to avoid triplex formation at shorter secondary sites.     

Spectroscopic and thermodynamic studies on the binding of sanguinarine and berberine to triple and double helical DNA and RNA structures

A comparative study on the interaction of sanguinarine and berberine with DNA and RNA triplexes and their parent duplexes was performed, by using a combination of spectrophotometric, UV thermal melting, circular dichroic and thermodynamic techniques. Formation of the DNA and RNA triplexes was confirmed from UV-melting and circular dichroic measurements. The interaction process was characterized by increase of thermal melting temperature, perturbation in circular dichroic spectrum and the typical hypochromic and bathochromic effects in the absorption spectrum. Scatchard analysis indicated that both the alkaloids bound to the triplex and duplex structures in a non-cooperative manner and the binding was stronger to triplexes than to parent duplexes. Thermal melting studies further indicated that sanguinarine stabilized the Hoogsteen base paired third strand of both DNA and RNA triplexes more tightly compared to their Watson-Crick strands, while berberine stabilized the third strand only without affecting the Watson-Crick strand. However, sanguinarine stabilized the parent duplexes while no stabilization was observed with berberine under identical conditions. Circular dichroic studies were also consistent with the observation that perturbations of DNA and RNA triplexes were more compared to their parent duplexes in presence of the alkaloids. Thermodynamic data revealed that binding of sanguinarine and berberine to triplexes (T.AxT and U.AxU) and duplexes (A.T and A.U) showed negative enthalpy changes and positive entropy changes but that of sanguinarine to C.GxC(+) triplex and G.C duplex exhibited negative enthalpy and negative entropy changes. Taken together, these results suggest that both sanguinarine and berberine can bind and stabilize the DNA and RNA triplexes more strongly than their respective parent 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.     

Human telomerase inhibition and cytotoxicity of regioisomeric disubstituted amidoanthraquinones and aminoanthraquinones

Telomerase is an attractive target for the rational design of new anticancer drugs due to its central role in the control of cellular proliferation. A number of 1,4-disubstituted amidoanthraquinones and 1,5-disubstituted aminoanthraquinones that are related to mitoxantrone and ametantrone have previously been prepared. The present study details the effects on human telomerase of these new classes of 1,4- and 1,5-difunctionalized tricyclic anthraquinone compounds. We have used cytotoxicity assay, reporter SEAP assay to monitor the hTERT expression, and TRAP-G4 assay to measure the relative activity of these compounds, and have examined how the attached substituents affect their ability to influence telomerase. Cytotoxicity levels in human tumor cell lines were at comparable levels for several compounds. Structural and activity relationships indicated that the position of disubstituent side chains is important for its inhibitory effect. Moreover, a primary amine or tertiary amine on the substitution group appears to be required for the telomerase inhibitory effect. There is no significant correlation between telomerase activity and cytotoxicity. These symmetrical disubstituted anthraquinones may represent useful leads for the development of human telomerase inhibitors as potential anticancer agents, and the exact mode of intercalative binding is dictated by the positional placement of substituent side chains for effective telomerase inhibition.     

Synthesis and biological evaluation of 2,7-Dihydro-3H-dibenzo[de,h]cinnoline-3,7-dione derivatives,a novel group of anticancer agents active on a multidrug resistant cell line

A series of anthrapyridazone derivatives with one or two basic side chains at various positions in the tetracyclic chromophore have been synthesized. The key intermediates in the synthesis are 2,7-dihydro-3H-dibenzo[de,h]cinnoline-3,7-diones 1, 12 and 15 monosubstituted at position 2 (4d, 16a-e), or 6 (2a-f) or disubstituted at positions 2 and 6 (4a-c) or 2 and 8 (17a-e) with appropriate alkylaminoalkylamines. All analogues showed in vitro cytotoxic activity against murine leukemia (L1210) and human leukemia (K562) cell lines. The compounds were also active against human leukemia multidrug resistant (K562/DX) cell line with resistance index (RI) in the range 1-3 depending on the compound's structure. Two of the most active in vitro compounds 4a and 11 were tested in vivo against murine P388 leukemia and displayed antileukemic activity comparable with that of Mitoxantrone. DNA-binding assays were performed and DNA affinity data were correlated with the structures of the compounds. The cytoplasmatic membrane affinity values (log k'(IAM)) have also been determined and the correlation with the resistance indexes discussed. The anthrapyridazones constitute a novel group of antitumor compounds that can overcome multidrug resistance.     

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.     

Spectroscopic studies on the interaction of aristololactam-beta-D-glucoside with DNA and RNA double and triple helices: A comparative study.

The interaction of aristololactam-beta-D-glucoside (ADG), a DNA intercalating alkaloid, with the DNA triplexes, poly(dT). poly(dA)xpoly(dT) and poly(dC).poly(dG)xpoly(dC+), and the RNA triplex poly(rU).poly(rA)xpoly(rU) was investigated by circular dichroic, UV melting profile, spectrophotometric, and spectrofluorimetric techniques. Comparative interaction with the corresponding Watson-Crick duplexes has also been examined under identical experimental conditions. Triplex formation has been confirmed from biphasic thermal melting profiles and analysis of temperature-dependent circular dichroic measurements. The binding of ADG to triplexes and duplexes is characterized by the typical hypochromic and bathochromic effects in the absorption spectrum, quenching of steady-state fluorescence intensity, a decrease in fluorescence quantum yield, an increase or decrease of thermal melting temperatures, and perturbation in the circular dichroic spectrum. Scatchard analysis indicates that ADG binds both to the triplexes and the duplexes in a noncooperative manner. Binding parameters obtained from spectrophotometric measurements are best fit by the neighbor exclusion model. The binding affinity of ADG to the DNA triplexes is substantially stronger than to the RNA triplex. Thermal melting study further indicates that ADG stabilizes the Hoogsteen base-paired third strand of the DNA triplexes whereas it destabilizes the same strand of RNA triplex but stabilizes its Watson-Crick strands. Comparative data reveal that ADG exhibits a stronger binding to the triple helical structures than to the respective double helical structures.     

Interactions of cryptolepine and neocryptolepine with unusual DNA structures

Cryptolepine, the main alkaloid present in the roots of Cryptolepis sanguinolenta, presents a large spectrum of biological properties. It has been reported to behave like a DNA intercalator with a preference for GC-rich sequences. In this study, dialysis competition assay and mass spectrometry experiments were used to determine the affinity of cryptolepine and neocryptolepine for DNA structures among duplexes, triplexes, quadruplexes and single strands. Our data confirm that cryptolepine and neocryptolepine prefer GC over AT-rich duplex sequences, but also recognize triplex and quadruplex structures. These compounds are weak telomerase inhibitors and exhibit a significant preference for triplexes over quadruplexes or duplexes.     

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.     

A Distinct Triplex DNA Unwinding Activity of ChlR1 Helicase

Mutations in the human ChlR1 (DDX11) gene are associated with a unique genetic disorder known as Warsaw breakage syndrome characterized by cellular defects in genome maintenance. The DNA triplex helix structures that form by Hoogsteen or reverse Hoogsteen hydrogen bonding are examples of alternate DNA structures that can be a source of genomic instability. In this study, we have examined the ability of human ChlR1 helicase to destabilize DNA triplexes. Biochemical studies demonstrated that ChlR1 efficiently melted both intermolecular and intramolecular DNA triplex substrates in an ATP-dependent manner. Compared with other substrates such as replication fork and G-quadruplex DNA, triplex DNA was a preferred substrate for ChlR1. Also, compared with FANCJ, a helicase of the same family, the triplex resolving activity of ChlR1 is unique. On the other hand, the mutant protein from a Warsaw breakage syndrome patient failed to unwind these triplexes. A previously characterized triplex DNA-specific antibody (Jel 466) bound triplex DNA structures and inhibited ChlR1 unwinding activity. Moreover, cellular assays demonstrated that there were increased triplex DNA content and double-stranded breaks in ChlR1-depleted cells, but not in FANCJ^−/− cells, when cells were treated with a triplex stabilizing compound benzoquinoquinoxaline, suggesting that ChlR1 melting of triple-helix structures is distinctive and physiologically important to defend genome integrity. On the basis of our results, we conclude that the abundance of ChlR1 known to exist in vivo is likely to be a strong deterrent to the stability of triplexes that can potentially form in the human genome.     

DNA triple-helix formation on nucleosome core particles. Effect of length of the oligopurine tract.

We have used DNase I footprinting to examine the formation of intermolecular triplexes on DNA fragments which have been complexed with nucleosome core particles. We have prepared five DNA fragments, based on the 160-bp tyrT sequence, which contain different length oligopurine tracts (up to 25 bp) at two different positions along the fragment, and have examined their availability for triple-helix formation after reconstituting onto nucleosome core particles. These results are compared with the formation of shorter triplexes in the same regions. In general we find that increasing the length of the complex does not facilitate nucleosomal triplex formation and that the most important factor affecting triplex formation is the position of the target site within the nucleosome-bound fragment. In some instances we find that longer oligonucleotides inhibit triplex formation. Although successful triplex formation was achieved on the longest nucleosome-bound oligopurine tracts, this was accompanied by changes in cleavage pattern that suggest oligonucleotide-induced changes in nucleosome structure.     

Stability of DNA triplexes on shuttle vector plasmids in the replication pool in mammalian cells

Triple helix-forming oligonucleotides may be useful as gene-targeting reagents in vivo, for applications such as gene knockout. One important property of these complexes is their often remarkable stability, as demonstrated in solution and in cells following transfection. Although encouraging, these measurements do not necessarily report triplex stability in cellular compartments that support DNA functions such as replication and mutagenesis. We have devised a shuttle vector plasmid assay that reports the stability of triplexes on DNA that undergoes replication and mutagenesis. The assay is based on plasmids with novel variant supF tRNA genes containing embedded sequences for triplex formation and psoralen cross-linking. Triple helix-forming oligonucleotides were linked to psoralen and used to form triplexes on the plasmids. At various times after introduction into cells, the psoralen was activated by exposure to long wave ultraviolet light (UVA). After time for replication and mutagenesis, progeny plasmids were recovered and the frequency of plasmids with mutations in the supF gene determined. Site-specific mutagenesis by psoralen cross-links was dependent on precise placement of the psoralen by the triple helix-forming oligonucleotide at the time of UVA treatment. The results indicated that both pyrimidine and purine motif triplexes were much less stable on replicated DNA than on DNA in vitro or in total transfected DNA. Incubation of cells with amidoanthraquinone-based triplex stabilizing compounds enhanced the stability of the pyrimidine triplex.     

Triplex hydration: nanosecond molecular dynamics simulation of the solvated triplex formed by mixed sequences

A theoretical model for the hydration pattern and motion of ions around the triple helical DNA with mixed sequences d(GACTGGTGAC)d(GTCACCAGTC)*d(GACTGGTGAC) in solution, during MD simulation, using the particle mesh Ewald sum method, is elaborated here. The AMBER 5.0 force field has been used during the simulation in solvent. The simulation studies support a dynamically stable atmosphere around the DNA triplex in solution over the entire length of the trajectory. The results have been compared with Hoogsteen triplexes and examined in the context of the observed behaviour of hydration in crystallographic data of duplexes. The dynamical organization of counterions and water molecules around the triplex formed by mixed sequences is described here. It has been observed that cations prefer to bind between two adjoining purines of the second and the third strands. The idea of localized complexes (mobile counterions in unspecific electronegative pockets around the DNA triplex with water molecules) may have important implications for understanding the specificity of the interactions of nucleic acids with proteins and other ligands.