Visualization of G-Quadruplexes, i-Motifs and Their Associates.

The non-canonical structures formed by G- or C-rich DNA regions, such as quadruplexes and i-motifs, as well as their associates, have recently been attracting increasing attention both because of the arguments in favor of their existence in vivo and their potential application in nanobiotechnology. When studying the structure and properties of non-canonical forms of DNA, as well as when controlling the artificially created architectures based on them, visualization plays an important role. This review analyzes the methods used to visualize quadruplexes, i-motifs, and their associates with high spatial resolution: fluorescence microscopy, transmission electron microscopy (TEM), and atomic force microscopy (AFM). The key approaches to preparing specimens for the visualization of this type of structures are presented. Examples of visualization of non-canonical DNA structures having various morphologies, such as G-wires, G-loops, as well as individual quadruplexes, i-motifs and their associates, are considered. The potential for using AFM for visualizing non-canonical DNA structures is demonstrated.

Aggregation of Influenza A Virus Nuclear Export Protein.

Influenza A virus nuclear export protein (NEP) plays an important role in the viral life cycle. Recombinant NEP proteins containing (His)6-tag at either N- or C-terminus were obtained by heterologous expression in Escherichia coli cells and their high propensity for aggregation was demonstrated. Dynamic light scattering technique was used to study the kinetics and properties of NEP aggregation in solutions under different conditions (pH, ionic strength, presence of low-molecular-weight additives and organic solvents). Using atomic force microscopy, the predominance of spherical aggregates in all examined NEP preparations was shown, with some amyloid-like structures being observed in the case of NEP-C protein. A number of structure prediction programs were used to identify aggregation-prone regions in the NEP structure. All-atom molecular dynamics simulations indicate a high rate of NEP molecule aggregation and reveal the regions preferentially involved in the intermolecular contacts that are located at the edges of the rod-like protein molecule. Our results suggest that NEP aggregation is determined by different types of interactions and represents an intrinsic property of the protein that appears to be necessary for its functioning in vivo.

A hypothetical hierarchical mechanism of the self-assembly of the Escherichia coli RNA polymerase σ(70) subunit.

Diverse morphology of aggregates of amyloidogenic proteins has been attracting much attention in the last few years, and there is still no complete understanding of the relationships between various types of aggregates. In this work, we propose the model, which universally explains the formation of morphologically different (wormlike and rodlike) aggregates on the example of a σ(70) subunit of RNA polymerase, which has been recently shown to form amyloid fibrils. Aggregates were studied using AFM in solution and depolarized dynamic light scattering. The obtained results demonstrate comparably low Young's moduli of the wormlike structures (7.8-12.3 MPa) indicating less structured aggregation of monomeric proteins than that typical for ß-sheet formation. To shed light on the molecular interaction of the protein during the aggregation, early stages of fibrillization of the σ(70) subunit were modeled using all-atom molecular dynamics. Simulations have shown that the σ(70) subunit is able to form quasi-symmetric extended dimers, which may further interact with each other and grow linearly. The proposed general model explains different pathways of σ(70) subunit aggregation and may be valid for other amyloid proteins.

Synthetic sialylglycopolymer receptor for virus detection using cantilever-based sensors.

We describe the rapid, label-free detection of Influenza A viruses using a cantilever transducer modified with a synthetic sialylglycopolymer receptor layer. Surface stresses induced by viruses binding to the receptor layer were used as the analytical signal. The synthetic sialylglycopolymer receptor layer can be used in nanoscale strain-gauge cantilever transducers for highly sensitive virus detection. Strain-gage transducers using such sensor layers exhibit long lifetimes, high sensitivities, and possible regeneration. Nanomechanical cantilever systems using optical detectors were used for the surface stress measurements. We demonstrated the positive, label-free detection of Influenza A at concentrations below 10(6) viruses per ml. In contrast to hemagglutination assays, cantilever sensors are label free, in situ, and rapid (less than 30 min), and they require minimal or nearly no sample preparation.

The Use of Atomic Force Microscopy for 3D Analysis of Nucleic Acid Hybridization on Microarrays.

Oligonucleotide microarrays are considered today to be one of the most efficient methods of gene diagnostics. The capability of atomic force microscopy (AFM) to characterize the three-dimensional morphology of single molecules on a surface allows one to use it as an effective tool for the 3D analysis of a microarray for the detection of nucleic acids. The high resolution of AFM offers ways to decrease the detection threshold of target DNA and increase the signal-to-noise ratio. In this work, we suggest an approach to the evaluation of the results of hybridization of gold nanoparticle-labeled nucleic acids on silicon microarrays based on an AFM analysis of the surface both in air and in liquid which takes into account of their three-dimensional structure. We suggest a quantitative measure of the hybridization results which is based on the fraction of the surface area occupied by the nanoparticles.

[Investigation of the Dependence of Escherichia coli RNA Polymerase σ70-Subunit Structure on Ionic Strength by Molecular Dynamics Simulation Method].

The σ70-subunit of E. coli RNA polymerase (a small protein, being a part of RNA holoenzyme, and responsible for initiation of transcription of constitutive genes) is modeled at different ionic strengths. Two variants of the location of C-end domain 4 are obtained. At low ionic strength domain 4 interacts with the region of high negative charge 190-210 AK within NCR domain. At high ionic strength this region was screened and domain 4 was free and set away from domain NCR. We suppose that this leads to the increase in polymerization rate. Simulation data do not confirm any hypothesis about a self-inhibition mechanism.

Statistical analysis of molecular nanotemplate driven DNA adsorption on graphite.

In this work, we have studied the conformation of DNA molecules aligned on the nanotemplates of octadecylamine, stearyl alcohol, and stearic acid on highly oriented pyrolytic graphite (HOPG). For this purpose, fluctuations of contours of adsorbed biopolymers obtained from atomic force microscopy (AFM) images were analyzed using the wormlike chain model. Moreover, the conformations of adsorbed biopolymer molecules were characterized by the analysis of the scaling exponent ν, which relates the mean squared end-to-end distance and contour length of the polymer. During adsorption on octadecylamine and stearyl alcohol nanotemplates, DNA forms straight segments, which order along crystallographic axes of graphite. In this case, the conformation of DNA molecules can be described using two different length scales. On a large length scale (at contour lengths l > 200-400 nm), aligned DNA molecules have either 2D compact globule or partially relaxed 2D conformation, whereas on a short length scale (at l ≤ 200-400 nm) their conformation is close to that of rigid rods. The latter type of conformation can be also assigned to DNA adsorbed on a stearic acid nanotemplate. The different conformation of DNA molecules observed on the studied monolayers is connected with the different DNA-nanotemplate interactions associated with the nature of the functional group of the alkane derivative in the nanotemplate (amine, alcohol, or acid). The persistence length of λ-DNA adsorbed on octadecylamine nanotemplates is 31 ± 2 nm indicating the loss of DNA rigidity in comparison with its native state. Similar values of the persistence length (34 ± 2 nm) obtained for 24-times shorter DNA molecules adsorbed on an octadecylamine nanotemplate demonstrate that this rigidity change does not depend on biopolymer length. Possible reasons for the reduction of DNA persistence length are discussed in view of the internal DNA structure and DNA-surface interaction.

[Implementation of scanning probe microscopy for the solution of molecular diagnostics tasks].

We present new approaches to improve the efficiency of DNA by scanning probe microscopy using a highly specific hybridization on affine surfaces and nanostructures of gold as a labels. Scanning probe microscopy allows to register of individual acts of hybridization by the detection of gold labels on the surface affinity followed by automatic calculation of the total.

[Nanoanalytics for medicine].

The applications of atomic force microscopy and the methods based on atomic force microscopy that can be useful in medical nanoanalytics have been reviewed. The main fields of possible application of scanning probe microscopy in medicine have been outlined. Among these are studying the resistance of bacterial cells to modern antibiotics and drugs, morphological analysis of blood components, trichology, nanotoxicology, DNA sequencing, and biocompatibility of medicinal materials. Examples of application of atomic force microscopy for studies in these fields have been considered, and prospects for its use in medicine have been demonstrated.

[Atomic force microscopy of living cells: advances and future outlooks].

The advances of the method of atomic force microscopy for investigating the animal cells and an analysis of its development have been reviewed, with much attention being given to studies of living cells. The features and problems of the method have been considered, and a number of special methods based on the use of atomic force microscopy have been analyzed. The problems of choosing the geometry of probes for studies of animal cells, determination of cell adhesion on substrate, mapping of the cell surface using chemically modified cantilevers, and the distribution of molecular components inside the cell with the use of micro- and nanosurgical approaches have been discussed. The problems of combining the atomic force microscopy with optical and laser scanning confocal microscopy have been considered. Possible applications of the method in biotechnology and medicine are discussed.

Characteristics of Artificial Virus-like Particles Assembled in vitro from Potato Virus X Coat Protein and Foreign Viral RNAs.

Potato virus X (PVX) and some other potexviruses can be reconstitutedin vitrofrom viral coat protein (CP) and RNA. PVX CP is capable of forming viral ribonucleoprotein complexes (vRNP) not only with homologous, but also with foreign RNAs. This paper presents the structure and properties of vRNP assembledin vitroupon incubation of PVX CP and RNAs of various plant and animal viruses belonging to different taxonomic groups. We have shown that the morphology and translational properties of vRNPs containing foreign (heterologous) RNA are identical to those of homological vRNP (PVX RNA - PVX CP). Our data suggest that the assembly of the "mixed" vRNPin vitrocould be started at the 5'-proximal region of the RNA, producing a helical structure of vRNPs with foreign nucleic acids. The formation of heterologous vRNPin vitrowith PVX CP appears not to require a specific 5' end RNA nucleotide sequence, and the PVX CP seems to be able to pack foreign genetic material of various sizes and compositions into artificial virus-like particles.

[Mixed monolayers of amphiphile-modified nucleic bases and diynoic acids. I. Phase states at air-water interface and in Langmuir-Blodgett films]. / Smeshannye monosloi amfifil'nykh proizvodnykh azotistykh osnovanii i diinovykh kislot. I. Fazovoe sostoianie na granitse voda-vozdukh i v plenkakh Lengmiura-Blodzhett.

Monolayers of amphiphile-modified nucleic bases with diynoic acid were obtained and characterized. The synthesized nucleic bases contained in the monolayer complementarily bind the nucleotide molecules contained in the aqueous subphase, and the structure of the resulting monolayers can be fixed by the photopolymerization of diynoic acid. The resulting monolayer exemplifies a novel type of model systems for investigating molecular recognition at the surface of biological membranes. Procedures for the transfer of the monolayers onto solid substrates and photopolymerization of the diynoic acid in mixtures with the derivatives of nucleic bases were developed. The films obtained were structurally characterized using atomic force microscopy. Compression isotherms of the mixed monolayers as well as individual components of monolayers at the air-water interface allowed one to determine the concentration range at which the diynoic acid form true mixtures or domain structures with the derivatives of nucleic base. A study of the films transferred to the solid substrate by atomic force microscopy indicated that this concentration dependence of miscibility behavior was conserved in the transferred films.