Targeting duplex DNA with chimeric α,ß-triplex-forming oligonucleotides.

Triplex-directed DNA recognition is strictly limited by polypurine sequences. In an attempt to address this problem with synthetic biology tools, we designed a panel of short chimeric α,ß-triplex-forming oligonucleotides (TFOs) and studied their interaction with fluorescently labelled duplex hairpins using various techniques. The hybridization of hairpin with an array of chimeric probes suggests that recognition of double-stranded DNA follows complicated rules combining reversed Hoogsteen and non-canonical homologous hydrogen bonding. In the presence of magnesium ions, chimeric TFOs are able to form highly stable α,ß-triplexes, as indicated by native gel-electrophoresis, on-array thermal denaturation and fluorescence-quenching experiments. CD spectra of chimeric triplexes exhibited features typically observed for anti-parallel purine triplexes with a GA or GT third strand. The high potential of chimeric α,ß-TFOs in targeting double-stranded DNA was demonstrated in the EcoRI endonuclease protection assay. In this paper, we report, for the first time, the recognition of base pair inversions in a duplex by chimeric TFOs containing α-thymidine and α-deoxyguanosine.

Fluorescent 2-pyrimidinone nucleoside in parallel-stranded DNA.

Stretches of parallel-stranded (ps) double-helical DNA can arise within antiparallel-stranded (aps) Watson-Crick DNA in looped structures or in the presence of sequence mismatches. Here we studied an effect of a pyrimidinone-G (PG) base pair on the stability and conformation of the ps DNA to explore whether P is useful as a structural probe.

Protein-free parallel triple-stranded DNA complex formation.

A 14 nt DNA sequence 5'-AGAATGTGGCAAAG-3' from the zinc finger repeat of the human KRAB zinc finger protein gene ZNF91 bearing the intercalator 2-methoxy,6-chloro,9-amino acridine (Acr) attached to the sugar-phosphate backbone in various positions has been shown to form a specific triple helix (triplex) with a 16 bp hairpin (intramolecular) or a two-stranded (intermolecular) duplex having the identical sequence in the same (parallel) orientation. Intramolecular targets with the identical sequence in the antiparallel orientation and a non-specific target sequence were tested as controls. Apparent binding constants for formation of the triplex were determined by quantitating electrophoretic band shifts. Binding of the single-stranded oligonucleotide probe sequence to the target led to an increase in the fluorescence anisotropy of acridine. The parallel orientation of the two identical sequence segments was confirmed by measurement of fluorescence resonance energy transfer between the acridine on the 5'-end of the probe strand as donor and BODIPY-Texas Red on the 3'-amino group of either strand of the target duplex as acceptor. There was full protection from OsO(4)-bipyridine modification of thymines in the probe strand of the triplex, in accordance with the presumed triplex formation, which excluded displacement of the homologous duplex strand by the probe-intercalator conjugate. The implications of these results for the existence of protein-independent parallel triplexes are discussed.

Parallel intramolecular DNA triple helix with G and T bases in the third strand stabilized by Zn(2+) ions.

We present evidence of formation of an intramolecular parallel triple helix with T*A.T and G*G.C base triplets (where * represents the hydrogen bonding interaction between the third strand and the duplex while. represents the Watson-Crick interactions which stabilize the duplex). The third GT strand, containing seven GpT/TpG steps, targets the polypurine sequence 5'-AGG-AGG-GAG-GAG-3'. The triple helix is obtained by the folding back twice of a 36mer, formed by three dodecamers tethered by hydroxyalkyl linkers (-L-). Due to the design of the oligonucleotide, the third strand orientation is parallel with respect to the polypurine strand. Triple helical formation has been studied in concentration conditions in which native gel electrophoresis experiments showed the absence of intermolecular structures. Circular dichroism (CD) and UV spectroscopy have been used to evidence the triplex structure. A CD spectrum characteristic of triple helical formation as well as biphasic UV and CD melting curves have been obtained in high ionic strength NaCl solutions in the presence of Zn(2+) ions. Specific interactions with Zn(2+) ions in low water activity conditions are necessary to stabilize the parallel triplex.

Parallel and antiparallel A*A-T intramolecular triple helices.

Intramolecular triple helices have been obtained by folding back twice oligonucleotides formed by decamers bound by non-nucleotide linkers: dA10-linker-dA10-linker-dT10 and dA10-linker-dT10-linker-dA10. We have thus prepared two triple helices with forced third strand orientation, respectively antiparallel (apA*A-T) and parallel (pA*A-T) with respect to the adenosine strand of the Watson-Crick duplex. The existence of the triple helices has been shown by FTIR, UV and fluorescence spectroscopies. Similar melting temperatures have been obtained in very different oligomer concentration conditions (micromolar solutions for thermal denaturation classically followed by UV spectroscopy, milimolar solutions in the case of melting monitored by FTIR spectroscopy) showing that the triple helices are intramolecular. The stability of the parallel triplex is found to be slightly lower than that of the antiparallel (deltaT(m) = 6 degrees C). The sugar conformations determined by FTIR are different for both triplexes. Only South-type sugars are found in the antiparallel triplex whereas both South- and North-type sugars are detected in the parallel triplex. In this case, thymidine sugars have a South-type geometry, and the adenosine strand of the Watson-Crick duplex has North-type sugars. For the antiparallel triplex the experimental results and molecular modeling data are consistent with a reverse-Hoogsteen like third-strand base pairing and South-type sugar conformation. An energetically optimized model of the parallel A*A-T triple helix with a non-uniform distribution of sugar conformations is discussed.

Regioselective immobilization of short oligonucleotides to acrylic copolymer gels.

Four types of polyacrylamide or polydimethyl-acrylamide gels for regioselective (by immobilization at the 3' end) of short oligonucleotides have been designed for use in manufacturing oligonucleotide microchips. Two of these supports contain amino or aldehyde groups in the gel, allowing coupling with oligonucleotides bearing aldehyde or amino groups, respectively, in the presence of a reducing agent. The aldehyde gel support showed a higher immobilization efficiency relative to the amino gel. Of all reducing agents tested, the best results were obtained with a pyridine-borane complex. The other supports are based on an acrylamide gel activated with glutaraldehyde or a hydroxyalkyl-functionalized gel treated with mesyl chloride. The use of dimethylacrylamide instead of acrylamide allows subsequent gel modifications in organic solvents. All the immobilization methods are easy and simple to perform, give high and reproducible yields, allow long durations of storage of the activated support, and provide high stability of attachment and low non-specific binding. Although these gel supports have been developed for preparing oligonucleotide microchips, they may be used for other purposes as well.

Stabilization of parallel (recombinant) triplex with propidium iodide.

Earlier we have shown that the oligonucleotide 5'-d(CATGCTAACT)-L-d(AGTTAGCATG)-L-d(CATGCTAACT)-3' [L = pO(CH2CH2O)3p] is able to fold back forming intramolecular RecA-independent triplex with identical strands oriented parallel to each other (parallel triplex) [A.K. Shchyolkina, E.N. Timofeev, O.F. Borisova, I.A. Il'icheva, E.E. Minyat, E.V. Khomyakova, V.L. Florentiev, FEBS Letters 339, 113-118 (1994) (1)]. In this study the propidium iodide (PI) was found to intercalate into the parallel triplex and increase its stability significantly (Tm increased from 21.4 up to 44.4 degrees C in 0.01 M Na phosphate buffer, pH 7, 0.1 M NaCl, when three PI molecules per triplex were bound). Fluorescence excitation and emission spectra, the quantum yield of fluorescence (q = 0.16) and the fluorescence lifetime of PI (tau = 24.5 ns at 3 degrees C) for the parallel triplex studied were shown to be similar to those for DNA. Scatchard binding plots indicated an anticooperative mode of PI binding to the parallel triplex. The association constant is close to that of PI binding to DNA. The fluorescence experiments revealed the maximum number of binding sites to be five PI molecules per one triplex molecule. Molecular mechanics calculation of possible structures for the parallel triplex-PI complex were performed.

Parallel purine-pyrimidine-purine triplex: experimental evidence for existence.

Oligonucleotides 5'-d(CT)5-L-d(AG)5-L-d(GA)5-3' and 5'-d(GA)5-L-d(TC)5-L-d(GA)5-3' [L = pO(CH2CH2O)3p] were studied by thermal denaturation, chemical modification and binding of fluorescent dyes. Both oligonucleotides are shown to fold back on itself twice forming at pH 7 a sufficiently stable triplex ether with antiparallel-oriented oligopurine strands (the first compound) or parallel-oriented oligopurine strands (the second compounds). The parallel triplex is significantly less stable than the antiparallel one. On the basis of conformational modeling, possible types of base tripling in the triplets are proposed. Thus our data provide the first convincingly evidence for the existence of a purine-pyrimidine-purine triplex with parallel orientation of identical strands.

A triple helix obtained by specific recognition of all 4 bases in duplex DNA can adopt a collapsed or an extended form.

It has been proposed that during homologous recombination promoted by RecA DNA triple helices can be formed between a Watson Crick duplex and a homologous third strand without any sequence constraint. A triple helix, obtained by targeting the d(AGTTAGCATG) sequence containing all 4 bases, in which both homologous strands are oriented in a parallel direction with respect to each other, stabilized by addition of Mn2+ ions has been studied by UV and FTIR spectroscopies. We have characterized the sugar conformations of this triplex. All strands are found to contain S type sugars (C2'endo, B family form). Progressive addition of propidium iodide induces a complete reorientation of the sugar geometry to a N type conformation (C3'endo, A family form). This sugar repuckering is consistent with a conformational transition from a collapsed to an extended DNA triple helical structure.

Trivaline molecular complexes with trinucleotides form in solution the extended, about 1000 A in length structures.

The fluorescence, flow linear dichroism and electron microscopy (EM) have shown the trivaline ability to interact in solution with certain molecules of trinucleotides. This interaction results in formation of extended structures up to several thousand angstroms in length. Such structures were observed for trivaline complexes with homopurine, homopyrimidine or random sequences of deoxyribo- and ribonucleotides, independently of the presence or absence of the terminal 5'-phosphate residue. A model of such a structural organization is proposed. An elementary structural unit consists of a trivaline beta-dimer and adsorbed trinucleotide. So, "dimeric" complex is formed. Two such "dimeric" complexes combine with each other by means of peptide-peptide contacts (as with beta-sandwich). So, "tetrameric" complex is formed. It has a dyad axis. Two such structural units combine with each other by means of Hoogsteen's hydrogen bonds. So, "octameric" complex is formed. It has three mutually perpendicular dyad axes. The "octameric" complexes appear to be able to combine with each other by means of stacking interactions, and to form the regular organized aggregates consisting of many dozens of elementary units. So, "stacking" structure is formed. The "octameric" complex is the symmetry translational unit of such a structure. The spatial position of the bases in all these structures is additionally fixed by the nucleo-peptide interactions. These aggregates have the appearance of extended structures on electron micrographs.

Dissociation of duplexes formed by hybridization of DNA with gel-immobilized oligonucleotides.

The method of DNA sequencing by hybridization with oligonucleotides matrix (SHOM) developed in this laboratory (1.2) uses the matrix of oligonucleotides immobilized within polyacrylamide gel. The particular feature of this matrix is that the apparent thermostability of the duplexes depends on the concentration of gel-immobilized oligonucleotides. This dependence is specific for oligonucleotides immobilized in the gel volume (3-D-immobilization) rather than on a flat surface of a filter or glass (2-D-immobilization). The theory has been developed that provides a quantitative description of temperature-dependent duplex dissociation within gel. The theory takes into account that the diffusion of dissociated DNA out of the gel is retarded by multiple acts of association-dissociation of DNA with immobilized oligonucleotides. It allows to calculate the apparent dissociation temperature of duplexes and describes quantitatively its growth upon increase in the enthalpy of duplex dissociation, concentration of immobilized oligonucleotides, gel thickness and decrease of dissociation entropy and washing time. Concentration of gel-immobilized oligonucleotides can be calculated for a normalized matrix in which GC-rich and AT-rich duplexes exhibit the same apparent thermostabilities and are washed off at the same temperature. This simplifies identification of perfect duplexes formed on the matrix which can be carried out for all duplexes at the same temperature. The gel-immobilized oligonucleotide matrix provides also a higher capacity for immobilization and therefore a higher sensitivity of measurements, resulting in a higher discrimination power for identification of perfect duplexes as compared with matrixes of oligonucleotides immobilized on a surface.

The R-form of DNA does exist.

Oligonucleotide 5'-d(CATGCTAACT)-L-d(AGTTAGCATG)-L-d(CATGCTAACT)-3' [L = pO(CH2CH2O)3p] is shown to fold back on itself twice forming at pH 7 a sufficiently stable triplex (Tm is about 30 degrees C) with parallel-orientated identical strands (the recombinant or R-form of DNA). Experimental evidence was obtained by studying thermal denaturation, chemical modification and binding of fluorescent probes. The stability of the R-triplex increases in the presence of divalent ions or spermidine. Its structure is characterized by a certain heterogeneity that causes the cooperativity of a triplex-to-duplex transition to decrease. On the basis of conformational modeling, the possible types of base tripling in all four triplets are proposed. The experimental data as well as the molecular mechanic calculations indicate that the stabilities of triplets in the R-triplex decrease in the order: G:C-G = A:T-A >> T:A-T > C:G-C.

Three-stranded clip of the oligonucleotide 5'-(dT)10pO(CH2CH2O)3p(dT)10pO(CH2CH2O)3p(dA)10-3'.

Temperature dependence of UV and CD spectra of the oligonucleotide 5'-(dT)10-L-(dT)10-L-(dA)10-3' [tripl(ATT)] [L = -pO(CH2CH2O)3p-] in phosphate buffer, pH 7, at various NaCl concentrations and in the presence or absence of 0.01 M MgCl2 has been studied. At low oligonucleotide concentrations (2.2 x 10(-5) M nucleotide concentration) all structural transitions proceed intramolecularly. Tripl(ATT) exists in three forms: as a three-stranded clip (at low temperatures), a double-stranded hairpin (at intermediate temperatures), and as an open strand (at high temperatures). Thermodynamic parameters of the triplex formation depending on the NaCl concentration were calculated. The CD spectra were assigned to the single-, double-, and three-stranded forms. Ethidium bromide (EtBr) binding to the three-stranded clip was studied. Ethidium bromide molecules were shown to intercalate into the triple helix with the stable complex formation (association constant is 10(6)). One molecule of three-stranded clip binds not more than three EtBr molecules. The proposed synthetic model (oligonucleotide blocks coupled by hydroxyalkyl chains) has been shown to be convenient for studies of the physical and chemical properties of the triplex and other multistranded complexes of DNA.

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.

Trivaline 'catalyzes' 5'-pdGTT oligomerization in solution.

We have found that the 5'-pdGTT molecules at a concentration of 10(-4) M are oligomerized in solution in the presence of 10(-4) M tripeptide-(L-Val)3-NH-NH-DNS.CF3COOH and the condensation reagents (carbodiimide and imidazole). Oligonucleotides not less than 12 bases long were formed in the yield which was over 15%. It is known that in the absence of peptide 10(-2) M mono- or dinucleotides are required. Thus trivaline can be considered as one of the simplest enzymes. This oligomerization seems to be an essential way for the synthesis of long enough oligonucleotides of the random GC-sequence, which could be used at the earliest steps of evolution.

Improved chips for sequencing by hybridization.

The SHOM method (Sequencing by Hybridization with Oligonucleotide Matrix) developed in 1988 is a new approach to nucleic acid sequencing by hybridization to an oligonucleotide matrix composed of an array of immobilized oligonucleotides. The original matrix proposed for sequencing by SHOM had to contain at least 65,536 octanucleotides. The present work describes a new family of matrices, which allows one to reduce the number of synthesized oligonucleotides 5-15 times without essentially decreasing the resolving power of the method.

A method for DNA sequencing by hybridization with oligonucleotide matrix.

A new technique of DNA sequencing by hybridization with oligonucleotide matrix (SHOM) which could also be applied for DNA mapping and fingerprinting, mutant diagnostics, etc., has been tested in model experiments. A dot matrix was prepared which contained 9 overlapping octanucleotides (8-mers) complementary to a common 17-mer. Each of the 8-mers was immobilized as individual dot in thin layer of polyacrylamide gel fixed on a glass plate. The matrix was hybridized with the 32P-labeled 17-mer and three other 17-mers differing from the first one by a single base change. The hybridization enabled us to distinguish perfect duplexes from those containing mismatches in 32 out of 35 cases. These results are discussed with respect to the applicability of the approach for sequencing. It was shown that hybridization of DNA with an immobilized 8-mer in the presence of a labeled 5-mer led to the formation of a stable duplex with the 5-mer only if the 5- and the 8-mers were in continuous stacking making a perfect nicked duplex 13 (5+8) base pairs long. These experiments and computer simulations suggest that continuous stacking hybridization may increase the efficiency of sequencing so that random or natural coding DNA fragments about 1000 bases long could be sequenced in more than 97% of cases. Miniaturized matrices or sequencing chips were designed, where oligonucleotides were immobilized within 100 x 100 micron dots disposed at 100 micron intervals. Hybridization of fluorescently labeled DNA fragments with microchips may simplify sequencing and ensure sensitivity of at least 10 attomoles per dot. The perspectives and limitations of SHOM are discussed.