[Characteristics of complex formation between monomeric and dimeric bisbenzimidazoles and AT-containing polynucleotide].

Double-stranded DNA is a one of the most important intracellular anticancer agent targets. Disturbance of DNA functions as well as DNA structure lead to disorder of such processes as transcription and/or translation thus inducing tumor cells death. Complex formation between novel dimeric bisbenzimidazole DB(7) and poly(dA-dT) duplex in comparison with known monomeric bisbenzimidazole MB(Ac) was investigated in this study. DB(7)-poly(dA-dT) binding constant was determined by fluorescence spectroscopy using Scatchard plot and it values 1.18 x 10(8) M(-1) that is two orders of magnitude larger than MB(Ac) one (2.06 x 10(6) M(-1)). Thus, from findings mentioned above it could be concluded that the presence of two bisbenzimidazole moieties in the ligand structure significantly increases its affinity to the polynucleotide which motivates the synthesis of new potential anticancer drugs based on dimeric bisbenzimidazoles.

[DNA sequence-specific ligands: XIV. Synthesis of fluorescent biologicaly active dimeric bisbenzimidazoles-DB(3, 4, 5, 7, 11)].

Five fluorescent symmetric dimeric bisbenzimidazoles DB(n) have been synthesized containing four 2,6-substited benzimidazole fragments and differ in length of oligomethylene linker (n=3, 4, 5, 7, 11) between the two bisbenzimidazole blocks. The ability of these dimeric bisbenzimidazoles to form complexes with double-stranded DNA (dsDNA) was shown by spectral methods. Upon binding to dsDNA DB(n) are localized in the minor groove. The DNA-methyltransferase Dnmt3a inhibition data are demonstrate the site-specific binding of dimeric bisbenzimidazoles DB(3) and DB(11) with oligonucleotide duplex.

[Synthesis and properties of a symmetric dimeric bisbenzimidazole, a DNA-specific ligand].

Dimeric bisbenzimidazole DB(5) fluorescing in the blue spectral area (lambda(em) 476 nm) within the DNA complex was synthesized. DB(5) is bound by a double-strand DNA and can differentially stain human and plant (flax) chromosomes. According to preliminary data, it provides considerably more intensely contrasting chromosome staining than DAPI and Hoechst 33258 dyes. It was also found that DB(5) is an in vitro inhibitor of HIV-1 integrase in the reaction of 3'-processing. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.

[DNA sequence-specific ligands: XIII. New Dimeric Hoechst 33258 molecules, inhibitors of HIV-1 integrase in vitro].

Dimeric Hoechst 33258 molecules [dimeric bisbenzimidazoles (DBBIs)] that, upon binding, occupy one turn of the B form of DNA in the narrow groove were constructed by computer simulation. Three fluorescent DBBIs were synthesized; they consist of two bisbenzimidazole units tail-to-tail linked to phenolic hydroxy groups via penta- or heptamethylene or tri(ethylene glycol) spacers and have terminal positively charged N.N-dimethylaminopropyl carboxamide groups in the molecule. The absorption spectra of the DBBIs in the presence of different DNA concentrations showed a hypochromic effect and a small shift of the absorption band to longer wavelengths, which indicated the formation of a complex with DNA. The presence of an isobestic point in the spectrum indicates the formation of one type of DBBI-DNA complexes. The interaction of DBBIs with DNA was studied by CD using a cholesteric liquid-crystalline dispersion (CLD) of DNA. The appearance of a positive band in the absorption region of ligand chromophores in the CD spectrum of the DNA CLD indicates the formation of a DBBI-DNA complex in which ligand chromophores are arranged at an angle close to 54 degrees relative to the helix axis of DNA, which suggests the localization of the DBBI in the narrow groove of DNA. All the DBBIs were found to be in vitro inhibitors of HIV-1 DNA integrase in the 3'-processing reaction, and, of the three DBBIs, two dimers inhibit HIV-1 integrase even in submicromolar concentrations.

[Ligands with affinity to specific sequence of DNA base pairs. XII. The synthesis and cytological studies of dimeric Hoechst 33258 molecules].

We synthesized dimeric Hoechst dye molecules composed of two moieties of the Hoechst 33258 fluorescent dye phenolic hydroxy groups of which were tethered via pentamethylene, heptamethylene, or triethylene oxide linkers. A characteristic pattern of differential staining of chromosome preparations from human premonocytic leukemia HL60 cells was observed for all the three fluorescent dyes. The most contrast pattern was obtained for the bis-Hoechst analogue with the heptamethylene linker; its quality was comparable with the picture obtained in the case of chromosome staining with 4',6-diamidino-2-phenylindole. The ability to penetrate into the live human fibroblasts was studied for the three bis-Hoechst compounds. The fluorescence intensity of nuclei of live and fixed cells stained with the penta- and heptamethylene-linked bis-Hoechst analogues was found to differ only slightly, whereas the fluorescence of the nuclei of live cells stained with triethylene oxide-linked bis-Hoechst was considerably weaker than that of the fixed cells. The bis-Hoechst molecules are new promising fluorescent dyes that can both differentially stain chromosome preparations and penetrate through cell and nuclear membranes and effectively stain cell nuclei.

[DNA-specific dimeric bisbenzimidazole]. / DNK-spetsifichnyi dimernyi bisbenzimidazol.

A dimeric analogue of the fluorescent dye Hoechst 33258 was synthesized. It was shown to differentially stain human chromosome preparations and bind to double-stranded DNAs.

[Effect of supporting substrates on the structure of DNA and DNA-trivaline complexes studied by atomic force microscopy]. / Vliianie podlozhek pri atomno-silovoi mikroskopii DNK i kompleksov DNK s trivalinom na ikh strukturu.

Linear DNA, circular DNA, and circular DNA complexes with trivaline (TV), a synthetic oligopeptide, were imaged by atomic force microscopy (AFM) using mica as a conventional supporting substrate and modified highly ordered pyrolytic graphite (HOPG) as an alternative substrate. A method of modifying the HOPG surface was developed that enabled the adsorption of DNA and DNA-TV complexes onto this surface. On mica, both purified DNA and DNA-TV complexes were shown to undergo significant structural distortions: DNA molecules decrease in height and DNA-TP displays substantial changes in the shape of its circular compact structures. Use of the HOPG support helps preserve the structural integrity of the complexes and increase the measured height of DNA molecules up to 2 nm. AFM with the HOPG support was shown to efficiently reveal the particular points of the complexes where, according to known models of their organization, a great number of bent DNA fibers meet. These results provide additional information on DNA organization in its complexes with TV and are also of methodological interest, since the use of the modified HOPG may widen the possibilities of AFM in studying DNA and its complexes with various ligands.

[Interaction of topotecan-a DNA topoisomerase I inhibitor-with dual-stranded polydeoxyribonucleotides. V. Topotecan is able to cause single- and double-strand breaks in ring superhelical DNA in the absence of enzyme]. / Vzaimodeistvie topotekana--ingibitora DNK-topoizomerazy I--s dvunitevymi polidezoksiribonukleotidami. V. Topotekan sposoben vyzyvat' odno- i dvunitevye razryvy kol'tsevoi superskruchennoi DNK v otsutstvie fermenta.

Temperature, concentration, and time dependence for the emergence of breaks in the sugar-phosphate backbone in a circular supercoiled DNA (scDNA) was studied for the first time in the presence of topotecan (TPT) and in the absence of human DNA topoisomerase I (topo I). Because TPT is a comptothecin (CPT) derivative, it is the first example for the ability of molecules of CPT family to cause double-stranded breaks in scDNA in the absence of the enzyme. The experiments were carried out in low ionic strength solutions (10 mM sodium cacodylate) at neutral pH (6.8). Incubation time necessary for the appearance of double-stranded breaks in scDNA in the presence of TPT correlated with the time of formation of strong TPT-DNA complex. A model was suggested for the complex composed of two crossed DNA duplexes bound through a bridge of two dimers of TPT lactone form. According to this model, two carbonyl groups of D rings of different TPT dimers form hydrogen bonds with 2-amino groups of guanines located in the neighboring base pairs of diverse strands of one of DNA duplexes. At the same time, two other carbonyl groups of D rings of TPT dimers form hydrogen bonds with 2-amino groups of guanines spaced five bp apart in the same strand of the second DNA duplex.

[Interaction of topotecan, DNA topoisomerase I inhibitor, with double-stranded polydeoxyribonucleotides. 4. Topotecan binds preferably to the GC base pairs of DNA]. / Vzaimodeistvie topotekana--ingibitora DNK-topoizomerazy I--s dvunitevymi polidezoksiribonukleotidami. IV. Topotekan sviazyvaetsia preimushchestvenno s GC-parami osnovanii DNK.

Interaction of topotecan (TPT) with synthetic double-stranded polydeoxyribonucleotides has been studied in solutions of low ionic strength at pH = 6.8 by linear flow dichroism (LD), circular dichroism (CD), UV-Vis absorption and Raman spectroscopy. The complexes of TPT with poly(dG-dC).poly(dG-dC), poly(dG).poly(dC), poly(dA-dC).poly(dG-dT), poly(dA).poly(dT) and previously studied by us complexes of TPT with calf thymus DNA and coliphage T4 DNA have been shown to have negative LD in the long-wavelength absorption band of TPT, whereas the complex of TPT with poly(dA-dT).poly(dA-dT) has positive LD in this absorption band of TPT. Thus, there are two different types of TPT complexes with the polymers. TPT has been established to bind preferably to GC base pairs because its affinity to the polymers of different GC composition decreases in the following order: poly(dG-dC).poly(dG-dC) > poly(dG).poly(dC) > poly(dA-dC).poly(dG-dT) > poly(dA).poly(dT). The presence of DNA has been shown to shift monomer-dimer equilibrium in TPT solutions toward dimer formation. Several duplexes of the synthetic polynucleotides bound together by the bridges of TPT dimers may participate in the formation of the studied type of TPT-polynucleotide complexes. Molecular models of TPT complex with linear and ring supercoiled DNAs and with deoxyguanosine have been considered. TPT (and presumably all camptothecin family) proved to be a representative of a new class of DNA-specific ligands whose biological action is associated with formation of dimeric bridges between two DNA duplexes.

[Interaction of topotecan, DNA topoisomerase I inhibitor, with double-stranded polydeoxyribonucleotides. III. Binding at the minor groove]. / Vzaimodeistvie topotekana--ingibitora DNK-topoizomerazy I--s dvunitevymi polidezoksiribonukleotidami. III. Mesto sviazyvaniia--uzkaia borozdka.

Interaction of topotecan (TPT) with calf thymus DNA, coliphage T4 DNA, and poly(dG-dC). poly(dG-dC) was studied by optical (linear flow dichroism, UV-vis spectroscopy) and quantum chemical methods. The linear dichroism (LD) signal of TPT bound to DNA was shown to have positive sign in the range 260-295 nm. This means that the plane of quinoline fragment (rings A and B) of TPT molecule form an angle lower 54 degrees with the long axis of DNA, and hence TPT molecule can not intercalate between DNA base pairs. TPT was established to bind to calf thymus DNA as readily as to coliphage T4 DNA whose all cytosines in the major groove were glycosylated at the 5th position. Consequently, the DNA major groove does not participate in TPT binding. TPT molecule was shown to compete with distamycin for binding sites in the minor groove of DNA and poly(dG-dC). poly(dG-dC). Thus, it was demonstrated for the first time that TPT binds to DNA at its minor groove.

[Thermodynamic model of the formation of bridges between nucleic acid molecules in a liquid crystal]. / Termodinamicheskaia model' obrazovaniia mostikov mezhdu molekulami nukleinovykh kislot v zhidkom kristalle.

A thermodynamic model of the formation of bridges consisting of alternating daunomycin molecules and copper ions and connecting neighboring nucleic acid molecules in a particle of a liquid crystalline dispersion was constructed. The model is based on the conception that ligands are adsorbed on lattices of reaction centers which are formed in a liquid crystal at a particular spatial arrangement of adjacent nucleic acid molecules (phasing). Equations were derived that describe typical experimentally obtained S-shaped dependences of bridge concentration on the concentration of copper ions and daunomycin molecules in an initial solution. It was shown that dependences of this kind take place in two variants of the adsorption model: when the binding of daunomycin with adjacent nucleic acid molecules is considered to be independent on the formation of bridges and when bridges compete with single daunomycin molecules for the sites on adjacent nucleic acid molecules.

[Interaction of topotecan--a DNA topoisomerase inhibitor--with dual-stranded polydeoxyribonucleotides. I. Dimerization of topotecan in solution]. / Vzaimodeistvie topotekana--ingibitora DNK-topoizomerazy I--s dvunitevymi polidezoksiribonukleotidami. I. Dimerizatsii topotekana v rastvore.

Behavior of topotecan, DNA topoisomerase I inhibitor, was studied in aqueous solutions by optical methods. Topotecan absorption spectra were recorded in the pH range 0.5-11.5 and its pKa were determined. Quantum chemical calculations were made for all charge states of the topotecan molecule in lactone and carboxylate form. The calculated absorption maxima agree well with the experimental data. Protonation of the topotecan D ring (pKa = 3.6) was revealed. Comparison of experimental and calculated data showed topotecan structure with a proton at the oxygen atom at C16a rather than N4 to be the most preferable. Topotecan molecules were shown to form dimers at concentrations above 10(-5) M. Topotecan dimerization is accompanied by an increase in the pKa of hydroxy group of the A ring from 6.5 ([TPT] = 10(-6) M) to 7.1 ([TPT] = 10(-4) M), which indicates participation of this group in dimer stabilization, perhaps due to intermolecular hydrogen bonding with N1 of the B ring of a neighboring molecule. Probable dimer structures were proposed. The topotecan dimerization constant was determined, K = (4.0 +/- 0.7) x 10(3) M-1.

[Interaction of topotecan--a DNA topoisomerase I inhibitor--with dual-stranded polydeoxyribonucleotides. II. Formation of a complex containing several DNA molecules in the presence of topotecan]. / Vzaimodeistvie topotekana--ingibitora DNK-topoizomerazy I--s dvunitevymi polidezoksiribonukleotidami. II. Obrazovanie kompleksa, soderzhashchego neskol'ko molekul DNK, v prisutstvii topotekana.

This study is a continuation of a series of papers dealing with topotecan interaction with double-stranded polydeoxyribonucleotides. We showed earlier that topotecan molecules form dimers in solution at concentration above 10(-5) (per base pair). Topotecan interaction with calf thymus DNA in solutions of low ionic strength was studied by fluorescence, circular dichroism, and linear flow dichroism. The data obtained indicate that topotecan forms two types of complex with DNA, DNA molecules combining with each other during formation of one of these complexes. The association constant of two topotecan-filled DNA molecules with each other was estimated at 10(4) M-1 (per base pair) in 1 mM sodium cacodylate buffer, pH 6.8, at 20 degrees C. A possibility of modulation of DNA topoisomerase I activity by topotecan due to complexation with several sites of a supercoiled DNA molecule is discussed.

[New physical interpretation of S-shaped curves: competition between various types of binding of ligands with DNA]. / Novaia fizicheskaia interpretatsiia S-obraznogo vida krivykh: konkurentsiia mezhdu raznymi tipami sviazyvaniia liganda s DNK.

In studies of interactions of ligands with nucleic acid molecules, S-shaped curves are often obtained. In this work we show that the sigmoidal shape of the curves is due to a competition of ligand molecules for DNA binding sites. If a ligand that binds to DNA can form two types of complexes with the binding constants K1 and K2 and binding site sizes L1 and L2, respectively, then in the event that K1 < K2 and L1 < L2, the dependence of the concentration of complexes of the first type on the concentration of the ligand added to solution can be S-shaped.

[Trivaline initiates formation of homo- and heteroquadruplex DNA structures]. / Trivalin initsiiruet obrazovanie gomo- i geterokvadruplesnoi struktur DNK.

Formation of heterologous (calf thymus double-stranded DNA) and homologous (linearized pBR322 plasmid double-stranded DNA) quadruplexes upon binding with the simple aliphatic tripeptide derivative (dansyl hydrazide trivaline) was examined by fluorimetry, flow linear and circular dichroism and electron microscopy. The morphology of the rod-like compact particles formed due to the association of double-stranded DNA segments proved to be the same for both DNAs, whereas the stability of the compact DNA structure upon tripeptide removal from the complex with DNA differed substantially for homologous versus non-homologous double-stranded DNA used. The increase of NaCl concentration in the solution up to 30 mM removes the peptide from both types of the complexes completely. At the same time at 20 mM NaCl calf thymus DNA quadruplexes readily dissociate, whereas the structures formed by plasmid DNA retain their morphology in the solution containing NaCl with concentrations up to 40 mM and are only partially disrupted at even higher NaCl concentration. These results provide the analogy between trivaline-DNA model complexes and RecA-DNA binding.

[The hierarchy of complexes and compact structures of trivaline with nucleic acids. III. Complexes of trivaline with trinucleotides forms a rod-like structure with length of about 1000 A in solution]. / Ob ierarkhii kompleksov i kompaktnykh struktur trivalina s nukleinovymi kislotami. III. Kompleksy trivalina s trinukleotidami obrazuiut v rastvore palochkoobraznye struktury dlinoi 1000 A]

We demonstrated the ability of trivaline in the course of interaction with certain trinucleotides in solution to form extended fibre-like structures with lengths of up to several thousand angstroms. Such structures were observed for complexes of trivaline with both deoxyribo- and ribonucleotides with homopurine, homopyrimidine, or random sequences, with or without terminal 5'-phosphate. A model of organization of such structures is proposed. It is based on tetramer complex of trivaline with short nucleotides, two structural units of which, consisting of trivaline tetramer and two trinucleotides, form the octamer complex. It has three perpendicular axes of symmetry of the second order. The spatial location of bases in this structure is additionally fixed by nucleopeptide interactions. The latter create favourable conditions for arranging hydrogen bonds between trinucleotides belonging to different tetramer complexes and stacking interactions between the bases of each nucleotide. Octamer complexes are able to form regular aggregates in the form of a "stack", consisting of dozens of elementary units. These aggregates can be electron microscopically visualized as extended fibre-like structures.

[The hierarchy of complexes and compact structures of trivaline with nucleic acids. I. Interaction of trivaline with double-stranded DNA]. / Ob ierarkhii kompleksov i kompaktnykh struktur trivalina s nukleinovymi kislotami. I. Vzaimodeistvie trivalina s dtsDNK.

It was shown that trivaline-poly(U) complexes have no appreciable fluorescence at any peptide concentration if the polymer concentration is less than critical. By electron microscopy it was shown that dsDNA molecules undergo a second step of compactization if polymer and peptide concentrations are high enough. The diameter of this rod-like particles is over 40 nm. If the polymer concentration is less than critical, dsDNA molecules form with trivaline extended structures of dissimilar morphology. We propose a scheme of trivaline-dsDNA complexes and compact structure formation, beginning from a dimer complex. It is consistent with the results of equilibrium dialysis, fluorescence, electron microscopy, flow linear and circular dichroism measurements.