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.

Efficiency of sequencing by hybridization on oligonucleotide matrix supplemented by measurement of the distance between DNA segments.

DNA sequencing by hybridization on oligonucleotide microchip (SHOM) allows the determination of a spectrum of overlapping oligonucleotides constituting a DNA fragment that hybridizes to form perfect duplexes with an array of immobilized oligonucleotides and, as a result, enables reconstitution of the nucleotide sequence of the fragment. In longer DNA fragments, unambiguous reconstitution of DNA sequence is often impeded by the presence of repetitive regions and simple sequence repeats. Here it is demonstrated that SHOM supplemented by measurement of the distance between certain sites (for example, restriction sites or priming sites for PCR) within the analyzed DNA enables sequencing of much longer DNA fragments, containing repeats of different complexity.

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.

Torsional flexibility of B-DNA as revealed by conformational analysis.

The thermal fluctuations of a regular double helix belonging to the B-family were studied by means of atom-atomic potentials method. The winding angle fluctuation was found to be 2.4 degrees for poly(dA):poly(dT) and 3.0 degrees for poly(dG):poly(dC). The reasonable agreement of these estimations with those obtained experimentally reveals the essential role of the small-amplitude torsional vibrations of atoms in the mechanism of the double helix flexibility. The calculated equilibrium winding angle, tau 0, essentially depends on the degree of neutralization of phosphate groups, being about 35.5 degrees for the full neutralization. The deoxyribose pucker is closely related to the tau angle: while tau proceeds from 30 degrees to 45 degrees the pseudorotation phase angle, P, increases from 126 degrees to 164 degrees. Fluctuations of the angles TL and TW, which specify inclination of the bases to the helix axis, were evaluated to be 5 degrees-10 degrees. Possible correlation between conformational changes in the adjacent nucleotides is discussed.

Anisotropic flexibility of DNA and the nucleosomal structure.

Potential energy calculations of the DNA duplex dimeric subunit show that the double helix may be bent in the direction of minor and major grooves much more easily than in other directions. It is found that the total winding angle of DNA decreases upon such bending. A new model for DNA folding in the nucleosome is proposed on the basis of these findings according to which the DNA molecule is kinked each fifth base pair to the side of the minor and major grooves alternatively. The model explains the known contradiction between a C-like circular dichroism for the nucleosomal DNA and the nuclease digestion data, which testify to the B-form of DNA.

Magnetic circular dichroism studies of myoglobin, hemoglobin and peroxidase at room and low temperatures. Ferrous high spin derivatives.

The magnetic circular dichroism spectra (MCD) recorded for the visible and near-UV regions of high-spin ferrous derivatives of myoglobin, hemoglobin, hemoglobin dimers and isolated chains as well as of horseradish peroxidase at pH 6.8 and 11.4 have been compared at the room and liquid nitrogen temperatures. The MCD of the Q00- and QV-bands have been shown to be sensitive to structural differences in the heme environment of these hemoproteins. The room temperature visible MCD of native hemoglobin differs from that of myoglobin, hemoglobin dimers and isolated chains as well as from that of model pentacoordinated complex. The MCD of hemoglobin is characterized by the greater value of the MCD intensity ratio of derivative shape A-term in the Q00-band to the A-term in the QV-band. The evidneces are presented for the existence of two pH-dependent forms of ferroperoxidase, the neutral peroxidase shows the "hemoglobin-like" MCD, while the alkaline ferroperoxidase is characterized by the "myoglobin-like" MCD spectrum in the visible region. The differences in the MCD of deoxyhemoglobin and neutral ferroperoxidase as compared with other high-spin ferrous hemoproteins are considered to result from the constraints on heme group imposed by quaternary and/or tertiary protein structure. The differences between hemoporteins which are seen at the room temperature become more pronounced at liquid nitrogen temperature. Except the peak at approximately 580 nm in the MCD of deoxymyoglobin and reduced peroxidase at pH 11.4 the visible MCD does not show appreciable temperature dependent C-terms. The nature of the temperature dependent effect at approximately 580 nm is not clear. The Soret MCD of all hemoproteins studied are similar and are predominantly composed of the derivative-shaped C-terms as revealed by the increase of the MCD peaks approximately in accordance with Boltzmann distribution. The interpretation of temperature-dependent MCD observed for the Soret band has been made in terms of porphyrin to Fe-iron charge-transfer electronic transition which may be assigned as b( pi) leads to 3d. This charge-transfer band is strongly overlapped with usual B(pi --pi*) band resulting in diffuse Soret band. Adopting that only two normal vibrations are sinphase with charge-transfer transition the extracted C-terms of the Soret MCD have been fitted by theoretical dispersion curves.