Cholesteric Liquid-crystalline Dispersions Research Articles

A Principal Approach to the Detection of Radiation-Induced DNA Damage by Circular Dichroism Spectroscopy and Its Dosimetric Application

Using cholesteric liquid-crystalline dispersion (CLCD) of DNA, we demonstrate that the molecularly organized systems may be used both for qualitative assessment of the degree of radiation-induced DNA damage, as well as for detection of radiation doses in a very wide range. The doses up to 500 Gy do not cause any significant changes in optical signals of DNA in solution. However, when irradiated molecules are used to prepare the CLCD by addition of crowding polymer, a clear correlation of its optical signals with an absorbed dose is observed. For example, at a dose of 500 Gy, a maximum drop in the circular dichroism (CD) signal for DNA solution and for CLCD formed from preliminary irradiated molecules is ≈20% and ≈700%, respectively. This approach can also be used to expand the dosimetric capabilities of DNA CLCD. Compared to the case of irradiation of ready-made DNA CLCD, formation of the dispersed system from irradiated DNA allows to increase its sensitivity by more than 2 orders of magnitude. A similar decrease in the CD signal (≈1.45-fold) is observed in these systems at the doses of 100 kGy and 200 Gy, respectively. This principal approach seems to be relevant for other biomolecules and molecularly organized systems.

Förster resonance energy transfer from Hoechst 33258 to SYBR Green I in cholesteric liquid-crystalline DNA

In the present work Förster resonance energy transfer (FRET) between DNA-specific fluorescent dyes Hoechst 33258 (Ht58; donor) and SYBR Green I (SG; acceptor) is compared in isotropic DNA solution and in its cholesteric liquid-crystalline dispersion (DNA CLCD). At a fixed donor concentration of 3.57 × 10−6 М in the spatially ordered system, FRET starts at lower acceptor concentration than in the isotropic system and at the SG concentration below 1.05 × 10−7 M, its efficiency in DNA CLCD is up to ≈4-fold higher than in DNA solution (3D-FRET vs. 1D-FRET). However, the maximum FRET efficiency is higher in isotropic solution (≈70% vs. ≈50%, respectively) apparently due to the absence of steric restrictions on intermolecular interactions imposed by the dense packaging of the DNA molecules. In isotropic DNA solution FRET from Ht58 to SG is accompanied by the displacement of Ht58 from the minor-groove complex as the SG concentration increased, while the displacement of Ht58 in DNA CLCD seems to be limited. Using FRET between Ht58 and SG, the formation of DNA CLCD particles via stopped-flow method was investigated and for the first time the subsecond stage of the DNA CLCD particle formation, generation of “precholesteric” optically inactive nuclei, was demonstrated.

Cholesteric liquid-crystalline DNA – a new type of chemical detector of ionizing radiation

ABSTRACT Liquid crystals (LCs) can be promising for detection of ionising radiation (IR). However, there is little information about their interaction with IR and there are no systemic studies of the dosimetric application of LCs. In this communication, we show for the first time the capabilities of a new type of radiation detector – cholesteric liquid-crystalline dispersion (CLCD) of DNA obtained using polyethylene glycol (PEG) and salt indued condensation. Irradiation of the DNA CLCD samples with 0–100 kGy of 7.6 MeV bremsstrahlung led to significant change in their circular dichroism (CD): ≈5% and ≈12% increase in the signal was observed at 10 and 20 kGy, while in the range of 20–90 kGy the CD signal decreased linearly. Thus, detectors based on DNA CLCD could be of interest for such application of IR as material modification or radiation sterilisation. At the same time irradiation can help to stabilise dispersion particles and can be used to prevent their sedimentation. Unirradiated samples degraded within 24 h after preparation, while the irradiated ones were able to maintain the intensity of CD signal for more than 168 h.Schematic representation of the dual effect of radiation exposure the DNA CLCD.

Comparison of quenching efficacy of SYBR Green I and PicoGreen fluorescence by ultrasmall gold nanoparticles in isotropic and liquid-crystalline DNA systems

Gold nanoparticles (GNPs) are promising for sensors based on fluorescence quenching. Conjunction of GNPs to liquid crystalline (LC) matrix may improve their quenching properties. In the present work the quenching of SYBR Green I (SG) and PicoGreen (PG) fluorescence by negatively charged 2.5 nm GNPs was investigated in an isotropic DNA solution and in its cholesteric liquid crystalline dispersion (DNA CLCD). These dyes have similar chromophore structures and spectral properties but different charges: +2 (SG) and +3 (PG). The most efficient static fluorescence quenching was observed at the lowest concentration of dyes: in the DNA CLCD for SG (KSV = 7.4 × 107 M−1) and in the isotropic DNA solution for PG (KSV = 1.54 × 108 M−1). Since in the DNA solution quenching was due to the formation of a non-fluorescent complex with GNPs, the quenching was up to ~70-fold more efficient for PG than for SG. In the DNA CLCD the quenching efficacy of SG fluorescence increased up to ~15-fold because of the energy transfer. At the same time, due to steric hindrance for the interaction of PG with GNPs in the DNA CLCD caused by tight packaging of DNA molecules, the fluorescence quenching efficacy was ~5-fold higher in the isotropic solution.

Fluorescence superquenching of SYBR Green I in crowded DNA by gold nanoparticles

Gold nanoparticles (GNPs) are known to efficiently quench fluorescence with high constants (107-1011 M−1). Quenching efficiency is determined by GNPs parameters, the properties of dye complex with biomolecular substrate may also affect this efficiency. In the present work quenching of SYBR Green I fluorescence by 2.5 nm GNPs was investigated in an isotropic DNA solution and on cholesteric (DNA CLCD) and hexagonal (DNA HLCD) liquid crystalline dispersion templates obtained with polymer and salt-induced condensation. Superquenching of SYBR Green I fluorescence was observed in all investigated models. The quenching efficiency increased as the DNA molecules were spatially ordered: the greatest quenching was observed for DNA HLCD where DNA molecules are packed most densely. In the condensed DNA systems, unlike DNA solution, the mechanism of quenching was not only the formation of a nonfluorescent complex, but also energy transfer between dye molecules and GNPs. The Stern-Volmer and Perrin models were insufficient for description of quenching processes. The Perrin equation was in an agreement with experiment only for DNA in solution. The model of microphase separation most adequately described the SYBR Green I fluorescence quenching by GNPs in DNA solution as well as in the molecularly organized DNA CLCD and DNA HLCD.

Erratum to: “DNA sequence-specific ligands: XVI. Series of the DBP(n) fluorescent dimeric bisbenzimidazoles with 1,4-piperazine-containing linkers”

A novel series of the DBP(n) fluorescent symmetric dimeric bisbenzimidazoles in which the bisbenzimidazole fragments were attached to an oligomeric linker with the 1,4-piperazine residue in its center were prepared. The DBP(n) molecules were distinguished by the number of methylene groups n (where n = 1, 2, 3, 4) in the linker. The DBP(n) synthesis was based on a condensation of the monomeric bisbenzimidazole (MB) with 1,4-piperazinedialkylcarbonic acids. The ability of the DBP(n) dimeric bisbenzimidazoles to form complexes with the double-stranded DNA was demonstrated by a complex of physicochemical methods, including spectroscopy in the visual UV-area, circular dichroism (CD), and fluorescence. The DBP(1–4) molecules were localized in the DNA minor groove by the CD method with the use of cholesteric liquid-crystalline dispersions (CLCD) of the double-stranded DNA. The DBP(n) dimeric bisbenzimidazoles were easily soluble in water, penetrated through cellular and nuclear membranes, and stained DNA in living cells distinct from the previously synthesized DB(n) series.

About the spatial organization of double-stranded DNA molecules in the cholesteric liquid-crystalline phase and dispersion particles of this phase

The answer to a question on the organization of molecules in a cholesteric phase is well enough proved in case of low molecular mass compounds. However, in case of double-stranded nucleic acids molecules the unequivocal answer to such question is a subject of discussions. In this work an attempt to generalize the well known literary data on the structure of the cholesteric phase formed by double-stranded DNA molecules was undertaken. Besides the experimental results of authors describing the packing of these molecules in the cholesteric liquid-crystalline dispersion particles are added to these data. Comparison of the results obtained offers the possibility to come out with an assumption of high probability of the existence of both the short-range positional and long-range orientational order in arrangement of double-stranded DNA molecules in a liquid-crystalline phase, and in the particles of dispersions of this phase generated under certain conditions. The occurrence of the orientational order, i.e. rotation of 'quasinematic' layers of double-stranded DNA molecules by a small angle, defines the formation of spatially twisted (cholesteric) structure with characteristic for it physical and chemical properties.

Gold nanoparticles and DNA liquid crystals

This brief review covers the effect of small-size gold nanoparticles on cholesteric liquid-crystalline dispersions formed by double-stranded high-molecular-weight DNA molecules. It is shown that, as in the case of liquid crystals formed by low-molecular-weight compounds, this effect results in the nematization of the spatial structure of the dispersion particles and the incorporation of small-size gold nanoparticles in the “free” space between DNA molecules, forming quasinematic layers of dispersion particles. As a result of these processes, an “integrated” structure (“rigid” spatial DNA structure) is formed, which possess unique properties.

Superparamagnetic cobalt ferrite nanoparticles “blow up” the spatial ordering of double-stranded DNA molecules

The formation of cholesteric liquid-crystalline dispersions formed by double-stranded DNA molecules, handled by positively charged superparamagnetic cobalt ferrite nanoparticles, as well as action of these nanoparticles on DNA dispersion, are considered. The binding of magnetic nanoparticles to the linear double-stranded DNA in solution of high ionic strength (0.3 M NaCl) and subsequent phase exclusion of these complexes from polyethylene glycol-containing solutions lead to their inability to form dispersions, whose particles do possess the spatially twisted arrangement of neighboring double-stranded DNA molecules. The action of magnetic nanoparticles on DNA dispersion (one magnetic nanoparticle per one double-stranded DNA molecule) results in such "perturbation" of DNA structure at sites of magnetic nanoparticles binding that the regular spatial structure of DNA dispersion particles "blows up"; this process is accompanied by disappearance of both abnormal optical activity and characteristic Bragg maximum on the small-angle X-ray scattering curve. Allowing with the fact that the physicochemical properties of the DNA liquid-crystalline dispersion particles reflect features of spatial organization of these molecules in chromosomes of primitive organisms, it is possible, that the found effect can have the relevant biological consequences.

The Physicochemical and Nanotechnological Approaches to Creation of “Rigid” DNA Nanoconstructions

Two different approaches to the creation of rigid (gel-like) DNA particles (or DNA nanoconstructions) are considered. The physicochemical approach is based on the formation of nanobridges between double-stranded DNA molecules ordered in quasinematic layers of cholesteric liquid-crystalline dispersion (CLCD) particles or their salting-out. This approach represents by itself a chemical gelation of DNA molecules realized inside the nanometric DNA particles. The formed rigid DNA particles have unique physicochemical properties. The nanotechnological approach is based on induction of physical gelation as a result of the formation of Au-clusters in the free space between double-stranded DNA molecules ordered in CLCD particles. This approach results in the formation of a new rigid DNA material. Its dis- tinctive peculiarity, in contrast to the chemically bonded rigid DNA particles, consists in a very weak binding affinity to the nuclear membrane filter used for atomic force microscopy. This opens up a possibility for easy manipulations with CLCD particles doped with gold nanoparticles.

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.

Structural effects induced by gold nanoparticles in particles of cholesteric liquid-crystalline dispersion of double-stranded nucleic acids

Gold (Au) nanoparticles of diameter ~2 nm (but not 5 or 15 nm) were capable of incorporating effectively into quasi-nematic layers of particles of cholesteric liquid-crystalline dispersions (CLCD) formed by double-stranded nucleic acids of various families [B-form DNA, A-form poly(I) × poly(C)]. A study of the properties of CLCD particles treated with Au nanoparticles by various physicochemical methods demonstrated that incorporation of Au nanoparticles into quasi-nematic layers of these particles resulted in two effects. First, it caused rearrangement of the spatial cholesteric structure of LC dispersion particles that was accompanied by a decrease in the amplitude of the anomalous band in the circular dichroism spectrum of the corresponding initial CLCD. Second, it induced Au cluster formation within the LC particles that was accompanied by the appearance of a surface plasmon resonance band in the visible spectral region. It was possible that these structural effects were responsible for the genotoxicity of the Au nanoparticles.

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.

Retention of gold nanoparticles in the structure of quasinematic layers formed by DNA molecules

Gold nanoparticles are shown to get incorporated into double-stranded DNA molecules forming quasinematic layers in the cholesteric liquid-crystalline dispersion particles. The process of nanoparticle incorporation results in distortion in an ordered arrangement of the neighboring dsDNA molecules in a layer and in global spatial structure of particles of the dispersion, which may be one of the possible causes of the genotoxicity of gold nanoparticles.

A Dual Effect of Au-Nanoparticles on Nucleic Acid Cholesteric Liquid-Crystalline Particles

Au-nanoparticles (size about 2 nm, but not 5 or 15 nm) are capable of effectively incorporating into quasinematic layers of particles of cholesteric liquid-crystalline dispersion formed by double-stranded nucleic acid molecules of various families (DNA and poly(I)xpoly(C)). This Au-size-dependent process is accompanied by a decrease in amplitudes of abnormal bands in the CD spectra specific to initial cholesteric liquid-crystalline dispersions and simultaneously by an appearance of plasmon resonance band in visible absorption spectrum. The study of properties of particles of cholesteric liquid-crystalline dispersion treated with Au-nanoparticles by means of various physico-chemical methods demonstrates that incorporation of Au-nanoparticles into quasinematic layers of these particles results in two effects: i) it facilitates reorganization of the spatial cholesteric structure of particles, and ii) it induces the formation of Au-clusters in the content of particles. It is not excluded that these effects represent a possible reason for genotoxicity of Au-nanopar- ticles.

SAXS-data-based structural modeling of DNA–gadolinium complexes fixed in particles of cholesteric liquid-crystalline dispersions

Structure of cholesteric liquid-crystalline dispersions (CLCDs) formed by double-stranded DNA molecules and treated with gadolinium salts was studied by small-angle X-ray scattering (SAXS). The obtained SAXS data open the way for structural modeling of these complexes to obtain a reasonable explanation for the correlated decrease in amplitude of an abnormal negative band in the circular dichroism (CD) spectra and the characteristic Bragg peak in the experimental small-angle X-ray scattering curves observed on treatment of CLCD by gadolinium salts. Model simulations of different kinds of structural organizations of the DNA-gadolinium complex were performed using novel SAXS data analysis methods in combination with several new, complementary modeling techniques, enabling us to build low-resolution three-dimensional structural models of DNA-gadolinium complexes fixed in CLCD particles. The obtained models allow us to suggest that a change takes place in the helical twist of quasinematic layers formed by these molecules at high concentrations of gadolinium salt. This change in the twist can be used to explain the experimentally observed increase in amplitude of an abnormal band in the CD spectra of DNA CLCD.

Liquid-crystalline dispersions formed by the 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.

A Transition in DNA Molecule's Spatial Ordering Due to Nano-Scale Structural Changes

The optical, X-ray- and AFM-properties of the particles of cholesteric liquid-crystalline dispersion (CLCD) formed by double-stranded DNA molecules and treated with gadolinium salts are studied. It is shown that under certain conditions this treatment results in two effects: i)amplification of the abnormal negative band in the CD spectra specific to the initial CLCD, and ii) disappearance of small-angle X-ray scattering specific of ordered DNA molecules. The explana- tion of these effects is based on a suggestion that gadolinium ions when present at high concentrations not only induce nano-scale changes in the DNA secondary structure, but also overcompensate the DNA negative charges, influencing the efficiency of DNA-DNA interactions in the content of the particles of CLCD. These processes lead to transformation of the spatial structure in particles of CLCD. The resulting structure of particles of DNA CLCD highly enriched in gadolin- ium is not liquid-crystalline; rather it is a rigid construction of practical importance.

Nanotechnology and Nucleic Acids

The peculiarities of two approaches in the nanodesign based on the double-stranded nucleic acid molecules are considered. Main attention is devoted to the formation and properties of nanoconstructions based on the double-stranded DNA molecules and the complexes with chitosan, fixed in the spatial structure of particles of their cholesteric liquidcrystalline dispersions, and cross-linked by artificial nanobridges consisting of alternating copper ions and daunomycin molecules. In contrast to initial DNA liquid-crystalline particles, the structure of resulting nanoconstruction is not “liquidcrystalline” any more; rather it is rigid, crystal-like, three-dimensional structure. The formed nanoconstructions possess the unique spatial and optical properties; this opens a gate for their application in biotechnology and medicine.

Regulation of the spatial organization of DNA molecules in particles of liquid-crystalline dispersions by means of biologically active compounds

Using the “structural conflict” method based on differences in the spatial organization of a liquid-crystalline dispersion (LCD) of DNA-polycation complexes and the DNA cholesteric LCD, it was shown that in a solvent with specific properties, the spatial structure of the LCD of DNA-polycation complexes can be controlled by biologically active compounds (BAC) that cause either dissociation of the DNA-polycation complex or destruction of the polycation molecules. In both cases, the cholesteric structure of LCD typical of the initial uncomplexed DNA molecules is recovered. The recovery of cholesteric LCD of DNA was proved by circular dichroism spectroscopy and confirmed by X-ray diffraction and polarization microscopy. The fact that spatial restructuring of LCD of the DNA-polycation complexes is induced by BAC in low concentrations opens up the possibility for using particles of these dispersions as sensing elements (sandwich-type biosensing units) of biosensor devices. A new method for determination of heparin based on the use of LCD of the DNA-polyconidine complexes was developed. The minimum heparin concentration determined in the sample equals ∼ 0.4–0.5 µg mL−1.