Circular dichroism and UV melting studies on formation of an intramolecular triplex containing parallel T*A:T and G*G:C triplets: netropsin complexation with the triplex.

We have used circular dichroism and UV absorption spectroscopy to characterize the formation and melting behaviour of an intramolecular DNA triple helix containing parallel T*A:T and G*G:C triplets. Our approach to induce and to stabilize a parallel triplex involves the oligonucleotide 5'-d(G4A4G4[T4]C4T4C4-[T4]G4T4G4) ([T4] represents a stretch of four thymine residues). In a 10 mM sodium cacodylate, 0.2 mM disodium EDTA (pH 7) buffer, we have shown the following significant results. (i) While in the absence of MgCl2 this oligonucleotide adopts an intramolecular hairpin duplex structure prolonged by the single strand extremity 5'-d([T4]G4T4G4), the presence of millimolar concentrations of MgCl2generates an intramolecular triplex (via double hairpin formation). (ii) In contrast to the antiparallel triplex formed by the oligonucleotide 5'-d(G4T4G4[T4]G4A4G4[T4]C4T4C4), the parallel triplex melts in a biphasic manner (a triplex to duplex transition followed by a duplex to coil transition) and is less stable than the antiparallel one. The enthalpy change associated with triplex formation (-37 kcal/mol) is approximately half that of duplex formation (-81 kcal/mol). (iii) The parallel triple helix is disrupted by increasing the concentration of KCl(>10 mM), whereas, under the same conditions, the antiparallel triplex remains stable. (iv) Netropsin, a natural DNA minor groove-binding ligand, binds to the central site A4/T4of the duplex or triplex in an equimolar stoichiometry. Its association constant K is smaller for the parallel triplex ( approximately 1 x 10(7) M-1) than for the antiparallel one ( approximately 1 x 10(8) M-1). In contrast to the antiparallel structure, netropsin binding has no apparent effect on thermal stability of the parallel triple helix.     

Unexpected formation of parallel duplex in GAA and TTC trinucleotide repeats of Friedreich's ataxia

The onset and progress of Friedreich's ataxia (FRDA) is associated with the genetic instability of the (GAA).(TTC) trinucleotide repeats located within the frataxin gene. The instability of these repeats may involve the formation of an alternative DNA structure. Poly-purine (R)/poly-pyrimidine (Y) sequences typically form triplex DNA structures which may contribute to genetic instability. Conventional wisdom suggested that triplex structures formed by these poly-purine (R)/poly-pyrimidine (Y) sequences may contribute to their genetic instability. Here, we report the characterization of the single-stranded GAA and TTC sequences and their mixtures using NMR, UV-melting, and gel electrophoresis, as well as chemical and enzymatic probing methods. We show that the FRDA GAA/TTC, repeats are capable of forming various alternative structures. The most intriguing is the observation of a parallel (GAA).(TTC) duplex in equilibrium with the antiparallel Watson-Crick (GAA).(TTC) duplex. We also show that the GAA strands form self-assembled structures, whereas the TTC strands are essentially unstructured. Finally, we demonstrate that the FRDA repeats form only the YRY triplex (but not the RRY triplex) at neutral pH and the complete formation of the YRY triplex requires the ratio of GAA to TTC strand larger than 1:2. The structural features presented here and in other studies distinguish the FRDA (GAA)?(TTC) repeats from the fragile X (CGG).CCG), myotonic dystrophy (CTG).(CAG) and the Huntington (CAG).(CTG) repeats.     

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.     

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.     

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.     

Unwinding of a DNA triple helix by the Werner and Bloom syndrome helicases

Bloom syndrome and Werner syndrome are genome instability disorders, which result from mutations in two different genes encoding helicases. Both enzymes are members of the RecQ family of helicases, have a 3' --> 5' polarity, and require a 3' single strand tail. In addition to their activity in unwinding duplex substrates, recent studies show that the two enzymes are able to unwind G2 and G4 tetraplexes, prompting speculation that failure to resolve these structures in Bloom syndrome and Werner syndrome cells may contribute to genome instability. The triple helix is another alternate DNA structure that can be formed by sequences that are widely distributed throughout the human genome. Here we show that purified Bloom and Werner helicases can unwind a DNA triple helix. The reactions are dependent on nucleoside triphosphate hydrolysis and require a free 3' tail attached to the third strand. The two enzymes unwound triplexes without requirement for a duplex extension that would form a fork at the junction of the tail and the triplex. In contrast, a duplex formed by the third strand and a complement to the triplex region was a poor substrate for both enzymes. However, the same duplex was readily unwound when a noncomplementary 5' tail was added to form a forked structure. It seems likely that structural features of the triplex mimic those of a fork and thus support efficient unwinding by the two helicases.     

Sticky DNA formation in vivo alters the plasmid dimer/monomer ratio

Our discovery that plasmids containing the Friedreich's ataxia (FRDA) expanded GAA.TTC sequence, which forms sticky DNA, are prone to form dimers compared with monomers in vivo is the basis of an intracellular assay in Escherichia coli for this unusual DNA conformation. Sticky DNA is a single long GAA.GAA.TTC triplex formed in plasmids harboring a pair of long GAA.TTC repeat tracts in the direct repeat orientation. This requirement is fulfilled by either plasmid dimers of DNAs with a single trinucleotide repeat sequence tract or by monomeric DNAs containing a pair of direct repeat GAA.TTC sequences. DNAs harboring a single GAA.TTC repeat are unable to form this type of triplex conformation. An excellent correlation was observed between the ability of a plasmid to adopt the sticky triplex conformation as assayed in vitro and its propensity to form plasmid dimers relative to monomers in vivo. The variables measured that strongly influenced these measurements are as follows: length of the GAA.TTC insert; the extent of periodic interruptions within the repeat sequence; the orientation of the repeat inserts; and the in vivo negative supercoil density. Nitrogen mustard cross-linking studies on a family of GAA.TTC-containing plasmids showed the presence of sticky DNA in vivo and, thus, serves as an important bridge between the in vitro and in vivo determinations. Biochemical genetic studies on FRDA containing DNAs grown in recA or nucleotide excision repair or ruv-deficient cells showed that the in vivo properties of sticky DNA play an important role in the monomer-dimer-sticky DNA intracellular intercon-versions. Thus, the sticky DNA triplex exists and functions in living cells, strengthening the likelihood of its role in the etiology of FRDA.     

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

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

Structure-dependent recombination hot spot activity of GAA.TTC sequences from intron 1 of the Friedreich's ataxia gene

The recombinational properties of long GAA.TTC repeating sequences were analyzed in Escherichia coli to gain further insights into the molecular mechanisms of the genetic instability of this tract as possibly related to the etiology of Friedreich's ataxia. Intramolecular and intermolecular recombination studies showed that the frequency of recombination between the GAA.TTC tracts was as much as 15 times higher than the non-repeating control sequences. Homologous, intramolecular recombination between GAA.TTC tracts and GAAGGA.TCCTTC repeats also occurred with a very high frequency (approximately 0.8%). Biochemical analyses of the recombination products demonstrated the expansions and deletions of the GAA.TTC repeats. These results, together with our previous studies on the CTG.CAG sequences, suggest that the recombinational hot spot characteristics may be a common feature of all triplet repeat sequences. Unexpectedly, we found that the recombination properties of the GAA.TTC tracts were unique, compared with CTG.CAG repeats, because they depended on the DNA secondary structure polymorphism. Increasing the length of the GAA.TTC repeats decreased the intramolecular recombination frequency between these tracts. Also, a correlation was found between the propensity of the GAA.TTC tracts to adopt the sticky DNA conformation and the inhibition of intramolecular recombination. The use of novobiocin to modulate the intracellular DNA topology, i.e. the lowering of the negative superhelical density, repressed the formation of the sticky DNA structure, thereby restoring the expected positive correlation between the length of the GAA.TTC tracts and the frequency of intramolecular recombination. Hence, our results demonstrate that sticky DNA exists and functions in E. coli.     

An alternating sheared AA pair and elements of stability for a single sheared purine-purine pair flanked by sheared GA pairs in RNA

A previous NMR structure of the duplex 5'GGU GGA GGCU/PCCG AAG CCG5' revealed an unusually stable RNA internal loop with three consecutive sheared GA pairs. Here, we report NMR studies of two duplexes, 5'GGU GGA GGCU/PCCA AAG CCG5' (replacing the UG pair with a UA closing pair) and 5'GGU GAA GGCU/PCCG AAG CCG5' (replacing the middle GA pair with an AA pair). An unusually stable loop with three consecutive sheared GA pairs forms in the duplex 5'GGU GGA GGCU/PCCA AAG CCG5'. The structure contrasts with that reported for this loop in the crystal structure of the large ribosomal subunit of Deinococcus radiodurans [Harms, J., Schluenzen, F., Zarivach, R., Bashan, A., Gat, S., Agmon, I., Bartels, H., Franceschi, F., and Yonath, A. (2001) Cell 107, 679-688]. The middle AA pair in the duplex 5'GGU GAA GGCU/PCCG AAG CCG5' rapidly exchanges orientations, resulting in alternative base stacking and pseudosymmetry with exclusively sheared pairs. The U GAA G/G AAG C internal loop is 2.1 kcal/mol less stable than the U GGA G/G AAG C internal loop at 37 degrees C. Structural, energetic, and dynamic consequences upon functional group substitutions within related 3 x 3 and 3 x 6 internal loops are also reported.     

Energetics of strand-displacement reactions in triple helices: a spectroscopic study.

DNA triple helices offer exciting new perspectives toward oligonucleotide-directed inhibition of gene expression. Purine and GT triplexes appear to be the most promising motifs for stable binding under physiological conditions compared to the pyrimidine motif, which forms at relatively low pH. There are, however, very little data available for comparison of the relative stabilities of the different classes of triplexes under identical conditions. We, therefore, designed a model system which allowed us to set up a competition between the oligonucleotides of the purine and pyrimidine motifs targeting the same Watson-Crick duplex. Several conclusions may be drawn: (i) a weak hypochromism at 260 nm is associated with purine triplex formation; (ii) delta H degree of GA, GT and TC triplex formation (at pH 7.0) was calculated as -0.1, -2.5 and -6.1 kcal/mol per base triplet, respectively. This unexpectedly low delta H degree for the purine triple helix formation implies that its delta G degree is nearly temperature-independent and it explains why these triplexes may still be observed at high temperatures. In contrast, the pyrimidine triplex is strongly favoured at lower temperatures; (iii) as a consequence, in a system where two third-strands compete for triplex formation, displacement of the GA or GT strand by a pyrimidine strand may be observed at neutral pH upon lowering the temperature. This original purine-to-pyrimidine triplex conversion shows a significant hypochromism at 260 nm and a hyperchromism at 295 nm which is similar to the duplex-to-triplex conversion in the pyrimidine motif. Further evidence for this triplex-to-triplex conversion is provided by mung bean-nuclease foot-printing assay.     

Cleavage of single stranded oligonucleotides by EcoRI restriction endonuclease.

The 31mer 5'-TCA ACG CTA GAA TTC GGA TCC ATC GCT TGG T, the complementary 33mer 5'-CCA AGC GAT GGA TCC GAA TTC TAG CGT TGA GAT, the 40mer 5'-GGC CAG GAT GGT GAA GAA TTC GAT CCG GTA CGT AGC TAA G, and the complementary 42mer 5'-TAC TTA GCT ACG TAC CGG ATC GAA TTC TTC ACC ATC CTG GCC were synthesized and their reactivity towards EcoRI was studied. It was found that the 31mer and the 40mer were cleaved at a comparable rate to the 31mer-33mer hybrid and the 40mer-42mer hybrid, respectively. The rate of cleavage of the 33mer and the 42mer was an order of magnitude lower. To rule out possible intermolecular duplex formation, the 33mer was immobilized on cellulose by ligation and labeled with alpha 32P-dCTP using Klenow fragment of E. coli DNA polymerase. EcoRI cleaved this immobilized oligomer into specific fragments.