Specific stabilization of DNA triple helices by indolo[2,1-b]quinazolin-6,12-dione derivatives

Derivatives of indolo[2,1-b]quinazolinone containing aminoalkylamino side chains were synthesized as specific DNA triplex stabilizing agents. The aminoalkylamino side chains are essential for triplex stabilization. The position-8 fluorine atom or a methyl group to the nitrogen adjacent to the planar core can enhance triplex stability by 6 degrees C and the effect is additive. Conformational analysis reveals that the orientation of the side chain underlies the ability of this compound to stabilize a DNA triplex.     

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.     

Quantitative analysis of the ion-dependent folding stability of DNA triplexes

A DNA triplex is formed through binding of a third strand to the major groove of a duplex. Due to the high charge density of a DNA triplex, metal ions are critical for its stability. We recently developed the tightly bound ion (TBI) model for ion-nucleic acids interactions. The model accounts for the potential correlation and fluctuations of the ion distribution. We now apply the TBI model to analyze the ion dependence of the thermodynamic stability for DNA triplexes. We focus on two experimentally studied systems: a 24-base DNA triplex and a pair of interacting 14-base triplexes. Our theoretical calculations for the number of bound ions indicate that the TBI model provides improved predictions for the number of bound ions than the classical Poisson-Boltzmann (PB) equation. The improvement is more significant for a triplex, which has a higher charge density than a duplex. This is possibly due to the higher ion concentration around the triplex and hence a stronger ion correlation effect for a triplex. In addition, our analysis for the free energy landscape for a pair of 14-mer triplexes immersed in an ionic solution shows that divalent ions could induce an attractive force between the triplexes. Furthermore, we investigate how the protonated cytosines in the triplexes affect the stability of the triplex helices.     

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.     

Differential effects of cyclopolyamines on the stability and conformation of triplex DNA

Linear polyamines are excellent promoters of triplex DNA formation. The effects of structural rigidization of polyamines on triplex DNA stability are not known at present. We wished to develop a series of polyamine analogs as secondary ligands for triplex DNA stabilization for antigene applications. To accomplish this goal, we synthesized cyclopolyamines by interconnecting the two amino or imino groups of linear polyamines with a --(CH2)n-bridge (n=3,4,5). Melting temperature (Tm) data showed that [4,3]-spermine and [4,4]-spermine stabilized poly(dA) x 2poly(dT) triplex at >25 microM concentrations (Tm = 71 degrees C at 100 microM). The dTm/dlog [polyamine] values for these compounds were 26 and 40, respectively. [4,3]-Spermine and [4,4]-spermine also stabilized triplex DNA formed by a purine-motif triplex-forming oligonucleotide, TG3TG4TG4TG3T with its target duplex, as determined by Tm, circular dichroism (CD) spectroscopy, and electrophoretic mobility shift assay (EMSA). In contrast, [4,4]-putrescine and [4,5]-putrescine as well as [4,5]-spermine had no triplex DNA stabilizing effect. CD spectra also showed triplex DNA aggregation and psi-DNA formation at >100 microM [4,3]-spermine. These data demonstrate that structural rigidization of linear polyamines has a profound effect on their ability to stabilize triplex DNA and provoke conformational transitions.     

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.     

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.     

Triplex formation at physiological pH by 5-Me-dC-N4-(spermine) [X] oligodeoxynucleotides: non protonation of N3 in X of X*G:C triad and effect of base mismatch/ionic strength on triplex stabilities.

Oligodeoxynucleotide (ODN) directed triplex formation has therapeutic importance and depends on Hoogsteen hydrogen bonds between a duplex DNA and a third DNA strand. T*A:T triplets are formed at neutral pH and C+*G:C are favoured at acidic pH. It is demonstrated that spermine conjugation at N4 of 5-Me-dC in ODNs 1-5 (sp-ODNs) imparts zwitterionic character, thus reducing the net negative charge of ODNs 1-5. sp-ODNs form triplexes with complementary 24mer duplex 8:9 show foremost stability at neutral pH 7.3 and decrease in stability towards lower pH, unlike the normal ODNs where optimal stability is found at an acidic pH 5.5. At pH 7.3, control ODNs 6 and 7 carrying dC or 5-Me-dC, respectively, do not show any triple helix formation. The stability order of triplex containing 5-Me-dC-N4-(spermine) with normal and mismatched duplex was found to be X*G:C approximately X*A:T > X*C:G > X*T:A. The hysteresis curve of sp-ODN triplex 3*8:9 indicated a better association with complementary duplex 8:9 as compared to unmodified ODN 6 in triplex 6*8:9. pH-dependent UV difference spectra suggest that N3 protonation is not a requirement for triplex formation by sp-ODN and interstrand interaction of conjugated spermine more than compensates for loss in stability due to absence of a single Hoogsteen hydrogen bond. These results may have importance in designing oligonucleotides for antigene applications.     

Sequence-dependent stability of intramolecular DNA triple helices

A set of 21 oligodeoxynucleotides were designed to fold into intramolecular triple helices of the pyrimidine motif under appropriate conditions. UV melting experiments on the triplexes which only differ in the number and distribution of third strand cytosines reveal the influence of sequence and pH on triplex stability and can be summarized as follows: (1) increasing the cytosine content in the third strand results in a higher thermal stability of the triplex at acidic pH but lowers the triplex to duplex melting temperature at neutral pH; (2) cytosines at terminal positions destabilize the triple helical structure as compared to non-terminal positions; (3) contiguous cytosines lead to a pH dependent destabilization of the triplex, the destabilizing effect being more pronounced at higher pH. Analysis of these effects in terms of the various interactions within a triple helical complex indicate that the sequence-dependent stabilities are largely determined by the extent of protonation for individual third strand cytosines.     

Intramolecular DNA triplexes in supercoiled plasmids. II. Effect of base composition and noncentral interruptions on formation and stability

The effects of interruptions in the homopurine bias and the G+C content of the homopurine.homopyrimidine (pur.pyr) sequences on intramolecular triplex formation and stability in supercoiled plasmids were evaluated. In addition, the interconversion of triplex and duplex, after altering the stabilizing factors (low pH or supercoiling), was studied. We conclude: (a) a 42-base pair pur.pyr sequence with three consecutive interruptions does not form a large triplex with three unpaired nucleotides in the stem. Instead, a mixture of two smaller (27- and 28-nucleotide) triplexes forms. (b) A 28-nucleotide sequence with a single interruption forms a triplex with one unpaired nucleotide in the stem. This interruption causes the triplex to be 7 degrees C less thermostable and requires more superhelical energy for formation than the control triplex. (c) As the G+C content of a pur.pyr sequence increases, the thermostability of the triplex increases and the triplex requires less supercoiling for formation. (d) The interconversion between duplex and triplex is fast. After negative supercoiling is removed, all triplex becomes duplex in about 3 min. When the pH is shifted from 8.0 to 5.2, the conversion of duplex to triplex in a negatively supercoiled plasmid is complete in less than 2 min. Hence, these kinetic properties are consistent with important biological roles for triplexes. In summary, the results from both this and the accompanying paper show that a substantial amount of sequence imperfections is tolerated for triplex formation and stability.     

A new approach to overcome potassium-mediated inhibition of triplex formation.

G,A-containing purine oligonucleotides of various lengths form extremely stable and specific triplexes with the purine-pyrimidine stretch of the vpx gene [Svinarchuk,F., Monnot,M., Merle,A., Malvy,C. and Fermandjian,S. (1995) Nucleic Acids Res., 22, 3742--3747]. The potential application of triple-helix-forming oligonucleotides (TFO) in gene-targeted therapy has prompted us to study triplex formation mimicking potassium concentrations and temperatures in cells. Triplex formation was tested by dimethyl sulphate (DMS) footprinting, gel-retardation, UV melting studies and electron microscopy. In the presence of 10 mM MgCl2, KCl concentrations up to 150 mM significantly lowered both efficiency (triplex : initial duplex) and rate constants of triplex formation. The KCl effect was more pronounced for 11mer and 20mer TFOs than for 14mer TFO. Since the dissociation half-life for the 11mer TFO decreases from 420 min in the absence of monovalent cations to 40 min in the presence of 150 mM KCI, we suggest that the negative effect could be explained by a decrease in triplex stability. In contrast, for the 20mer TFO no dissociation of the triplex was observed during 24 h of incubation either in the absence of monovalent cations or in the presence of 150 mM KCl. We suppose that in the case of the 20mer TFO the negative effect of KCI on triplex formation is probably due to the self-association of the oligonucleotide in competitive structures such as parallel duplexes and/or tetraplexes. This negative effect may be overcome by the prior formation of a short duplex either on the 3'- or 5'-end of the 20mer TFO. We refer to these partial duplexes as 'zipper' TFOs. It was demonstrated that a 'zipper' TFO can form a triplex over the full length of the target, thus unzipping the short complementary strand. The minimal single-stranded part of the 'zipper' oligonucleotide which is sufficient to initiate triplex formation can be as short as three nucleotides at the 3'-end and six nucleotides at the 5'-end. We suggest that this type of structure may prove useful for in vivo applications.     

Sequence-Dependent Stability of Intramolecular DNA Triple Helices

Abstract

A set of 21 oligodeoxynucleotides were designed to fold into intramolecular triple helices of the pyrimidine motif under appropriate conditions. UV melting experiments on the triplexes which only differ in the number and distribution of third strand cytosines reveal the influence of sequence and pH on triplex stability and can be summarized as follows: (1) increasing the cytosine content in the third strand results in a higher thermal stability of the triplex at acidic pH but lowers the triplex to duplex melting temperature at neutral pH; (2) cytosines at terminal positions destabilize the triple helical structure as compared to non-terminal positions; (3) contiguous cytosines lead to a pH dependent destabilization of the triplex, the destabilizing effect being more pronounced at higher pH. Analysis of these effects in terms of the various interactions within a triple helical complex indicate that the sequence-dependent stabilities are largely determined by the extent of protonation for individual third strand cytosines.     

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.     

Triple helices formed at oligopyrimidine*oligopurine sequences with base pair inversions: effect of a triplex-specific ligand on stability and selectivity.

Oligonucleotide-directed triple helix formation is mostly restricted to oligopyrimidine*oligopurine sequences of double helical DNA. An interruption of one or two pyrimidines in the oligopurine target strand leads to a strong triplex destabilisation. We have investigated the effect of nucleotide analogues introduced in the third strand at the site opposite the base pair inversion(s). We show that a 3-nitropyrrole derivative (M) discriminates G*C from C*G, A*T and T*A in the presence of a triplex-specific ligand (a benzo[e]pyridoindole derivative, BePI). N6-methoxy-2,6-diaminopurine (K) binds to an A*T base pair better than a T*A, G*C or C*G base pair. Some discrimination is still observed in the presence of BePI and triplex stability is markedly increased. These findings should help in designing BePI-oligonucleotide conjugates to extend the range of DNA sequences available for triplex formation.     

Recognition of CG interrupting site by W-shaped nucleoside analogs (WNA) having the pyrazole ring in an anti-parallel triplex DNA

We have previously developed W-shaped nucleoside analogs (WNA) for recognition of TA and CG interrupting sites, which are the intrinsic limitation for the formation of a stable triplex DNA by the natural triplex-forming oligonucleotide (TFO). However, the stabilization effect of WNA is dependent on the neighboring nucleobases at both sides of the WNA analogs within the TFO. Considering that the base is located at the hindered site constructed of three bases of the target duplex and the TFO, it was expected that replacement of the pyrimidine base of the WNA analog with a smaller pyrazole ring might avoid steric repulsion to produce a greater stability for the triplex. In this study, the new WNA analogs bearing the pyrazole ring, 3-aminopyrazole (AP), and 4-methyl-3-pyrazole-5-on (MP) were synthesized, incorporated into the TFOs, then their stabilizing effects on the triplexes were evaluated. A remarkable success was illustrated by the fact that the TFO containing WNA-βAP in the 3′G-WNA-G-5′ sequence formed a stable triplex with selectivity to the CG interrupting site where the previous WNA-βC did not induce the triplex formation.     

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

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

Structural diversity of target-specific homopyrimidine peptide nucleic acid-dsDNA complexes

Sequence-selective recognition of double-stranded (ds) DNA by homopyrimidine peptide nucleic acid (PNA) oligomers can occur by major groove triplex binding or by helix invasion via triplex P-loop formation. We have compared the binding of a decamer, a dodecamer and a pentadecamer thymine–cytosine homopyrimidine PNA oligomer to a sequence complementary homopurine target in duplex DNA using gel-shift and chemical probing analyses. We find that all three PNAs form stable triplex invasion complexes, and also conventional triplexes with the dsDNA target. Triplexes form with much faster kinetics than invasion complexes and prevail at lower PNA concentrations and at shorter incubation times. Furthermore, increasing the ionic strength strongly favour triplex formation over invasion as the latter is severely inhibited by cations. Whereas a single triplex invasion complex is formed with the decameric PNA, two structurally different target-specific invasion complexes were characterized for the dodecameric PNA and more than five for the pentadecameric PNA. Finally, it is shown that isolated triplex complexes can be converted to specific invasion complexes without dissociation of the Hoogsteen base-paired triplex PNA. These result demonstrate a clear example of a ‘triplex first’ mechanism for PNA helix invasion.