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.     

Base triplet nonisomorphism strongly influences DNA triplex conformation: effect of nonisomorphic G* GC and A* AT triplets and bending of DNA triplexes

Structural understanding of DNA triplexes is grossly inadequate despite their efficacy as therapeutic agents. Lack of structural similarity (isomorphism) of base triplets that figure in different DNA triplexes brings in an added complexity. Recently, we have shown that the residual twist (Deltat degrees ) and the radial difference (Deltar A) adequately define base triplet nonisomorphism in structural terms and allow assessment of their role in conferring stability as well as sequence-dependent structural variations in DNA triplexes. To further corroborate these, molecular dynamics (MD) simulations are carried out on DNA triplexes comprising nonisomorphic G* GC and A* AT base triplets under different sequential contexts. Base triplet nonisomorphism between G* GC and A* AT triplets is dominated by Deltat degrees (9.8 degrees ), in view of small Deltar (0.2 A), and is in contrast to G* GC and T* AT triplets where both Deltat degrees (10.6 degrees ) and Deltar (1.1A) are prominent. Results show that Deltat degrees alone enforces mechanistic influence on the triplex-forming purine strand so as to favor a zigzag conformation with alternating conformational features that include high (40 degrees ) and low (20 degrees ) helical twists, and high anti(G) and anti(A) glycosyl conformation. Higher thermal stability of this triplex compared to that formed with G* GC and T* AT triplets can be traced to enhanced base-stacking and counterion interactions. Surprisingly, it is found for the first time that the presence of a nonisomorphic G* GC or A* AT base triplet interrupting an otherwise mini A* AT or G* GC isomorphic triplex can induce a bend/curvature in a DNA triplex. These observations should prove useful in the design of triplex-forming oligonucleotides and in the understanding the binding affinities of this triplex with proteins.     

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.     

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.     

Stability of G,A triple helices.

In this work we selected double-stranded DNA sequences capable of forming stable triplexes at 20 or 50 degrees C with corresponding 13mer purine oligonucleotides. This selection was obtained by a double aptamer approach where both the starting sequences of the oligonucleotides and the target DNA duplex were random. The results of selection were confirmed by a cold exchange method and the influence of the position of a 'mismatch' on the stability of the triplex was documented in several cases. The selected sequences obey two rules: (i) they have a high G content; (ii) for a given G content the stability of the resulting triplex is higher if the G residues lie in stretches. The computer simulation of the Mg2+, Na+and Cl-environment around three triplexes by a density scaled Monte Carlo method provides an interpretation of the experimental observations. The Mg2+cations are statistically close to the G N7 and relatively far from the A N7. The presence of an A repels the Mg2+from adjacent G residues. Therefore, the triplexes are stabilized when the Mg2+can form a continuous spine on G N7.     

Chemical modification of the third strand: differential effects on purine and pyrimidine triple helix formation.

DNA triple helices offer exciting perspectives toward oligonucleotide-directed control of gene expression. Oligonucleotide analogues are routinely used with modifications in either the backbone or the bases to form more stable triple-helical structures or to prevent their degradation in cells. In this article, different chemical modifications are tested in a model system, which sets up a competition between the purine and pyrimidine motifs. For most modifications, the DeltaH degrees of purine triplex formation is close to zero, implying a nearly temperature-independent affinity constant. In contrast, the pyrimidine triplex is strongly favored at lower temperatures. The stabilization induced by modifications previously known to be favorable to the pyrimidine motif was quantified. Interestingly, modifications favorable to the GT motif (propynyl-U and dU replacing T) were also discovered. In a system where two third strands compete for triplex formation, replacement of the GA or GT strand by a pyrimidine strand may be observed at neutral pH upon lowering the temperature. This purine-to-pyrimidine triplex conversion depends on the chemical nature of the triplex-forming strands and the stability of the corresponding triplexes.     

Pyrimidine oligodeoxyxylonucleotides form triplexes with purine DNA in neutral media

Interactions of oligonucleotides comprising (1-beta-D-2'-deoxy-threo-pentafuranosyl)thymine and (1-beta-D-2'-deoxy-threo-pentafuranosyl)cytosine residues (oligodeoxyxylonucleotides or OXNs) with complementary single-stranded DNA fragments were investigated. Using nondenaturing gel electrophoresis, footprinting, and melting assays, pyrimidine OXNs were shown to form triplexes with the purine DNA template, which are stable at neutral pH and comparable in heat stability with the corresponding natural polypurine-polypyrimidine DNA duplexes. In such triplexes, the N3 of cytosines in one of the OXNs are protonated. As revealed by CD spectroscopy in the 210-340 nm range, the form of the triple helix depends on the nucleotide composition and sequence of the DNA template, and is intermediate between A and B.     

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.     

Physico-chemical studies on DNA triplexes containing an alternate third strand with a non-nucleotide linker

Differential scanning calorimetric (DSC), circular dichroism (CD) and molecular mechanics studies have been performed on two triple helices of DNA. The target duplex consists of 16 base pairs in alternate sequence of the type 5′-(purine)_m(pyrimidine)_m-3′. In both the triplexes, the third oligopyrimidine strand crosses the major groove at the purine–pyrimidine junction, with a simultaneous binding of the adjacent purine tracts on alternate strands of the Watson–Crick duplex. The switch is ensured by a non-nucleotide linker, the 1,2,3 propanetriol residue, that joins two 3′–3′ phosphodiester ends. The third strands differ from each other for a nucleotide in the junction region. The resulting triple helices were termed 14-mer-PXP and 15-mer-PXP (where P=phosphate and X=1,2,3-propanetriol residue) according to the number of nucleotides that compose the third strand. DSC data show two independent processes: the first corresponding to the dissociation of the third strand from the target duplex, the second to the dissociation of the double helix in two single strands. The two triple helices show the same stability at pH 6.6. At pH 6.0, the 15-mer-PXP triplex is thermodynamically more stable than the 14-mer-PXP triplex. Thermodynamic data are discussed in relation to structural models. The results are useful when considering the design of oligonucleotides that can bind in an antigene approach to the DNA for therapeutic purposes.     

Single-Stranded DNA and RNA Targeted Triplex-Formation: UV,CD and Molecular Modeling Studies of Foldback Triplexes Containing Different RNA, 2′-OMe-RNA and DNA Strand Combinations

Abstract

We studied the influence of different 2′-OMe-RNA and DNA strand combinations on single strand targeted foldback triplex formation in the Py.Pu:Py motif using ultraviolet (UV) and circular dichroism (CD) spectroscopy, and molecular modeling. The study of eight combinations of triplexes (D D:D, R* D:D, D D:R*, R* D:R*, D R:D, R* R:D, DR:R*, and R*-R:R*; where the first, middle, and last letters stand for the Hoogsteen Pyrimidine, Watson-Crick [WC] purine and WC pyrimidine strands, respectively, and D, R and R* stand for DNA, RNA and 2′-OMe-RNA strands, respectively) indicate more stable foldback triplex formation with a DNA purine strand than with an RNA purine strand. Of the four possible WC duplexes with RNA/DNA combinations, the duplex with a DNA purine strand and a 2′-O-Me-RNA pyrimidine strand forms the most thermally stable triplex, although its thermal stability is the lowest of all four duplexes. Irrespective of the duplex combination, a 2′-OMe-RNA Hoogsteen pyrimidine strand forms a stable foldback triplex over a DNA Hoogsteen pyrimidine strand confirming the earlier reports with conventional and circular triplexes. The CD studies suggest a B-type conformation for an all DNA homo-foldback triplex (D.D.D), while hetero-foldback triplex spectra suggest intermediate conformation to both Atype and B-type structures. A novel molecular modeling study has been carried out to understand the stereochemical feasibility of all the combinations of foldback triplexes using a geometric approach. The new approach allows use of different combinations of chain geometries depending on the nature of the chain (RNA vs. DNA).     

Triple helix forming TRIPside molecules that target mixed purine/pyrimidine DNA sequences

A new strategy to form stable and sequence-specific triple helical DNA structures at mixed purine/pyrimidine sequences using a combination of four C-glycosides (TRIPsides) has been described [Li et al. (2003) J. Am. Chem. Soc. 125, 2084]. The partial realization of the approach is demonstrated by incorporating two of the four TRIPsides into oligomers that can potentially fold into intramolecular triplexes that contain one or two major groove crossovers of the purine Hoogsteen H-bond information. Using temperature-dependent electronic and fluorescence spectroscopy and differential scanning calorimetry, it is demonstrated that stable triplexes form at physiological conditions at non-homopurine targets. In addition, triplexes using the TRIPsides form in a highly sequence specific manner.     

Influence of loop sequence on relative stability of bimolecular triplex DNA

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Single-base discrimination mediated by proofreading inert allele specific Primers

The role of 3' exonuclease excision in DNA polymerization was evaluated for primer extension using inert allele specific primers with exonuclease-digestible ddNMP at their 3' termini. Efficient primer extension was observed in amplicons where the inert allele specific primers and their corresponding templates were mismatched. However, no primer-extended products were yielded by matched amplicons with inert primers. As a control, polymerase without proofreading activity failed to yield primer-extended products from inert primers regardless of whether the primers and templates were matched or mismatched. These data indicated that activation was undertaken for the inert allele specific primers through mismatch proofreading. Complementary to our previously developed SNP-operated on/off switch, in which DNA polymerization only occurs in matched amplicon, this new mutation detection assay mediated by exo(+) DNA polymerases has immediate applications in SNP analysis independently or in combination of the two assays.     

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.     

Recognition of Double-Stranded Dna by Peptide Nucleic Acid

Abstract

Peptide nucleic acid (PNA) is an oligonucleotide mimic in which the backbone of DNA has been replaced by a pseudopeptide. We here show that there are distinct variations as to how PNA oligomers interact with double-stranded DNA depending on choice of nucleobases. Thymine-rich homopyrimidine PNA oligomers recognise double-stranded polynucleotides by forming PNA₂-DNA triplexes with the DNA purine strand. By contrast, cytosine-rich homopyrimidine PNAs add to double-stranded polynucleotides as Hoogsteen strands, forming PNA-DNA₂ triplexes, while homopurine, or alternating thymine-guanine, PNA oligomers invade DNA to form PNA-DNA duplexes.     

Pyrimidine motif triplexes containing polypurine RNA or DNA with oligo 2'-O-methyl or DNA triplex forming oligonucleotides

Triplex forming oligonucleotides (TFOs) are potentially useful in targeting RNA for antisense therapeutic applications. To determine the feasibility of targeting polypurine RNA with nuclease-resistant oligonucleotides, TFOs containing 2'-deoxy or 2'-O-methyl (2'-OMe) backbones, designed to form pyrimidine motif triplexes with RNA, were synthesized. TFOs were made which can form trimolecular triplexes, or bimolecular, 'clamp' triplexes with polypurine RNA and DNA. It was found that the relative stabilities of the triplexes formed followed the order: M.DM(clamp)>D.DD approximately M.DD>M. RM>D.DM>M.RD approximately M.DM, where M is a 2'-OMe, D is a DNA and R is an RNA backbone. The third strand is listed first, separated by a dot from the purine strand of the Watson-Crick duplex, followed by the pyrimidine strand of the duplex. The results described here provide insight into the feasibility of using TFOs containing a 2'-OMe backbone as antisense agents.     

Linear nicking endonuclease-mediated strand-displacement DNA amplification

We describe a method for linear isothermal DNA amplification using nicking endonuclease-mediated strand displacement by a DNA polymerase. The nicking of one strand of a DNA target by the endonuclease produces a primer for the polymerase to initiate synthesis. As the polymerization proceeds, the downstream strand is displaced into a single-stranded form while the nicking site is also regenerated. The combined continuous repetitive action of nicking by the endonuclease and strand-displacement synthesis by the polymerase results in linear amplification of one strand of the DNA molecule. We demonstrate that DNA templates up to 5000 nucleotides can be linearly amplified using a nicking endonuclease with 7-bp recognition sequence and Sequenase version 2.0 in the presence of single-stranded DNA binding proteins. We also show that a mixture of three templates of 500, 1000, and 5000 nucleotides in length is linearly amplified with the original molar ratios of the templates preserved. Moreover, we demonstrate that a complex library of hydrodynamically sheared genomic DNA from bacteriophage lambda can be amplified linearly.