[Dynamics of penetration of "solid" nanoconstructions based on double-stranded DNA complexed with gadolinium into CHO cells].

Currently, neutron capture therapy is a promising cancer treatment. This method is based on the reaction of the thermal neutron capture by some non-radioactive elements (e.g., Gds57), which results in subsequent emission of electrons and gamma rays. An effective instrument for delivery of gadolinium into the tumor tissue are the particles of the "rigid" nanostructures (NS) based on double-stranded DNA complexes with gadolinium (NS-Gd). The local concentration of Gd in such nanostructures may reach 40%. To optimize the process of neutron capture therapy it is very important to investigate possible penetration mechanisms of NS-Gd particles into the tumor cells. In this work, the dynamics of interaction NS-Gd with cultivated chinese hamster ovary cells (CHO) was studied by confocal and electron microscopy. It is shown that NS-Gd are able to enter CHO cells. This process begins in about 1 hour after the start ofincubation. After 6 h NS-Gd particles were detected in almost all cells. A further increase of the incubation time does not lead to significant changes in cell morphology, although the number NS-Gd inside the cells increases. The plasma membrane of the cells remains intact. The NS-Gd particles, which entered the cells, remain inside the cells for a long time. The data obtained show that NS-Gd are relatively low-toxic and suggest that the presence of NS-Gd in the tumor cells does not prevent their division. The data obtained are important for improving the efficiency of the neutron capture therapy method.

[Structural nucleic acid nanotechnology: liquid-crystalline approach].

The properties of the particles of cholesteric liquid-crystalline dispersions formed by double-stranded DNA molecules obtained as a result of phase exclusion of these molecules from water-salt polymer-containing solutions are briefly described. Physicochemical properties of quasinematic layers of dispersion particles and double-stranded DNA molecules in their content are taken into account in the course of developing fundamental background of the liquid-crystalline approach to the DNA structural nanotechnology. According to different versions of this approach, which is based on intraparticle gelation of cholesteric liquid-crystalline dispersions, spatial structures (DNA nanoconstructions, "rigid" DNA particles) with unique properties, are created. By means of atomic force microscopy images of "rigid" DNA particles of different type are registered. Specific properties of metallic nanoparticles (in particular, gold nanoparticles) are considered while developing the other approach to DNA structural nanotechnology, which provides the basis for "metallized" DNA nanoconstructions.

[Linear clusters of gold nanoparticles in quasinematic layers of DNA liquid-crystalline dispersion particles].

The effects of small size (approximately 2 nm) gold nanoparticles on the properties of particles of cholesteric liquid-crystalline dispersions formed by double-stranded DNA molecules were analyzed. It has been shown that gold nanoparticles induce two different processes. First, they facilitate reorganization of the spatial cholesteric structure of dispersion particles to nematic one. This process is accompanied by the fast decrease in the amplitude of abnormal band in the CD spectrum. Second, they can form ensembles consisting of gold nanoparticles. This process is accompanied by the development and displacement of surface plasmon resonance band in the visible region of the absorption spectrum. The appearance of this band is analyzed by considering two different models of the formation of ensembles consisting of gold nanoparticles. By small-angle X-ray scattering we performed structural analysis of phases formed by DNA cholesteric liquid-crystalline dispersion particles treated with gold nanoparticles. As a result of this study it was possible to prove the formation of linear clusters of gold nanoparticles in the "free space" between the adjacent DNA molecules fixed in the quasinematic layers of liquid-crystalline particles. It has been hypothesized that the formation of linear clusters of gold nanoparticles is most likely related to DNA molecules, ordered in the spatial structure of quasinematic layers, and the toxicity of these nanoparticles in biological systems hypothesized.

[Bovine sperm chromatin is not protected from the effects ultrasmall gold nanoparticles].

The response of ejaculated bovine spermatozoa to gold nanoparticles was studied by the standard method of nuclear chromatin decondensation in vitro. After the treatment of semen samples with a hydrosol containing gold nanoparticles with an average diameter of 3.0 nm and a concentration of 1 x 10(15) particles/mL, the ability of sperm nuclei to decondense in the presence of sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) dramatically changed compared to the control. The frequencies of gametes with nondecondensed ("intact"), partially decondensed, and completely decondensed nuclei correlated as 40 : 32 : 28% and 0 : 36 : 64% in the experiment and the control, respectively. Moreover, the appearance of a sufficiently large number of gametes with destructed and almost completely destroyed nuclei was noticed in the spermatozoa treated with gold nanoparticles. This article suggests the putative mechanisms of action of ultrasmall gold nanoparticles on the structural and functional integrity of the deoxyribonucleoprotein (DNP) complex of mature male gametes.

[Effect of gold nanoparticles on mouse spermatogenesis].

The response of the mouse male germ cells exposed to gold nanoparticles (approximately 2.5 nm) was studied. Our investigation demonstrates that treatment with Au nanoparticles for four days does not impair the architecture of the spermatogenic epithelium. Cytogenetic evaluation using micronucleus assay showed that gold nanoparticles can affect the chromosomes of early primary spermatocytes. However, gold nanoparticles did not induce chromosome abnormalities in spermatogonial stem cells. Further, the cauda epididymal sperm was isolated on the 14th day after treatment and was incubated in SDS solution (Na sodium dodecyl) and then in a solution containing DTT (dithiothreitol) to induce nuclear chromatin decondensation. Observations showed that after four days of treatment of spermiogenic (postmeiotic) cells with gold nanoparticles the decondensation process had no differences from the control. On the contrary, in the experiment with the same cells and period of fixation but with a single exposure to gold nanoparticles, the number of mature gametes with totally decondensed nuclei reached 100% as opposed to 44% in the controls.

[From liquid crystals to nanoconstructions of DNA].

This paper reviews data obtained in our Laboratory in the field of the liquid-crystalline dispersions of the double-stranded nucleic acids (DNA, RNA) and results of the analysis of behavior of these biopolymeric molecules in the quasinematic layers. The fundamental data obtained in this area were used as a background for creation of the DNA nanoconstructions, containing different "guest" (biologically active or/and chemical substances) molecules. Two theoretically possible ways for creation of the DNA nanoconstructions were compared. The unique properties of the nanoconstructions dictating the scope of their practical application are described.

[Liquid-crystalline dispersions formed by complexes of linear double-stranded DNA molecules with dendrimers].

The formation of liquid-crystalline dispersions as a result of interaction of linear, double-stranded DNA molecules with poly(propyleneimine) dendrimers in water-salt solutions was studied. It was shown, that this process does not depend on the ionic strength of solution and molecular structure of dendrimer. By means of the atomic force microscopy, it was established, that in the case of the dendrimer molecules of the 4th generation (G4), the mean size of particles of (DNA-dendrimer G4) liquid-crystalline dispersion is equal to 300-400 nm. The "boundary" conditions (ionic strength of solutionand molecular mass of dendrimer) of formation of optically active (cholesteric) and optically inactive of the (DNA-dendrimer) dispersions were determined using circular dichroism spectroscopy. The interaction of dendrimers of 1st, 2nd, 3rd and 5th generations with DNA molecules results in the obtaining of the optically inactive dispersions. Dendrimer molecules of 4th generation induce the formation of two types dispersions: in solutions of high ionic strength (micro > 0.4) they induce the formation of cholesteric liquid-crystalline dispersions, and in solutions of low or intermediate ionic strength (micro < 0.4) they can form the optically inactive one. The "molecular crowding" affects both the efficiency of binding of dendrimer molecules of 4th generation to DNA, and the mode of spatial packing of (DNA-dendrimer G4) complexes in particles of liquid-crystalline dispersion. The possible reasons capable of explaining the structural polymorphism of (DNA-dendrimer) liquid-crystalline dispersions are discussed.

[Description of the binding of chitosan to DNA at different ionic strengths in terms of the theories of ion condensation and adsorption].

The binding of chitosan molecules to DNA in solutions of different ionic strength has been studied. The data were analyzed in terms of the model of ion condensation and the thermodynamic theory of the binding of protracted ligands to DNA. Combining these approaches made it possible to estimate the sterical and energetic characteristics of chitosan-DNA binding and establish the dependence of the chitosan-DNA binding constant on the ionic strength of solution.

[Complexes of gadolinium with linear and liquid-crystalline forms of DNA].

The binding of Gd3+ ions to linear double-stranded DNA molecules in water-salt solutions or in liquid-crystalline dispersions is accompanied by sharp changes in their optical and X-ray characteristics. Depending on the initial conditions of complex formation, the binding of Gd3+ ions either to DNA bases or phosphate groups occurs, which leads to changes in the properties of the liquid-crystalline dispersions. The packing of neighboring DNA molecules in particles of the liquid-crystalline dispersion of the complex DNA-Gd3+ depends strongly on the concentration of Gd3+ ions. This process is accompanied by a decrease in the amplitude of Bragg's reflection maximum. The unique properties of the developed material open the possibilities for its practical use.

[Structural polymorphism of the liquid-crystalline dispersions formed from the double-stranded DNA molecules complexed with synthetic polycations].

The double-stranded, linear DNA molecules form the liquid-crystalline dispersions (LCD) in water-salt solutions containing positively charged polyconidin molecules. It was established from the analysis of the absorption spectra of the LCDs formed from (DNA-polyconidin) complexes, that the mean size of the particles of these dispersions is equal to -6000 angstroms. The small-angle X-ray data show, that in the LCD particles different density of packing of the (DNA-polycation) complexes is realized. The comparison of the X-ray data of the liquid-crystalline phases of (DNA-polyconidin) complexes formed under various conditions with the phase diagram, that reflects the polymorphism of the linear double-stranded DNA liquid crystals, demonstrates that the hexagonal mode of the LCD packing is existing in 0.15-0.4 M NaCl solutions, whereas in 0.4-0.55 M NaCl solutions-- the cholesteric one. As a result of specific spatial organization the cholesteric LCD possesses of an abnormal optical activity in the CD spectrum. The similar situation takes place in the case of another synthetic polycation--poly(2,5-ionen), whose chemical structure differs from that of polyconidin. Thus, the structural polymorphism of the (DNA-polyconidine) LCDs was evidenced. It means that change of NaCl concentration opens a gate to control the spatial packing of the molecules of (DNA-polycation) complexes in the particles of LCDs. The supposition about mechanism of formation of the DNA cholesteric liquid-crystalline state in the narrow interval of NaCl concentrations was suggested.

[The thermodynamic stability of nanoconstructions based on the double-stranded DNA molecules].

The temperature stability of nanoconstructions formed by double-stranded DNA molecules fixed in the structure of their liquid crystalline dispersions and cross-linked by nanobridges was determined. It was shown that the heating of nanoconstructions is accompanied by a decrease in the amplitude of the negative bands in the CD spectrum both at lambda approximately 310 and lambda approximately 505 nm. Temperature "melting curves" were derived and characterzed by T(M) values. The T(M) values at lambda approximately 310 and lambda approximately 505 nm coincided with each other but differed from the T(M) value characteristic of the DNA cholesteric liquid crystalline dispersion.

[A compact form of DNA in solution. 2. Peculiarities of acidic titration of double-stranded DNA in PEG-containing water-salt solutions]. / Kompaktnaia forma DNK v rastvore

The formation of compact double-stranded DNA molecules in PEG-containing watersalt solutions (0.3 M NaCl) may be observed within the pH-range 3-10; i.e. under conditions at which parameters of double-stranded DNA helices are not strongly different from those of B-form. At pH less than 3, when regularity of double helices is significantly changed, the formation of the specific compact particles of DNA in PEG-containing solutions does not take place. Denaturation of the compact form of DNA in PEG-containing solution is accompanied by disappearance of the negative band in CD spectrum. Hyperchromic effect of denaturation of DNA compact form is uninformative because of the influence of the light-scattering by compact DNA molecules.

[Formation of various types of complexes upon the interaction of anthraquinones with DNA]. / Obrazovanie kompleksov raznogo tipa pri vzaimodeistvii antrakhinonov s DNK.

The optical properties (circular and linear dichroism) of the 11 anthraquinone antibiotics complexed with DNA molecules under conditions giving rise to cholesteric liquid-crystalline dispersions were investigated. Complexes of compounds A911 and A912 with double-stranded linear or circular DNA molecules differ strongly in their properties from those of other anthraquinones. The possible reasons of these differences and their possible correlation with the biological activity of the A911 and A912 are discussed.

[Enzymatic cleavage of superhelical DNA in a liquid crystal state]. / Fermentativnoe rasshcheplenie superspiral'noi DNK v usloviiakh ee zhidkokristallicheskoi upakovki.

Superhelical pBR322 DNA molecules form liquid-crystalline dispersions in water-salt solutions containing poly(ethyleneglycol). The formation of the liquid-crystalline dispersions from superhelical DNA molecules results in the appearance of two sites inside the DNA molecules that are split by Micrococcal nuclease. The first site of digestion does not differ from the standard site split by this enzyme in water-salt solutions, whereas the second one represents a new site specific only for the DNA molecules forming liquid-crystalline dispersions. Splitting of the DNA molecule through the first site is accompanied by formation of its linear form; splitting of a new site results in the formation of two linear DNA fragments with molecular masses equal to half of the initial DNA molecules. Enzyme digestion of superhelical DNA molecules forming liquid-crystalline dispersions induces a reformation of the "nonspecific" space organization of dispersions to the cholesteric one. A hypothetic model for packing of the superhelical DNA molecules inside liquid-crystalline dispersions and its transformation under enzyme action is suggested.

[Compact form of synthetic polynucleotides. Relationship between secondary structure and circular dichroism spectra]. / Kompaktnaia forma sinteticheskikh polinukleotidov. Sviaz' sektrov krugovogo dikhroizma so vtorichnoi strukturoi.

The formation of compact particles from synthetic double- and triplestranded polynucleotides in water-salt solutions, containing poly(ethylene glycol) (PEG) has been investigated. CD spectra of compact particles are characterized by intense bands (positive or negative) in the region of 270 nm, compact particles being divided into two families--psi- and psi+--according to the CD band sign. The amplitude of the CD band at 270 nm increases with the increase of CPEG. Heating of a solution, containing compact particles, results in a disappearance of the CD band, the "melting" of compact particles as revealed by the CD method occuring prior to the melting of the secondary structure of the corresponding polynucleotide. It is concluded that intense CD bands, which are characteristic of the compact form of synthetic polynucleotides, arise (similar to the case of DNA or dsRNA) from regular arrangement of polynucleotide chains in compact particles. The question, concerning the relation between parameters of the secondary structure of polynucleotides and their belonging either to psi- or to psi+ family is discussed. The factors, which could account for the appearance of intense bands in CD spectra of compact particles are also considered.

[A compact form of DNA in solution. III. Influence of the ion composition of the solution on the compactization process of double-stranded DNA in the presence of peg]. / Kompaktnaia forma DNK v rastvore III. Vliianie ionnogo sostava rastvora na protsess kompaktizatsii dvukhtsepochechnoi DNK v prisutstvii polietilenglikolia

The data showing the features of the DNA compactization process in PEG-containing solutions of chlorides of different alkaline metals (LiCl, KCl, RbCl and CsCl) and an ammonium salt (CH3-(CH2)17-N-(CH3)3Br) are presented. The data indicate that the formation of a compact form of the double-stranded DNA in PEG-containing water-salt solutions depends not only on the PEG concentration and ionic strength but on tha cation nature as well. The compactization occurs most easily in the presence of Na+-ions. This indicates a specific character of interaction between Na+-ions and DNA phosphate groups which may be due to an optimum structural fit between the hydrated Na+-ions and orientation of the phosphate groups in the DNA molecule. The nature of forces involved in the processes of the intramolecular compactization and intermolecular aggregation of double-stranded DNA molecules in water-salt solution is discussed. The difference between the effect of Na+ and that of K+-ions on the compactization process at the ionic strengths close to physiological values makes it possible to suggest that the changes of the tertiary structure of double-stranded DNA which accompany its function in vivo may take place under conditions of a decreased water activity at the expense of relatively slight changes in ion composition of the water surrounding DNA.

[Some peculiarities of the kinetics of DNA structural transition after thermal jump registered by hyperchromic effect]. / Obosobennostiakh kinetiki strukturnogo perekhoda DNK Posle teplovogo udara, registriruemoi po giperkhromnomy effektu.

Structural transition of DNA is characterized by complex optical kinetic curve. The kinetic version of Felsenfeld's method is used for estimation of concentrations of "denatured" AT- and GC-base pairs in DNA molecules, and the rate constants of the accumulation of "denatured" AT as well as GC pairs have been estimated. The possible interpretation of the kinetic data obtained may be done by assumption that the fast stage of the process reflects the accumulation of the "denatured" AT pairs, which is due to the melting of AT-rich regions in the DNA molecules, whereas the slow stage of the process is limited by the melting of GC-rich regions. In contrast to the fast stage, slow stage takes place at conditions, when unwinded AT-rich regions (loops) may exist in DNA molecules and cause the delay in the process of denaturation.

[Liquid crystals and liquid crystal dispersions of DNA-daunomycin complexes]. / Zhidkie kristally i zhidkokristallicheskie dispersii kompleksov DNK-daunomitsin.

The X-ray and optical properties as well as the textures of liquid crystals formed of DNA molecules bound in complex with an anthracycline antibiotic daunomycin were investigated. It was shown that DNA.daunomycin complexes, depending on the extent of alteration of the DNA secondary structure by daunomycin, could yield two types of cholesterics differing in the direction of their spatial twisting as well as nematics. Liquid-crystalline dispersions of DNA.daunomycin complexes have CD spectra with "mirror" bands depending on daunomycin concentration. "Testing" of the orientation of daunomycin molecules on the DNA in liquid-crystalline particles suggested two types of binding.

[Nucleic acids as a basis for creating biosensors]. / Nukleonovye kisloty kak osnova dlia sozdaniia biosensorov.

Here we present a brief conception of biosensors. Structural peculiarities and properties of single- and double-stranded nucleic acids that are to be taken into account when creating biosensors on the basis of these biomolecules are considered. On the example of two biologically active compounds a possibility is shown for constructing biosensors on the basis of liquid-crystalline dispersions of low molecular mass DNA and on the basis of liquid-crystalline DNA dispersions immobilized due to their inclusion into the synthetic polymeric matrix.

[Compact particles of double-stranded polyribonucleotides. I. Conditions for the formation of optically active compact double-stranded RNA particles]. / Kompaktnye chastitsy dvukhtsepochnykh poliribonuckleotidov. I. Usloviia formirovaniia opticheski aktivnykh kompaktnykh chastits dvukhtsepochechnoi RNK.

The conditions (mol. weight of PEG, ionic strength) of formation of double-stranded RNA (dsRNA) compact particles in water-salt solutions, containing poly(ethylene glycol) (PEG) have been determined. It has been shown, that in solutions of mild ionic strength (approximately 0,3) the compact particles of dsRNA are characterized by an intense positive CD-band (lambda 270 nm), but in solutions of high ionic strength (1,0-1,5)--by intense positive or negative CD-bands (lambda 270 nm). The heating of the solutions of a high ionic strength, containing the compact particles with negative CD-bands is accompanied by the change of sign of CD-band. The same effect is observed in the case of decrease of the ionic strength of the solutions. "Melting" of compact particles as revealed by the CD-method occurs prior to the melting of the secondary structure of dsRNA. These data show, that the intense CD-bands reflect the ordered arrangement of the chromophores of polynucleotide chain in compact particles. The reasons of change of the sign of the CD-bands are discussed.