DNA triple helix stabilisation by a naphthylquinoline dimer

We have used DNase I footprinting to examine the effect of a novel naphthylquinoline dimer, designed as a triplex-specific bis-intercalator, on the stability of intermolecular DNA triplexes. We find that this compound efficiently promotes triplex formation between the 9-mer oligonucleotide 5'-TTTTTTCTT and its oligopurine duplex target at concentrations as low as 0.1 microM, enhancing the triplex stability by at least 1000-fold. This compound, which is the first reported example of a triplex bis-intercalator, is about 30 times more potent than the simple monofunctional ligand.     

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.     

Protection of DNA sequences by triplex-bridge formation.

We have demonstrated that the DNA sequence between two triplex-forming polypurine.polypyrimidine (Pu.Py) tracts was protected from DNA modifying enzymes upon formation of triplex DNA structures with an oligodeoxyribonucleotide in which two triplex-forming Pu or Py tracts were placed at the termini (triplex-bridge formation). In model experiments, when two triplex structures were formed between double-stranded DNA with the sequence (AG)17-(N)18-(T)34, and an oligodeoxyribonucleotide, (T)34-(N)18-(GA)17, not only the Pu.Py tracts but also the 18 bp non-Pu.Py sequence in the duplex DNA between the tracts was protected from restriction enzymes, HpaII methylase and DNase I. This protection occurred only when both of the Pu.Py tracts were involved as triplexes. The length of the tracts could be as short as 21 bp, while the difference in length between the non-Pu.Py sequences on the duplex and the oligodeoxyribonucleotide should be within 10 nucleotides. The efficiency of protection was enhanced in the presence of a cationic detergent, cetyltrimethylammonium bromide, during triplex formation. Protection was also observed with another type of the triplex bridge formed between (G)34 and (T)34 tracts with an oligodeoxyribonucleotide, (T)34-(N)20-(G)34. These findings suggest that the protection of specific DNA sequences from enzymes by triplex-bridge formation can be applied to any DNA sequence by placing it between two triplex-forming sequences.     

Oligonucleotide-directed triple helix formation at adjacent oligopurine and oligopyrimidine DNA tracts by alternate strand recognition.

A significant limitation to the practical application of triplex DNA is its requirement for oligopurine tracts in target DNA sequences. The repertoire of triplex-forming sequences can potentially be expanded to adjacent blocks of purines and pyrimidines by allowing the third strand to pair with purines on alternate strands, while maintaining the required strand polarities by combining the two major classes of base triplets, Py.PuPy and Pu.PuPy. The formation of triplex DNA in this fashion requires no unusual bases or backbone linkages on the third strand. This approach has previously been demonstrated for target sequences of the type 5'-(Pu)n(Py)n-3' in intramolecular complexes. Using affinity cleaving and DNase I footprinting, we show here that intermolecular triplexes can also be formed at both 5'-(Pu)n(Py)n-3' and 5'-(Py)n(Pu)n-3' target sequences. However, triplex formation at a 5'-(Py)n(Pu)n-3' sequence occurs with lower yield. Triplex formation is disfavored, even at acid pH, when a number of contiguous C+.GC base triplets are required. These results suggest that triplex formation via alternate strand recognition at sequences made up of blocks of purines and pyrimidines may be generally feasible.     

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.     

Construction of nucleosome cores from defined DNA sequences of prokaryotic origin.

A procedure for the de novo construction of nucleosome core particles from defined DNA sequences of prokaryotic origin is described. Efficient de novo reconstitution without added carrier DNA is demonstrated. DNase I and exonuclease III analysis of a nucleosome core prepared from a 154 base pair fragment extending from base 853 to base 1006 of pBR322 indicates a non-random positioning of the histone core along the DNA. As bacteria have no histones, their DNA cannot be expected to have a histone core positioning signal encoded in it, the efficient formation of a uniquely positioned core particle is not self evident. The possibility that a phosphate end group positions DNA fragments on the histone is considered. The de novo reconstitution of carrier-less defined nucleosome core particles should facilitate the physicochemical study of nucleosomes on the fine structural level.     

Left-handed Z-DNA and intramolecular triplex formation at the site of an unequal sister chromatid exchange

An unequal sister chromatid exchange (USCE) in the mouse myeloma cell line MPC-11 between 3' regions of the C gamma 2a and C gamma 2b heavy chain genes results in duplication of the C gamma 2a heavy chain gene and generation of a novel recombination joint. The USCE occurs between (TC)n tracts adjacent to alternating purine-pyrimidine tracts. We have investigated the capacity of both the donor regions and the recombinant product involved in this event to adopt left-handed Z-DNA and intramolecular triplexes. The results of chemical probing with diethylpyrocarbonate and osmium tetroxide at the base pair level demonstrate that under the influence of negative supercoiling the alternating purine-pyrimidine regions of these plasmids can adopt Z-DNA at neutral pH, and the oligopurine.oligopyrimidine (pur.pyr) regions of these regions can adopt intramolecular triplexes at low pH (less than or equal to pH 6.0). At intermediate pH values, mixtures of both structures are present. Increasing the negative superhelical density of the plasmid does not increase the amount of triplex present at neutral pH indicating that the presence of long Z-DNA segments adjacent to pur.pyr tract prevents intramolecular triplex formation. In summary, we conclude that the sequences involved in the USCE can form either an intramolecular triplex in the (TC)n tract or Z-DNA in the alternating purine-pyrimidine tract and that Z-DNA will predominate under physiological conditions. The presence of segments which adopt Z-DNA at a site of USCE suggests that formation of this structure may enhance recombination between adjacent pur.pyr tracts.     

Alternate strand recognition of double-helical DNA by (T,G)-containing oligonucleotides in the presence of a triple helix-specific ligand.

Triple helix formation requires a polypurine- polypyrimidine sequence in the target DNA. Recent works have shown that this constraint can be circumvented by using alternate strand triplex-forming oligonucleotides. We have previously demonstrated that (T,G)-containing triplex- forming oligonucleotides may adopt a parallel or an antiparallel orientation with respect to an oligopurine target, depending upon the sequence and, in particular, upon the number of 5'-GpT-3' and 5'-TpG-3' steps [Sun et al. (1991) C.R. Acad. Sci. Paris Ser III, 313, 585-590]. A single (T,G)-containing oligonucleotide can therefore interact with two oligopurine stretches which alternate on the two strands of the target DNA. The (T,G) switch oligonucleotide contains a 5'-part targeted to one of the oligopurine sequences in a parallel orientation followed by a 3'-part that adopts an antiparallel orientation with respect to the second oligopurine sequence. We show that a limitation to the stability of such a triplex may arise from the instability of the antiparallel part, composed of reverse-Hoogsteen C.GxG and T.AxT base triplets. Using DNase I footprinting and ultraviolet absorption experiments, we report that a benzo[e]pyridoindole derivative [(3-methoxy- 7H-8-methyl-11-[(3'-amino-propyl) amino] benzo[e]pyrido [4,3-b]indole (BePI)], a drug interacting more tightly with a triplex than with a duplex DNA, strongly stabilizes triplexes with reverse-Hoogsteen C.GxG and T.AxT triplets thus allowing a stabilization of the triplex-forming switch (T,G) oligonucleotide on alternating oligopurine- oligopyrimidine 5'-(Pu)14(Py)14-3' duplex sequences. These results lead to an extension of the range of oligonucleotide sequences for alternate strand recognition of duplex DNA.     

Extension of the range of DNA sequences available for triple helix formation: stabilization of mismatched triplexes by acridine-containing oligonucleotides.

Triple helix formation usually requires an oligopyrimidine*oligopurine sequence in the target DNA. A triple helix is destabilized when the oligopyrimidine*oligopurine target contains one (or two) purine*pyrimidine base pair inversion(s). Such an imperfect target sequence can be recognized by a third strand oligonucleotide containing an internally incorporated acridine intercalator facing the inverted purine*pyrimidine base pair(s). The loss of triplex stability due to the mismatch is partially overcome. The stability of triplexes formed at perfect and imperfect target sequences was investigated by UV thermal denaturation experiments. The stabilization provided by an internally incorporated acridine third strand oligonucleotide depends on the sequences flanking the inverted base pair. For triplexes containing a single mismatch the highest stabilization is observed for an acridine or a propanediol tethered to an acridine on its 3'-side facing an inverted A*T base pair and for a cytosine with an acridine incorporated to its 3'-side or a guanine with an acridine at its 5'-side facing an inverted G*C base pair. Fluorescence studies provided evidence that the acridine was intercalated into the triplex. The target sequences containing a double base pair inversion which form very unstable triplexes can still be recognized by oligonucleotides provided they contain an appropriately incorporated acridine facing the double mismatch sites. Selectivity for an A*T base pair inversion was observed with an oligonucleotide containing an acridine incorporated at the mismatched site when this site is flanked by two T*A*T base triplets. These results show that the range of DNA base sequences available for triplex formation can be extended by using oligonucleotide intercalator conjugates.     

Interaction of Hoechst 33258 and echinomycin with nucleosomal DNA fragments containing isolated ligand binding sites

We have examined the interaction of Hoechst 33258 and echinomycin with nucleosomal DNA fragments which contain isolated ligand binding sites. A 145 base pair fragment was prepared on the basis of the sequence of tyrT DNA, which contained no CpG or (A/T)(4) binding sites for these ligands. Isolated binding sites were introduced into this fragment at discrete locations where the minor groove is known to face toward or away from the protein core when reconstituted onto nucleosome core particles. The interaction of ligands with target sites on these nucleosomal DNA fragments was assessed by DNase I footprinting. We find that Hoechst 33258 can bind to single nucleosomal sites which face both toward and away from the protein core, without affecting the nucleosome structure. Hoechst binding is also observed on nucleosomal fragments which contain two or more drug binding sites, though in these cases the footprints are accompanied by the presence of new cleavage products in positions which suggest that the ligand has caused a proportion of the DNA molecules to adopt a new rotational positioning on the protein surface. Hoechst 33258 does not affect nucleosome reconstitution with any of these fragments. In contrast, the bifunctional intercalating antibiotic echinomycin is not able to bind to single nucleosomal CpG sites. Echinomycin footprints are observed on nucleosomal fragments containing two or more CpG sites, but there are no changes in the cleavage patterns in the remainder of the fragment. Echinomycin abolishes nucleosome reconstitution when included in the reconstitution mixture.     

Intramolecular DNA triplexes in supercoiled plasmids. I. Effect of loop size on formation and stability

The capacity of four oligopurine.oligopyrimidine (pur.pyr) sequences with different lengths of interruptions in the center [GAA)4(N)n(GAA)4G) (n = 3, 5, 7, and 9) to adopt intramolecular DNA triplexes was evaluated in recombinant plasmids. The hyperreactive patterns of the pur.pyr inserts to specific chemical probes (OsO4, diethyl pyrocarbonate, and dimethyl sulfate) at the base pair level demonstrate that intramolecular triplexes with identical 12-base triads in the stem but with different loop sizes (4, 6, 8, and 10 bases) can form in supercoiled plasmids. Furthermore, the extent of OsO4 modification was measured as a function of temperature and of average negative supercoil density. In addition, the transition free energy of B-DNA to triplexes at pH 4.5 was determined by two-dimensional electrophoresis. These comparative studies show that longer loops require more supercoil energy for triplex formation and are less thermostable than triplexes with shorter loops. Also, it may be that not only the loop size but the base composition of the loop region affects the structural transition and triplex stability. Thus, these results significantly broaden the range of natural pur.pyr sequences that may adopt triplexes.     

Metal ions cause the isomerization of certain intramolecular triplexes.

The influence of cations on the capacity of five oligopurine.oligopyrimidine mirror repeat sequences to adopt intramolecular triplexes (the usual H-y3 isomer and/or the rare H-y5 isomer) in recombinant plasmids was investigated. Unexpectedly, the presence of certain metal ions (magnesium, zinc, manganese, and calcium) stabilized the (GAA)4TTCGC(GAA)4 insert in the rare H-y5 when cloned into either of two different sequence backgrounds. Alternatively, either shortening or lengthening the sequence at the central interruption, which becomes the loop of the triplex, led to the formation of the canonical H-y3 under all conditions tested. Similarly, other oligopurine.oligopyrimidine mirror repeat sequences (i.e. (GAA)8 or (GGA)8) formed only the H-y3 under all experimental conditions. All triplexes were stabilized by negative supercoiling at pH 5.0; chemical probe and primer extension analyses served as critical structural tools. Hence, the nature of the central interruption sequence (the loop) of the mirror repeat is important for the stabilization of the H-y5. This region may be important for metal ion binding in the initial stages of triplex formation and thus may play a critical role in determining which isomer is formed. The possible biological role of a DNA sequence adopting three different conformations is discussed.     

Photofootprinting of DNA triplexes.

We have used a photofootprinting assay to study intermolecular and intramolecular DNA triplexes. The assay is based on the fact that the DNA duplex is protected against photodamage (specifically, against the formation of the (6-4) pyrimidine photoproducts) within a triplex structure. We have shown that this is the case for PyPuPu (YRR) as well as PyPuPy (YRY) triplexes. Using the photofootprinting assay, we have studied the triplex formation under a variety of experimentally defined conditions. At acid pH, d(C)n.d(G)n.d(C)n and d(CT)n.d(GA)n.d(CT)n triplexes are detected by this method. The d(CT)n.d(GA)n.d(CT)n triplexes are additionally stabilized by divalent cations and spermidine. PyPuPu triplexes are pH-independent and are stabilized by divalent cations, such as Mg++ and Zn++. The effect depends on the type of cation and on the DNA sequence. The d(CT)n.d(GA)n.d(GA)n triplex is stabilized by Zn++, but not by Mg++, whereas the d(C)n.d(G)n.d(G)n triplex is stabilized by Mg++. In H-DNA, virtually the entire pyrimidine chain is protected against photodimerization, whereas only half of the pyrimidine chain participating in a triplex is protected in the CGG intramolecular triplex.     

lac Operator nucleosomes. 2. lac Nucleosomes can change conformation to strengthen binding by lac repressor

We have shown previously that lac repressor binds specifically and quantitatively to lac operator restriction fragments which have been complexed with histones to form artificial nucleosomes (203 base pair restriction fragment) or core particles (144 base pair restriction fragment. We describe here a quantitative method for determining the equilibrium binding affinities of repressor for these lac reconstitutes. Quantitative analysis shows that the operator-histone reconstitutes may be grouped into two affinity classes: those with an affinity for repressor close to that of naked DNA and those with an affinity 2 or more orders of magnitude less than that of naked DNA. All particles in the lac nucleosome preparations bind repressor with high affinity, but the lac core particle preparations contain particles of both high and low affinities for repressor. Formaldehyde cross-linking causes all high-affinity species to suffer a 100-fold decrease in binding affinity. In contrast, there is no effect of cross-linking on species of low affinity. Therefore, the ability of a particle to be bound tightly by repressor depends on a property of the particle which is eliminated by cross-linking. Control experiments have shown that chemical damage to the operator does not accompany cross-linking. Therefore, the property sensitive to cross-linking must be the ability of the particle to change conformation. We infer that the particles of low native affinity, like cross-linked particles, are of low affinity because of an inability to facilitate repressor binding by means of this conformational change. Dimethyl suberimidate cross-linking experiments show that histone-histone cross-linking is sufficient to preclude high-affinity binding. Thus, the necessary conformational change involves a nucleosome histone core event. We find that the ability of a particle to undergo a repressor-induced facilitating conformational change appears to depend on the position of the operator along the DNA binding path of the nucleosome core. We present a general model which proposes that nucleosomes are divided into domains which function differentially to initiate conformational changes in response to physiological stimuli.     

Wrapping of genomic polydA.polydT tracts around nucleosome core particles.

Five human clones containing genomic regions of polydA have been isolated by their ability to form intermolecular triple helices with agarose cross-linked polyU. All of these clones contain Alu repetitive DNA sequences. End-labelled DNA fragments containing these sequences have been successfully reconstituted onto nucleosome core particles by salt exchange. The structure of these has been examined by digesting with DNase I, hydroxyl radicals or diethylpyrocarbonate. DNase I cleavage of the polydA tracts is poor in the free DNA but is markedly enhanced at certain positions when complexed with nucleosome cores. Phased digestion patterns are observed which continue through the (A)n blocks and reveal an average helical periodicity of about 10 base pairs. The distance between adjacent maxima varies between 8-12 base pairs, suggesting that the exact helical repeat is not necessarily constant. One fragment containing the sequence (TA)11T34 reveals a 12 base pair repeat within the (AT)n region. A pUC19 polylinker fragment containing a block of A69.T69 cloned into the Smal site could also be reconstituted onto nucleosome cores and reveals the same phased DNaseI digestion pattern. The DNase I cleavage pattern is not identical at each of the maxima, suggesting that the structural distortions imposed by the core particles are not constant along the DNA.     

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.     

Effect of length, supercoiling, and pH on intramolecular triplex formation. Multiple conformers at pur.pyr mirror repeats

The conformations adopted by five oligopurine.oligopyrimidine (pur.pyr) inserts of various lengths and sequence repeats in recombinant plasmids were evaluated as a function of pH and negative super-helicaldensity. Patterns of chemical reactivity (OsO4 and diethylpyrocarbonate) indicate that long (greater than 36 base pairs) pur.pyr segments can adopt intramolecular triplexes and that increasing the length of the pur.pyr tract reduces the dependence on low pH for structure formation, such that (GA)37 adopts an intramolecular triplex under moderate levels of negative superhelical stress (-sigma = 0.049) at neutral pH. This demonstrates that long pur.pyr segments, which are abundant in eukaryotic genomes, have the potential to adopt triplexes in vivo. Two-dimensional gel electrophoresis of the plasmids combined with chemical probing indicates that for longer sequences, multiple conformers of the intramolecular triplex exist at low pH. These conformers result from nucleation at various positions on the polypurine stretch, giving rise to different extents of relaxation at the same linking number. In addition, the metal ions Co2+, Mn2+, and Mg2+ have profound effects on the pattern of chemical reactivity displayed by long pur.pyr segments at both neutral and low pH, indicating that quite different structures may form in the presence of divalent metal ions. Thus, the types and extent of unusual structures adopted by long pur.pyr segments are complex and heterogeneous, and are dependent on pH, supercoiling, and the presence of divalent cations.     

Metalloregulation of triple helix formation by control of the loop conformation

The flexible polypyridine ligand, 2,2':6',2(')-terpyridine (terpy), was built into the backbone of oligonucleotides to form DNA conjugates. The terpy unit functioned as a good loop when the conjugates formed the bimolecular triplexes with complementary oligopurine. The triplex structure was destabilized by the specific interaction with divalent transition metal ions (Cu(2+), Zn(2+), and Fe(2+)), in particular Cu(2+) ions. This ion destabilized one of the triplexes by 4.2 kcalmol(-1) or made the triplex formation constant less than 1/10(3) at 298 K. This result is attributed to the substantial turbulence of the terminal structure of the triplexes.