Evidence for the tetraplex structure of the d(GT)n repetitive sequences in solution.

The ability of oligonucleotides 3'-d(GT)5pO(CH2)6Opd(GT)5-5' (anti[d(GT)]) and 3'-d(GT)5pO(CH2)6Opd(GT)5-3' (par[par[d(GT)]) to form tertiary structures has been studied. Circular dichroism (CD) as well as the fluorescence of the ethidium bromide (EtBr) complexes with oligonucleotides and hydrodynamic volume measurements in solutions containing 0.01 M phosphate buffer, pH 7 and NaCl in concentrations from 0.1 M to 1 M, have been used. The data obtained in the temperature interval from 3 degrees C to 10 degrees C are in good agreement with the structure suggested earlier where the par[d(GT)] and anti[d(GT)] form structures with four parallel strands in which layers of four G-residues alternate with unpaired bulged-out T-residues. Ethidium bromide interacts with the structure in a cooperative manner. Two ethidium bromide molecules intercalate between two layers of four G-residues.     

Parallel-Stranded Oligonucleotides with Alternating d(A-isoG)/d(T·C) and d(A·G)/d(T·m5isoC) Sequences

Abstract

Parallel-stranded (ps) DNA hairpins with alternating d(A-isoG)/d(T·C) (designated as ps-t1) and d(A·G)/d(T·m⁵isoC) (ps-t2) sequences were studied by means of UV, CD and fluorescence spectroscopy. The thermostability of d(A·G)/d(T·m⁵isoC) sequence was close to that of aps d(G·A)/d(T·C). The stability of the ps d(A·isoG)/d(T·C) sequence was even higher than that of a related anti-parallel-stranded (aps) d(G·A)/d(T·C) sequence, being unique for ps DNAs studied so far.     

Ethidium Probing of the Parallel Double- and Four-stranded Structures Formed by the Telomeric DNA Sequences dG(GT)4G and d(GT)5

Abstract

Oligonucleotides 3′-d(GT)₅-(CH₂CH₂O)₃-d(GT)₅-3′ (parGT), containing GT repeats present in the telomeric DNA from Saccharomyces cerevisiae, had been demonstrated to form bimolecular structure, GT-quadruplex (qGT) [O. F. Borisova et al. FEBS Letters 306, 140–142 (1992)]. Four d(GT)₅ strands of the GT-quadruplex are parallel and form five G-quartets while thymines are bulged out. The four GT repeats when flanked by guanines, 3′-dG(TG)₄G-(CH₂CH₂O)₃- dG(GT)₄G-3′ (hp-GT), had been shown to form a novel parallel-stranded (ps) double helix with G·G and T·T base pairs (hp-GT ps-DNA) [A. K. Shchyolkina et al. J. Biomol. Struct. Dynam. 18, 493–503 (2001)]. In the present study the intercalator ethidium bromide (Et) was used for probing the two structures. The mode of Et binding and its effect on thermostability of qGT and hp-GT were compared. The quantum yield (q) and the fluorescence lifetime (τ) of Et:qGT (q = 0.15 ±0.01 and τ = 24 ±1 ns) and Et:hp-GT (q = 0.10 ± 0.01 and τ = 16.5 ± 1 ns) indicative of intercalation mode of Et binding were determined. Et binding to qGT was found to be cooperative with corresponding coefficient ω = 3.9 ± 0.1 and the binding constant K= (6.4 ± 0.1)·10M^?1. The maximum number of Et molecules intercalating into GT-quadruplex is as high as twice the number of inerspaces between G-quartets (eight in our case). The data conform to the model of Et association with GT-quadruplex suggested earlier [O. F. Borisova et al. Mol. Biol. (Russ) 35, 732–739 (2001)]. The anticooperative type of Et binding was observed in case of hp- GT ps-DNA, with the maximum number of bound Et molecules, N = 4 ÷ 5, and the association constant K = (1.5 ± 0.1)·10⁵ M^?1. Thermodynamic parameters of formation of Et:qGT and EtBr:hp-GT complexes were calculated from UV thermal denaturation profiles.     

Complexes of antiparallel telomeric G-quadruplex d(TTAGGG)4 with carboxymethyl tetracationic porphyrins

Porphyrins are a chemical class that is widely used in drug design. Cationic porphyrins may bind to DNA guanine quadruplexes. We report the parameters of the binding of 5,10,15,20-tetrakis(N-carboxymethyl-4-pyridinium) porphyrin (P1) and 5,10,15,20-tetrakis(N-etoxycarbonylmethyl-4-pyridinium) porphyrin (P2) to antiparallel telomeric G-quadruplex formed by d(TTAGGG)₄ sequence (TelQ). The binding constants (K _i ) and the number of binding sites (N _j ) were determined from absorption isotherms generated from the absorption spectra of complexes of P1 and P2 with TelQ. Compound P1 demonstrated a high affinity to TelQ (K _i = (40 ± 6) × 10⁶ M^?1, N ₁ = 1; K ₂ = (5.4 ± 0.4) × 10⁶ M^?1, N ₂ = 2). In contrast, the binding constants of P2-TelQ complexes (K ₁ = (3.1 ± 0.2) × 10⁶ M^?1, N ₁ = 1; K ₂ = (1.2 ± 0.2) × 10⁶ M^?1, N ₂ = 2) were one order of magnitude smaller than the corresponding values for P2-TelQ complexes. Measurements of the quantum yield and fluorescence lifetime of the drug’s TelQ complexes revealed two types of binding sites for P1 and P2 on the quadruplex oligonucleotide. We concluded that strong complexes can result from the interaction of the porphyrins with TTA loops whereas the weaker complexes are formed with G-quartets. The altered TelQ conformation detected by the circular dichroism spectra of P1-TelQ complexes can be explained by the disruption of the G-quartet. We conclude that peripheral carboxy groups contribute to the high affinity of P1 for the antiparallel telomeric G-quadruplex.     

Structural polymorphism of oligo(dC) with mixed alpha,beta-anomeric backbone

Oligonucleotides with mixed alpha,beta-anomeric backbone have been proposed recently for the recognition of random DNA sequence via new triplex motif (Doronina and Behr, Chem. Soc. Reviews 26, 63-71 (1997)). In the present work we examined alpha- and beta- anomers of cytidine as possible candidates to recognize AT and TA base pairs of the double stranded DNA. The binding properties of beta-oligo(dC) were studied on a series of synthetic oligodeoxynucleotides by UV absorbtion spectroscopy, measurements of bound EtBr fluorescence polarization, circular dichroism (CD) and non-denaturing gel electrophoresis. The UV thermal denaturation, polarization studies and CD experiments with three stranded oligonucleotide 5'-((dCalpha) (dCbeta))5-L-(dAT)5-L-(dAT)5 (L = triethyleneglycol linker) and other oligonucleotide models showed that the formation of semiprotonated oligocytidilic complexes takes place at low temperatures and neutral pH, rather than folding of the clip into intramolecular triplex. The low-temperature transition was observed in denaturation profiles of any oligonucleotide containing beta- or mixed alpha,beta- cytidine stretches at the concentration of 1 microM. Self-association of alpha,beta-oligo(dC) was additionally confirmed by the appearance of two CD bands (at 290 and 265 nm) characteristic of CC+ base pairs. Despite the effective ability of alpha,beta-oligo(dC) to form self-associates, we succeeded in targeting 30-bp AT containing random DNA duplex by a 30-nt alpha,beta-oligocytidilate as evidenced by non-denaturing gel electrophoresis. A complete binding of the duplex was observed at a 5-fold excess of the third strand at 15 degrees C. Along with the formation of the three-stranded complex, self-association of mixed backbone oligo(dC) strands occurred.     

Diverse modes of 5′‐[4‐(aminoiminomethyl)phenyl]‐[2,2′‐bifuran]‐5‐carboximidamide (DB832) interaction with multi‐stranded DNA structures

The modes of binding of 5′‐[4‐(aminoiminomethyl)phenyl]‐[2,2′‐Bifuran]‐5‐carboximidamide (DB832) to multi‐stranded DNAs: human telomere quadruplex, monomolecular R‐triplex, pyr/pur/pyr triplex consisting of 12 T*(T·A) triplets, and DNA double helical hairpin were studied. The optical adsorption of the ligand was used for monitoring the binding and for determination of the association constants and the numbers of binding sites. CD spectra of DB832 complexes with the oligonucleotides and the data on the energy transfer from DNA bases to the bound DB832 assisted in elucidating the binding modes. The affinity of DB832 to the studied multi‐stranded DNAs was found to be greater (K_ass ≈ 10⁷M^?1) than to the duplex DNA (K_ass ≈ 2 × 10⁵M^?1). A considerable stabilizing effect of DB832 binding on R‐triplex conformation was detected. The nature of the ligand tight binding differed for the studied multi‐stranded DNA depending on their specific conformational features: recombination‐type R‐triplex demonstrated the highest affinity for DB832 groove binding, while pyr/pur/pyr TTA triplex favored DB832 intercalation at the end stacking contacts and the human telomere quadruplex d[AG₃(T₂AG₃)₃] accommodated the ligand in a capping mode. Additionally, the pyr/pur/pyr TTA triplex and d[AG₃(T₂AG₃)₃] quadruplex bound DB832 into their grooves, though with a markedly lesser affinity. DB832 may be useful for discrimination of the multi‐sranded DNA conformations and for R‐triplex stabilization. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 8–20, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Parallel-Stranded DNA with Natural Base Sequences

Noncanonical parallel-stranded DNA double helices (ps-DNA) of natural nucleotide sequences are usually less stable than the canonical antiparallel-stranded DNA structures, which ensures reliable cell functioning. However, recent data indicate a possible role of ps-DNA in DNA loops or in regions of trinucleotide repeats connected with neurodegenerative diseases. The review surveys recent studies on the effect of nucleotide sequence on preference of one or other type of DNA duplex. (1) Ps-DNA of mixed AT/GC composition was found to have conformational and thermodynamic properties drastically different from those of a Watson–Crick double helix. Its stability depends strongly on the specific sequence in a manner peculiar to the ps double helix, because of the energy disadvantage of the AT/GC contacts. The AT/GC boundary facilitated flipping of A and T out of the ps double helix. Proton acceptor groups of bases are exposed into both grooves of the ps-DNA and are accessible to solvent and ligands, including proteins. (2) DNA regions containing natural minor bases isoguanine and isomethylisocytosine were shown to form ps-DNA with transAT-, trans isoGC, and transiso^5meCG pairs exceeding in stability a related canonical duplex. (3) Nucleotide sequence dG(GT)₄G from yeast telomeres and microsatellites was demonstrated to form novel ps-DNA with GG and TT base pairing. Unlike d(GT) _n - and d(G _n T _m ) sequences able to form quadruplexes, the dG(GT)₄G sequence formed no alternative double- or multistranded structures in a wide range of experimental conditions, thus suggesting that the nucleotide context governs the observed structural polymorphism of the d(GT) _n sequence. The possible biological role of ps-DNA and the prospects of its study are discussed.     

Four-stranded DNA helices: conformational analysis of regular poly(dT).poly(dA).poly(dA).poly(dT) helices with various types of base binding

The paper presents results obtained in conformational analysis of homopolymeric four-stranded poly(dT).poly(dA).poly(dA).poly(dT) DNA helices in which the pairs of strands with identical bases are parallel and have a two-fold symmetry axis. All possible models of base binding to yield a symmetric complex have been considered. The dihedral angles of sugar-phosphate backbones and helix parameters, which are consistent with the minima of conformational energy for four-stranded DNAs, have been determined using the results of optimization of conformational energy calculated at atom-atom approximation. Potential energy is shown to depend on the structure of base complexes and on the mutual orientation of unlike strands. Possible biological functions of four-stranded helices are discussed.