[Conformational polymorphysm of G-rich fragments of DNA Alu-repeats. II. the putative role of G-quadruplex structures in genomic rearrangements]. / Konformatsionnyi polimorfizm G-bogatykh fragmentov ALU-povtorov DNK. II. potentsial'naia rol' G-kvadrupleksnykh struktur v genomnykh perestroikakh.

Three evolutionary conserved sites of Alu repeats (PQS2, PQS3 and PQS4) were shown to form stable inter- and intramolecular G-quadruplexes (GQs) in vitro. Structures and topologies of these GQs were elucidated using spectral methods. Self-association of G-rich Alu fragments was studied. Dimeric GQ formation from two distal identical or different putative quadruplex sites - (PQS2)2, (PQS3)2 or PQS2-PQS3 - within one lengthy DNA strand was demonstrated by a FRET-based method. Oligomer PQS4 (folded into a parallel intramolecular GQ) was shown to form stacks of quadruplexes that are stabilized by stacking interactions of external G-tetrads (this was confirmed by DOSY NMR, AFM microscopy and differential CD spectroscopy). Comparative analysis of the properties of various GQs allowed us to put forward a hypothesis of two general mechanisms of intermolecular GQ-dependant genomic rearrangements: 1) formation of a dimeric GQs; 2) association of pre-folded intramolecular parallel GQs from different strands into GQ-stacks. Thus, the observed co-localization of G-rich motifs of Alu elements with double-strand break hotspots and rearrangement hotspots may be accounted for by the specific secondary structure of these motifs. At the same time, this is likely primarily due to high abundance of such G-rich Alu fragments in the genome.

[Conformational polymorphysm of G-rich fragments of DNA ALU-repeats. I. Potential noncanonical structures]. / Konformatsionnyi polimorfizm G-bogatykh fragmentov ALU-povtorov DNK. I. Nekanonicheskie struktury.

In this paper, we report results of systematic studies of conformational polymorphism of G-rich DNA fragments from Alu repeats. Alu retrotransposones are primate-specific short interspersed elements. Using the Alu sequence from the prooncogen bcl2 intron and the consensus AluSx sequence as representative examples, we determined characteristic Alu sites that are capable of adopting G-quadruplex (GQ) conformations (i.e., potential quadruplex sites - PQSAlu), and demonstrated by bioinformatics methods that those sites are Alu-specific in the human genome. Genomic frequencies of PQSAlu were assessed (~1/10000 b.p.). The sites were found to be characteristic of young (active) Alu families (Alu-Y). A recombinant DNA sequence bearing the Alu element from the human bcl2 gene (304 b.p.) and its PQS-mutant (Alu-PQS) were constructed. The formation of noncanonical structures in Alubcl2 dsDNA and the absence of such structures in the case of Alu-PQS were shown using DMS-footprinting and AFM microscopy. Expression vectors bearing wild-type and mutant Alu insertions in the promoter regions were obtained, and the effects of these insertions on the expression of the reporter gene in НЕК293 and HeLa cell lines were compared. Our findings on the spatial organization of Alu repeats may provide insight into the mechanisms of genomic rearrangements which underlie many oncological and neurodegenerative diseases.

[2'-Modified oligoribonucleotides, containing 1,2-diol and aldehyde groups. Synthesis and properties].

1,2-Diol-oligoribonucleotides were prepared using fully protected 2'-O-[2-(2,3-dihydroxypropyl)amino-2-oxoethyl]uridine 3'-phosphoramidite. Incorporation of the 2'-modified uridine residue into oligonucleotide chains does not significantly affect the thermal stability of RNA and RNA-DNA duplexes. Periodate oxidation of the 1,2-diol results in reactive 2'-aldehyde oligoribonucleotides. Further application of these oligonucleotides for cross-linking with bacterial ribonuclease P was investigated.

Anticoagulant effects of thioanalogs of thrombin-binding DNA-aptamer and their stability in the plasma.

In order to create effective therapeutically significant oligonucleotide structures, a series of analogs of thrombin-binding aptamer d(GGTTGGTGTGGTTGG) containing thiophosphoryl substitutions were synthesized. The anticoagulation effects of the resultant thrombin-binding aptamer analogs were evaluated and the effects of local thiomodifications on their structure and function were studied, including the effects on stability in blood plasma and resistance to DNA nucleases. A promising modified oligonucleotide (LL11) was found with the structure modified only in TT loops. It retains antithrombin properties of thrombin-binding aptamer, but is more resistant to biodegradation.

Byproduct with altered fluorescent properties is formed during standard deprotection step of hexachlorofluorescein labeled oligonucleotides.

HEX-labeled oligonucleotides obtained via typical synthetic protocols may contain more than 15% of material with altered spectral characteristics. We discovered hexachlorofluorescein residue transformation unknown earlier for standard DNA ammonolysis step. HEX residue reacts with ammonium hydroxide yielding acridine derivative, which has differed UV-VIS and fluorescent properties compared to HEX. Therefore, for critical bioassays where sensitivity and/or fluorescent signal differentiation (e.g., in quantitative or multiplexed assays) are essential, the careful RP-HPLC purification step is required.

[Oligodeoxynucleotides containing substituted 4-nitroindoles: synthesis and study of their DNA duplexes].

The synthesis of oligonucleotides containing 1-(2-deoxy-beta-D-ribofuranosyl)-2-methyl-4-nitroindole and 1-(2-deoxy-beta-D-ribofuranosyl)-2-phenyl-4-nitroindole is described. The synthesized modified oligonucleotides were used for studying the stability of intermolecular DNA duplexes with one unnatural strand and for evaluation of discriminating potential of 2-methyl- and 2-phenyl-4-nitroindoles toward nucleic bases. For comparison, an unmodified oligonucleotide and oligonucleotides bearing 5-nitroindole were used. It was shown that 2-methyl-4-nitroindole was only insignificantly inferior in stability to 5-nitroindole and characterized by a similar discriminating potential. 2-Phenyl-4-nitroindole provided a more pronounced duplex destabilization, the discrimination toward natural bases being decreased. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http:// www.maik.ru.

Chaos-induced intensification of wave scattering.

Sound-wave propagation in a strongly idealized model of the deep-water acoustic waveguide with a periodic range dependence is considered. It is investigated how the phenomenon of ray and wave chaos affects the sound scattering at a strong mesoscale inhomogeneity of the refractive index caused by the synoptic eddy. Methods derived in the theory of dynamical and quantum chaos are applied. When studying the properties of wave chaos we decompose the wave field into a sum of Floquet modes analogous to quantum states with fixed quasi-energies. It is demonstrated numerically that the "stable islands" from the phase portrait of the ray system reveal themselves in the coarse-grained Wigner functions of individual Floquet modes. A perturbation theory has been derived which gives an insight into the role of the mode-medium resonance in the formation of Floquet modes. It is shown that the presence of a weak internal-wave-induced perturbation giving rise to ray and wave chaos strongly increases the sensitivity of the monochromatic wave field to an appearance of the eddy. To investigate the sensitivity of the transient wave field we have considered variations of the ray travel times--arrival times of sound pulses coming to the receiver through individual ray paths--caused by the eddy. It turns out that even under conditions of ray chaos these variations are relatively predictable. This result suggests that the influence of chaotic-ray motion may be partially suppressed by using pulse signals. However, the relative predictability of travel time variations caused by a large-scale inhomogeneity is not a general property of the ray chaos. This statement is illustrated numerically by considering an inhomogeneity in the form of a perfectly reflecting bar.

Wave chaos and mode-medium resonances at long-range sound propagation in the ocean.

We study how the chaotic ray motion manifests itself at a finite wavelength at long-range sound propagation in the ocean. The problem is investigated using a model of an underwater acoustic waveguide with a periodic range dependence. It is assumed that the sound propagation is governed by the parabolic equation, similar to the Schrodinger equation. When investigating the sound energy distribution in the time-depth plane, it has been found that the coexistence of chaotic and regular rays can cause a "focusing" of acoustic energy within a small temporal interval. It has been shown that this effect is a manifestation of the so-called stickiness, that is, the presence of such parts of the chaotic trajectory where the latter exhibit an almost regular behavior. Another issue considered in this paper is the range variation of the modal structure of the wave field. In a numerical simulation, it has been shown that the energy distribution over normal modes exhibits surprising periodicity. This occurs even for a mode formed by contributions from predominantly chaotic rays. The phenomenon is interpreted from the viewpoint of mode-medium resonance. For some modes, the following effect has been observed. Although an initially excited mode due to scattering at the inhomogeneity breaks up into a group of modes its amplitude at some range points almost restores the starting value. At these ranges, almost all acoustic energy gathers again in the initial mode and the coarse-grained Wigner function concentrates within a comparatively small area of the phase plane.

Suppression of alpha-cyano-4-hydroxycinnamic acid matrix clusters and reduction of chemical noise in MALDI-TOF mass spectrometry.

Progress in high-throughput MALDI-TOFMS analysis, especially in proteome applications, requires development of practical and efficient procedures for the preparation of proteins and peptides in a form suitable for high acquisition rates. These methods should improve successful identification of peptides, which depends on the signal intensity and the absence of interfering signals. Contamination of MALDI samples with alkali salts results in reduced MALDI peptide sensitivity and causes matrix cluster formation (widely reported for CHCA matrix) observed as signals dominating in the range below m/z 1200 in MALDI spectra. One way to remove these background signals, especially for concentrations of peptides lower than 10 fmol/microL, is to wash matrix/sample spots after peptide cocrystallization on the MALDI plate with deionized water prior to analysis. This method takes advantage of the low water solubility of the CHCA compared to its alkali salts. We report here that the application of some ammonium salt solutions, such as citrates and phosphates, instead of deionized water greatly improves the efficiency of this washing approach. Another way to reduce matrix cluster formation is to add ammonium salts as a part of the MALDI matrix. The best results were obtained with monoammonium phosphate, which successfully suppressed matrix clusters and improved sensitivity. Combining both of these approaches-the addition of ammonium salts in the CHCA matrix followed by one postcrystallization washing step with ammonium buffer-provided a substantial ( approximately 3-5-fold) improvement in the sensitivity of MALDI-MS detection compared to unwashed sample spots. This sample preparation method resulted in improved spectral quality and was essential for successful database searching for subnanomolar concentrations of protein digests.

[Effect of bacterial infection on the lipid composition of carp blood lymphocytes]. / Lipidnyi sostav limfotsitov krovi karpa pri bakterial'noi infektsii.

The initial stage of bacterial infection is characterized by an increase in the level of total lipids and polyenoic fatty acids in membrane phospholipids of blood lymphocytes of carp (Cyprinus carpio L.). In the fish infected with aeromonads, changes in the ratios of fatty acids in phospholipids are similar for either lymphocytes, liver, or whole blood. The extent to which these changes are pronounced depends on the original physiological status of the fish.

Sensitivity of ray travel times.

Ray in a waveguide can be considered as a trajectory of the corresponding Hamiltonian system, which appears to be chaotic in a nonuniform environment. From the experimental and practical viewpoints, the ray travel time is an important characteristic that, in some way, involves an information about the waveguide condition. It is shown that the ray travel time as a function of the initial momentum and propagation range in the unperturbed waveguide displays a scaling law. Some properties of the ray travel time predicted by this law still persist in periodically nonuniform waveguides with chaotic ray trajectories. As examples we consider few models with special attention to the underwater acoustic waveguide. It is demonstrated for a deep ocean propagation model that even under conditions of ray chaos the ray travel time is determined, to a considerable extent, by the coordinates of the ray endpoints and the number of turning points, i.e., by a topology of the ray path. We show how the closeness of travel times for rays with equal numbers of turning points reveals itself in ray travel time dependencies on the starting momentum and on the depth of the observation point. It has been shown that the same effect is associated with the appearance of the gap between travel times of chaotic and regular rays. The manifestation of the stickiness (the presence of such parts in a chaotic trajectory where the latter exhibits an almost regular behavior) in ray travel times is discussed. (c) 2002 American Institute of Physics.

Theory and applications of ray chaos to underwater acoustics.

Chaotic ray dynamics in deep sea propagation models is considered using the approaches developed in the theory of dynamical chaos. It has been demonstrated that the mechanism of emergence of ray chaos due to overlapping of nonlinear ray-medium resonances should play an important role in long range sound propagation. Analytical estimations, supported by numerical simulations, show that for realistic values of spatial periods and sound speed fluctuation amplitudes associated with internal-wave-induced perturbations, the resonance overlapping causes stochastic instability of ray paths. The influence of the form of the smooth unperturbed sound speed profile on ray sensitivity to the perturbation is studied. Stability analysis has been conducted by constructing the Poincaré maps and examining depth differences of ray trajectories with close take-off angles. The properties of ray travel times, including fractal properties of the time front fine structures, under condition of ray chaos have been investigated. It has been shown that the coexistence of chaotic and regular rays, typical for dynamical chaos, leads to the appearance of gaps in ray travel time distributions, which are absent in unperturbed waveguides. This phenomenon has a prototype in theory of dynamical chaos called the stochastic particle acceleration. It has been shown that mesoscale inhomogeneities with greater spatial scales than that of internal waves, create irregular local waveguide channels in the vicinity of the axis (i.e., sound speed minimum) of the unperturbed waveguide. Near-axial rays propagating at small grazing angles, "jump" irregularly between these microchannels. This mechanism determines chaotic behavior of the near-axial rays.

Application of DNA-binding polymers for preparation of DNA for analysis by matrix-assisted laser desorption/ionization mass spectrometry.

The susceptibility of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to the presence of salts in a sample, especially salts of alkali metals, requires careful and often tedious desalting procedures which complicate and slow the throughput of MS-based methods. A novel approach to sample preparation was developed based on the extraction of DNA out of solution onto a solid surface with an attached DNA-binding polymer, such as polyethyleneimine or polyvinylpyrrolidone. The observed binding is strong enough to sustain washing, and, as a result, desalting and concentration can be performed in a single fast step. After DNA has been immobilized on the surface and supernatant solution removed, subsequent addition of MALDI matrix releases material from the surface, which co-crystallizes with matrix. The mass spectrometric analysis is then performed directly from this support. Analysis of oligonucleotides and three-fold multiplexed SNP typing reactions performed by this method shows improved sensitivity and excellent resolution for various DNA fragments, together with high tolerance to various buffer components, such as alkali metals and surfactants. Simplicity and speed make it attractive for high-throughput sample preparation and analysis of oligonucleotide mixtures by MALDI-MS.

[Role of thrombolytic therapy in the treatment of patients with unstable angina]. / O meste tromboliticheskoi terapii v lechenii bol'nykh nestabil'noi stenokardiei.

Thrombolytic therapy (TT) was performed in 39 of 81 patients with unstable angina pectoris (UAP) resistant to conventional treatment. TT was found to improve prognosis of UAP, to quicken UAP stabilization. Predictors of UAP unfavorable course were discovered. TT is indicated in patients suffering from resistant UAP with markers of poor prognosis.

Multiplex genotyping of PCR products with MassTag-labeled primers.

A simple mass spectrometric based assay, the PinPoint assay, has previously been described for typing single nucleotide polymorphisms. The identity of a polymorphism is determined by mass differences of single base extended genotyping primers as determined by MALDI-TOF mass spectrometry. A simple method for multiplexing the assay is described, employing multiple primers with 5'oligo(dT) sequences (MassTags) which serve to mass discriminate the peaks of multiple extended and non-extended primers. The assay is extremely rapid and requires no labeling reagents.

Single-nucleotide polymorphism identification assays using a thermostable DNA polymerase and delayed extraction MALDI-TOF mass spectrometry.

We report a simple method, the PinPoint assay, for detecting and identifying single-base variations (polymorphisms) at specific locations within DNA sequences. An oligonucleotide primer is annealed to the target DNA immediately upstream of the polymorphic site and is extended by a single base in the presence of all four dideoxynucleotide triphosphates and a thermostable DNA polymerase. The extension products are desalted, concentrated, and subjected to delayed-extraction MALDI-TOF mass spectrometry. The base at the polymorphic site is identified by the mass added onto the primer. Heterozygous targets produce two mass-resolved species that represent the addition of both bases complementary to those at the polymorphic site. The assay is suitable for double-stranded PCR products without purification or strand separation. More than one primer can be simultaneously extended and then mass-analyzed. The mass spectrometric method thus shows promise for high-volume diagnostic or genotyping applications.

DNA sequence analysis by matrix-assisted laser desorption ionization MS.

The major advantage of this procedure is that, as detailed in Figure 5, the entire sequence of an oligonucleotide is determined in less than 1 h using inexpensive reagents. DE MS, because of its significantly higher resolving power, permits analysis of oligonucleotides as long as 50 bases, about twice as long as that which is possible without DE. Phosphorothioates can be sequenced after oxidation to the phosphodiester state. We have also found that the enzymes will digest through a number of modifying and protecting groups on DNA and RNA, including 2'-amino- and 2'-alkyl-substituted hydroxy groups. The resultant spectra can confirm not only the presence, but also the position, of modifying groups on both DNA and RNA molecules.

Sequencing oligonucleotides by exonuclease digestion and delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

A protocol was developed for sequencing oligonucleotides by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Oligonucleotides were partially hydrolyzed in separate time-course digestions with 3' --> 5' and 5' --> 3' acting phosphodiesterases in a MALDI-compatible buffer or in the MALDI matrix itself. The partial digests were analyzed by MALDI-TOF mass spectrometry employing an instrument equipped with delayed ion extraction and the sequence was inferred from the mass differences between adjacent peaks. Resolution, mass accuracy, and sensitivity were considerably enhanced with delayed extraction, in comparison with the standard MALDI technique. Much longer lengths of DNA can be unambiguously sequenced with delayed extraction MALDI compared with standard MALDI-MS.