Exclusion of RNA strands from a purine motif triple helix.

Research concerning oligonucleotide-directed triple helix formation has mainly focused on the binding of DNA oligonucleotides to duplex DNA. The participation of RNA strands in triple helices is also of interest. For the pyrimidine motif (pyrimidine.purine.pyrimidine triplets), systematic substitution of RNA for DNA in one, two, or all three triplex strands has previously been reported. For the purine motif (purine.purine.pyrimidine triplets), studies have shown only that RNA cannot bind to duplex DNA. To extend this result, we created a DNA triple helix in the purine motif and systematically replaced one, two, or all three strands with RNA. In dramatic contrast to the general accommodation of RNA strands in the pyrimidine triple helix motif, a stable triplex forms in the purine motif only when all three of the substituent strands are DNA. The lack of triplex formation among any of the other seven possible strand combinations involving RNA suggests that: (i) duplex structures containing RNA cannot be targeted by DNA oligonucleotides in the purine motif; (ii) RNA strands cannot be employed to recognize duplex DNA in the purine motif; and (iii) RNA tertiary structures are likely to contain only isolated base triplets in the purine motif.     

Hydrated Water Molecules of Pyrimidine/Purine/Pyrimidine DNA Triple Helices as Revealed by FT-IR Spectroscopy: A Role of Cytosine Methylation

Abstract

Hydrated water molecules of pyrimidine/purine/pyrimidine DNA hairpin triplex was studied by a comparison of triplex (CC·AG6) formed by a host oligodeoxypyrimidine of 5′- d(TC)₃T₄(CT)₃ (CC) with a target hexadeoxypurine 5′-d(AG)₃ (AG6) strand and by triplexes (MM·AG6, MC·AG6, and CM·AG6) formed by oligonucleotides with the exact sequences as above except 5-methylcytosine replaced all (MM), 5′ end half (MC), and 3′ end half (CM) cytosine bases in CC via FT-IR spectroscopy in hydrated film. Results revealed that: (i) all these triplexes have a similar hydration pattern, in which water molecules probably bound in the N₇ sites of adenines and guanines in the Crick-Hoogsteen groove, and to the methyl group of thymidines in the Watson-Hoogsteen groove. There are also some bound water molecules found at the O₂ sites of thymines in both Watson-Crick and Crick-Hoogsteen grooves, (ii) In the CC·AG6 triplex the S-type sugars are always dominant in all hydrated states, whereas in MM·AG6 triplex the relative population of the N-type sugars is very close to that of the S-type between 86% and 66% of humidity. Furthermore, the sugar conformation in two partially modified triplexes (CM·AG6, and MC·AG6) are dominant by the N-type at lower humidity. This phenomenon might reflect that the degree of bound water varies among the binding sites of bases, (iii) The effect of introducing a methyl group on cytosine is to generates spine of hydrophobic region in MM (MC and MC). The enlarging hydrophobic area not only increase the stability in solution, and also the stability in sodium hydrated films of the pyrimidine/purine/pyrimidine hairpin triplexes.     

Pyrimidine morpholino oligonucleotides form a stable triple helix in the absence of magnesium ions

Oligonucleotides can be used as sequence-specific DNA ligands by forming a local triple helix. In order to form more stable triple-helical structures or prevent their degradation in cells, oligonucleotide analogues that are modified at either the backbone or base level are routinely used. Morpholino oligonucleotides appeared recently as a promising modification for antisense applications. We report here a study that indicates the possibility of a triple helix formation with a morpholino pyrimidine TFO and its comparison with a phosphodiester and a phosphoramidate oligonucleotide. At a neutral pH and in the presence of a high magnesium ion concentration (10 mM), the phosphoramidate oligomer forms the most stable triple helix, whereas in the absence of magnesium ion but at a physiological monovalent cation concentration (0.14 M) only morpholino oligonucleotides form a stable triplex. To our knowledge, this is the first report of a stable triple helix in the pyrimidine motif formed by a noncharged oligonucleotide third strand (the morpholino oligonucleotide) and a DNA duplex. We show here that the structure formed with the morpholino oligomer is a bona fide triple helix and it is destabilized by high concentrations of potassium ions or divalent cations (Mg(2+)).     

Comparative Studies of Duplex and Triplex Formation of 2′-5′ and 3′-5′ Linked Oligoribonucleotides

Abstract

We have studied double and triple helix formation between 2′–5′ or 3–5′ linked oligoriboadenylates and oligoribouridylates with chain length 7 or 10 by CD spectrometry. The complex formation depends on the type of linkage of oligoribonucleotides, chain length, concentration and molar ratio of the strands, temperature and the cationic concentration. Mixture of any linkage isomers of oligo(rA) and oligo(rU) in 1:1 molar ratio form duplex at 0.1 M NaCl. The duplex stability largely depends on the type of the linkages and is in the following order; [35′] oligo(rA)·[3′-5′] oligo(rU) > [2′-5′] oligo(rA)'[3′-5′] oligo(rU) > [3′-5′] oligo(rA)·[2′-5′] oligo(rU) > [2–5′] oligo(rA)*[2′-5′] oligo(rU). The higher cationic concentrations, 0.5 M MgCl₂, stabilize the complex and either duplex or triplex is formed depending on the input strand ratio and the type of linkage. Thermodynamic parameters, DH and DS, for the complex formation between linkage isomers of oligo(rA) and oligo(rU) showed a linear relationship indicating an enthalpy-entropy compensation phenomena. The duplex and triplex composed of [2′-5′] oligo(rA) and [2′-5′] oligo(rU) exhibit different CD spectra compared to those of any others containing 3–5′ linkage, suggesting that the fully 2–5′ duplex and triplex may possess a unique conformation. We describe prebiological significance of the linkage isomers of RNA and selection of the 3–5′ linkage against 2′-5 linkage.     

Kinetics of deamination and Cu(II)/H2O2/Ascorbate-induced formation of 5-methylcytosine glycol at CpG sites in duplex DNA

Mutation in p53 tumor suppressor gene is a hallmark of human cancers. Six major mutational hotspots in p53 contain methylated CpG (mCpG) sites, and C →T transition is the most common mutation at these sites. It was hypothesized that the formation of 5-methylcytosine glycol induced by reactive oxygen species, its spontaneous deamination to thymine glycol and the miscoding property of the latter may account, in part, for the ubiquitous C →T mutation at CpG site. Here, we assessed the kinetics of deamination for two diastereomers of 5-methylcytosine glycol in duplex DNA. Our results revealed that the half-lives for the deamination of the (5S,6S) and (5R,6R) diastereomers of 5-methylcytosine glycol in duplex DNA at 37°C were 37.4 ± 1.6 and 27.4 ± 1.0 h, respectively. The deamination rates were only slightly lower than those for the two diastereomers in mononucleosides. Next, we assessed the formation of 5-methyl-2′-deoxycytidine glycol in the form of its deaminated product, namely, thymidine glycol (Tg), in methyl-CpG-bearing duplex DNA treated with Cu(II)/H₂O₂/ascorbate. LC-MS/MS quantification results showed that the yield of Tg is similar as that of 5-(hydroxymethyl)-2′-deoxycytidine. Together, our data support that the formation and deamination of 5-methylcytosine glycol may contribute significantly to the C →T transition mutation at mCpG dinucleotide site.     

Systematic Mutation in the Third Strand of a Purine Motif DNA Triple Helix: a Story of a Molecule Which Hides Its Tail

Abstract

A DNA triple helix formed according to the Purine-motif can accommodate both purines and pyrimidines in the third strand in a pH independent manner. This motif is thus a more versatile means of targeting double stranded DNA than the pH dependent Pyrimidine motif. In this paper we assess the impact of systematically replacing thymine with adenine, inosine or cytosine in the third strand. To this aim we have designed a double length, 22—mer “purine” strand to target a 9-mer pyrimidine strand such that the extending tail acts as the third strand (reversed-Hoogsteen strand) which is antiparallel to the purine strand of the underlying WC duplex. By systematically replacing thymines with adenines in the reversed-Hoogsteen strand there is an increase in the stability (T _m) of the triplex, particularly when the sequence closest to the loop consists of a stack of purines. Further substitution towards the 3′ end of the third strand reverses the stability. Systematic mutations in the third strand next to the loop reveal that the stability of the triads can be ranked according to their effect on T_m in the following order. A-AT > T-AT = I-AT. > C-AT where C is considered a mismatch.

     

A Parallel Stranded Linear DNA Duplex Incorporating dG · dC Base Pairs

Abstract

DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA · dT base pairs. We have substituted four dA · dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1 · D2) with dG · dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG · dC base pairs (ps-D5 · D6) is 10-16°C lower and the van't Hoff enthalpy difference Δ_vH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl₂) compared to that of ps-Dl · D2. Based on energy minimizations of a ps-[d(T₅GA₅) · d(A₅CT₅)] duplex using force field calculations we propose a model for the conformation of a trans dG · dC base pair in a ps helix.     

Molecular mechanics modeling of oligonucleotide adducts of the antitumor drug cis-diamminedichloroplatinum(II)

In order to assess the geometric changes caused when the antitumor drug cis-diammine-dichloroplatinum(II) (cis-DDP) binds to DNA, molecular mechanics calculations were performed on two double-stranded and two single-stranded oligonucleotides and their adducts with cis-{Pt(NH₃)₂}²⁺. For the platinated duplexes, three model structures have been derived, one involving only local disruption of base pairing with retention of the helix directionality, and two models showing pronounced kinking of the double helix. One of the kinked models is stabilized by bridging sodium ions. The other kinked duplex model shows retention of all Watson–Crick base pairing, including that of the coordinated guanines. All models exhibit hydrogen bonds connecting one ammine ligand of platinum with one or two phosphate groups located at the 5′ side of the platinated strand.     

Molecular mechanics studies of parallel and antiparallel phosphate-methylated DNA

Methylation of phosphate groups in oligo-dT strands leads to a parallel duplex with T · T base pairs. Molecular mechanics calculations on parallel d(TTTTTT)₂ show it to be a symmetric right-handed helix with B-DNA conformational characteristics. Phosphate methylation stabilizes the duplex by ca. 41 kcal/mol, due to removal of the interstrand phosphate electrostatic repulsions. The chirality introduced with phosphate methylation is important for the molecular geometry, since R_P methylation predominantly influences the conformation around the ζ bond (P? O_3′), while S_P methylation mostly changes the α conformation (P? O_5′). This is also true in antiparallel helices with methylated phosphates, as is shown by molecular mechanics calculations on d(GCGCGC)₂. These results may be of relevance to protein–DNA interactions, where phosphate charges are also shielded. As the pro-S_P oxygen is most available in a right-handed helix, we suggest changes around the α bond to occur upon protein complexation, leading to a widening of the major groove in the d(GCGCGC)₂ duplex (from 12 to 13 Å) and reduced minor groove (from 6 to 5 Å).     

Synthesis and Triplex Binding Properties of Oligonucleotides Containing a Novel Nucleobase

Abstract

The thiazolo-indole compound 1 bearing the complementary donor-acceptor-donor sites (dad) was designed for specific recognition of an AT inverted base pair in pyrimidine triple helix motif. It was successfully incorporated into 14-mer oligonucleotide using a serinol unit as sugar derivative. The triple helix hybridization studies were examined by means of thermal denaturation experiments with a 26-mer DNA duplex containing the AT inverted base pair.     

DESIGN AND SIMPLE ROUTES OF SYNTHESIS OF OLIGONUCLEOTIDE CONJUGATES FOR STUDIES OF DNA TRIPLE HELIX FORMATION

A series of oligonucleotides conjugated to intercalators, as well as fluorescent and lipophilic substances, minor groove binders and photoactive molecules were synthesized for studies of their ability to form a stable triple helix. Purine-rich short double stranded DNA fragments from HIV-1 genome and pyrimidine 16-mer oligodeoxyribonucleotide were used as models. A conjugate of a dipyrido[3,2-a:2′,3′-c]phenazine-ruthenium (II) complex and a triple helix-forming oligonucleotide was constructed. Upon sequence-specific duplex and triplex formation of the conjugate, the ruthenium complex becomes highly fluorescent. The attached ruthenium complex induces a stabilization of the DNA triple helix and a significant increase of the time of residence of the third strand on the duplex.     

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).     

A directional nucleation-zipping mechanism for triple helix formation

A detailed kinetic study of triple helix formation was performed by surface plasmon resonance. Three systems were investigated involving 15mer pyrimidine oligonucleotides as third strands. Rate constants and activation energies were validated by comparison with thermodynamic values calculated from UV-melting analysis. Replacement of a T·A base pair by a C·G pair at either the 5′ or the 3′ end of the target sequence allowed us to assess mismatch effects and to delineate the mechanism of triple helix formation. Our data show that the association rate constant is governed by the sequence of base triplets on the 5′ side of the triplex (referred to as the 5′ side of the target oligopurine strand) and provides evidence that the reaction pathway for triple helix formation in the pyrimidine motif proceeds from the 5′ end to the 3′ end of the triplex according to the nucleation-zipping model. It seems that this is a general feature for all triple helices formation, probably due to the right-handedness of the DNA double helix that provides a stronger base stacking at the 5′ than at the 3′ duplex–triplex junction. Understanding the mechanism of triple helix formation is not only of fundamental interest, but may also help in designing better triple helix-forming oligonucleotides for gene targeting and control of gene expression.     

Site-Specific RNA Cleavage Using Terpyridine·Cu(II)-Linked 2′-O-Methyloligonucleotides

Abstract

2′-Deoxy- and 2′-O-methyl-5′-O-terpyridyl derivatives of adenosine and cytidine were synthesized and used to construct 5′-end-modified oligonucleotides. These antisense agents complexed with Cu(II) exclusively cleaved a complementary RNA oligomer at the site opposite the terpyridine-nucleoside residue. We also found that the terpyridine·Cu(II) moiety stabilizes 2′-O-methyl RNA duplex. These suggest that after RNA hybridization, the terpyridine moiety is close to the RNA strand, presumably in an end capping manner.     

Studies on Alternate Strand Triple Helix Formation by Oligodeoxyribonucleotides Containing A 3′-3′ Phosphodiester Bond

Abstract

ODNs containing a 3′-3′ phosphodiester linkage as inversion of polarity motif have been shown to cooperatively bind to 5′-(purine)_m(pyrimidine)_n-3′ type duplexes by specific alternate strand recognition of the adjacent oligopurine domains. An NMR study has been undertaken to investigate the role of the 3′-3′ linked nucleosides and their nearest neighbours in the stabilization of the triple helical complexes.     

Nucleotide clusters in deoxyribonucleic acids. XII. The distribution of 5-methylcytosine in pyrimidine oligonucleotides of mouse L-cell satellite DNA and main band DNA.

Mouse L-cell DNA radioactively labeled in the 5-methylcytosine (5-MeC) residue was fractionated into satellite and main band DNA. Satellite DNA was found to contain about four times the molar concentration of 5-MeC than the main band DNA. Based on the known 5-MeC content of total L-cell DNA it was calculated that satellite DNA contains 3.5 – 4.6% 5-MeC. Both DNA fractions were depurinated and the pyrimidine oligonucleotides released separated by ionophoresis-homochromatography. In satellite DNA 5-MeC is distributed non-randomly. About 40% of the total 5-MeC is present in the sequence Pu - 5-MeC - Pu. The remainder occurs in the oligonucleotides CT, CT₃, C₂T₄, C₂T₅ and C₃T₅ only. The distribution of 5-MeC in main band DNA differs from that in satellite DNA indicating that two different fractions of the same nuclear DNA are methylated in different sequences.     

Inhibition of HIV-1 Integration by Mono- & Bifunctionalized Triple Helix Forming Oligonucleotides

Abstract

HIV-1 DNA integration is carried out by integrase, a viral protein which binds to specific sequences located on both extremities of the HIV-1 DNA LTR. Inhibition of integration was observed with submicromolar concentrations of mono- or bifunctionalized 11-mer oligonucleotide-intercalators, which were designed to form an alternate strand triple helix with the U5 LTR end containing two adjacent purine tracts on opposite strands 5′-GGAAAATCTCT-3′/3′-CCTTTTAGAGA-5′.     

Triple helical DNA in a duplex context and base pair opening

It is fundamental to explore in atomic detail the behavior of DNA triple helices as a means to understand the role they might play in vivo and to better engineer their use in genetic technologies, such as antigene therapy. To this aim we have performed atomistic simulations of a purine-rich antiparallel triple helix stretch of 10 base triplets flanked by canonical Watson–Crick double helices. At the same time we have explored the thermodynamic behavior of a flipping Watson–Crick base pair in the context of the triple and double helix. The third strand can be accommodated in a B-like duplex conformation. Upon binding, the double helix changes shape, and becomes more rigid. The triple-helical region increases its major groove width mainly by oversliding in the negative direction. The resulting conformations are somewhere between the A and B conformations with base pairs remaining almost perpendicular to the helical axis. The neighboring duplex regions maintain a B DNA conformation. Base pair opening in the duplex regions is more probable than in the triplex and binding of the Hoogsteen strand does not influence base pair breathing in the neighboring duplex region.