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A world-wide COVID-19 pandemic intensified strongly the studies of molecular mechanisms related to the coronaviruses. The origin of coronaviruses and the risks of human-to-human, animal-to-human, and human-to-animal transmission of coronaviral infections can be understood only on a broader evolutionary level by detailed comparative studies. In this paper, we studied ribonucleocapsid assembly-packaging signals (RNAPS) in the genomes of all seven known pathogenic human coronaviruses, SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-OC43, HCoV-HKU1, HCoV-229E, and HCoV-NL63 and compared them with RNAPS in the genomes of the related animal coronaviruses including SARS-Bat-CoV, MERS-Camel-CoV, MHV, Bat-CoV MOP1, TGEV, and one of camel alphacoronaviruses. RNAPS in the genomes of coronaviruses were evolved due to weakly specific interactions between genomic RNA and N proteins in helical nucleocapsids. Combining transitional genome mapping and Jaccard correlation coefficients allows us to perform the analysis directly in terms of underlying motifs distributed over the genome. In all coronaviruses RNAPS were distributed quasi-periodically over the genome with the period about 54 nt biased to 57 nt and to 51 nt for the genomes longer and shorter than that of SARS-CoV, respectively. The comparison with the experimentally verified packaging signals for MERS-CoV, MHV, and TGEV proved that the distribution of particular motifs is strongly correlated with the packaging signals. We also found that many motifs were highly conserved in both characters and positioning on the genomes throughout the lineages that make them promising therapeutic targets. The mechanisms of encapsidation can affect the recombination and co-infection as well., Comment: 40 pages, 12 figures

The genomic ssRNA of coronaviruses is packaged within a helical nucleocapsid. Due to transitional symmetry of a helix, weakly specific cooperative interaction between ssRNA and nucleocapsid proteins leads to the natural selection of specific quasi-periodic assembly/packaging signals in the related genomic sequence. Such signals coordinated with the nucleocapsid helical structure were detected and reconstructed in the genomes of the coronaviruses SARS-CoV and SARS-CoV-2. The main period of the signals for both viruses was about 54 nt, that implies 6.75 nt per N protein. The complete coverage of ssRNA genome of length about 30,000 nt by the nucleocapsid would need 4,400 N proteins, that makes them the most abundant among the structural proteins. The repertoires of motifs for SARS-CoV and SARS-CoV-2 were divergent but nearly coincided for different isolates of SARS-CoV-2. We obtained the distributions of assembly/packaging signals over the genomes with non-overlapping windows of width 432 nt. Finally, using the spectral entropy, we compared the load from point mutations and indels during virus age for SARS-CoV and SARS-CoV-2. We found the higher mutational load on SARS-CoV. In this sense, SARS-CoV-2 can be treated as a "newborn" virus. These observations may be helpful in practical medical applications and are of basic interest., 31 pages, 6 figures, 3 tables

In many processes, the variations in underlying characteristics can be approximated by noisy multi-periodic patterns. If large-scale patterns are superimposed by a noise with long-range correlations, the detection of multi-periodic patterns becomes especially challenging. To solve this problem, we developed a discrete double Fourier transform (DDFT). DDFT is based on the equidistance property of harmonics generated by multi-periodic patterns in the discrete Fourier transform (DFT) spectra. As the large-scale patterns generate long enough equidistant series, they can be detected by the iteration of the primary DFT. DDFT is defined as Fourier transform of intensity spectral harmonics or of their functions. It comprises widely used cepstrum transform as a particular case. We present also the relevant analytical criteria for the assessment of statistical significance of peak harmonics in DDFT spectra in the presence of noise. DDFT technique was tested by extensive numerical simulations. The practical applications of DDFT technique are illustrated by the analysis of variations in solar wind speed related to solar rotation and by the study of large-scale multi-periodic patterns in DNA sequences. The latter application can be considered as generic example for the general spectral analysis of symbolic sequences. The results are compared with those obtained by the cepstrum transform. The mutual combination of DFT and DDFT provides an efficient technique to search for noisy large-scale multi-periodic patterns., 45 pages, 15 figures

HIV-1 is one of the most variable viruses. The development of gene therapy technology using RNAi for AIDS/HIV-1 treatment is a potential alternative for traditional anti-retroviral therapy. Anti-HIV-1 siRNA should aim to exploit the most conserved viral targets. Using the deep sequencing of potential RNAi targets in 100-nt HIV-1 genome fragments from the clinical HIV-1 subtype A isolates in Russia, we found that the frequencies of all possible transversions and transitions in certain RNAi targets are 3–38 times lower than in adjacent sequences. Therefore, these targets are conserved. We propose the development of these RNAi targets for AIDS/HIV-1 treatment. Deep sequencing also enables the detection of the characteristic mutational bias of RT during the replication of viral RNA.

Gene therapy for AIDS based on RNA interference (RNAi) is currently looked upon as a promising alternative to conventional antiretroviral chemotherapy. The high variability of HIV-1 is the main challenge in developing new approaches to AIDS therapy. To date, about 18 million HIV-1 infected individuals receive antiretroviral therapy worldwide. As of 2017, about 44% of individuals with AIDS received antiretroviral therapy in Russia. Since the RNAs used for efficient RNAi and the corresponding targets in the viral transcript should be perfectly complementary to each other, it is necessary to continuously monitor the nucleotide sequences of clinical HIV-1 isolates obtained from blood and cells of naive patients and patients receiving antiretroviral therapy. Comprehensive analysis of the mutation frequencies in the viral genome is only possible with deep sequencing approaches. The present paper reports on an analysis of the mutation frequencies in six 100 bp genome regions in clinical HIV-1 isolates obtained from blood plasma of four Russian AIDS patients who have been receiving antiretroviral therapy for several years. These regions contain efficient RNAi targets. The average frequencies of all possible transversions and transitions within the RNAi targets and in their proximity have been estimated. It has been demonstrated that reverse transcriptase inhibition decreases the frequency of a number of reverse mutations. It has been found that mutations in RNAi targets are rarer (5-75 times lower than the mutation frequency for different nucleotide substitutions) than in the adjacent sequences. Our findings speak in favor of these conservative targets for developing new approaches to gene therapy of AIDS.

RNAi has been suggested for use in gene therapy of HIV/AIDS, but the main problem is that HIV-1 is highly variable and could escape attack from the small interfering RNAs (siRNAs) due to even single nucleotide substitutions in the potential targets. To exhaustively check the variability in selected RNA targets of HIV-1, we used ultra-deep sequencing of six regions of HIV-1 from the plasma of two independent cohorts of patients from Russia. Six RNAi targets were found that are invariable in 82%–97% of viruses in both cohorts and are located inside the domains specifying reverse transcriptase (RT), integrase, vpu, gp120, and p17. The analysis of mutation frequencies and their characteristics inside the targets suggests a likely role for APOBEC3G (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G, A3G) in G-to-A mutations and a predominant effect of RT biases in the detected variability of the virus. The lowest frequency of mutations was detected in the central part of all six targets. We also discovered that the identical RNAi targets are present in many HIV-1 strains from many countries and from all continents. The data are important for both the understanding of the patterns of HIV-1 mutability and properties of RT and for the development of gene therapy approaches using RNAi for the treatment of HIV/AIDS. Keywords: HIV-1, RNAi targets, gene therapy, ultra-deep sequencing, conserved HIV-1 sequences

Human rDNA clusters form numerous contacts with different chromosomal regions as evidenced by chromosome conformation capture data. Heterochromatization of rDNA genes leads to heterochromatization in different chromosomal regions coupled with the activation of the transcription of genes related to differentiation. These data suggest a role for rDNA clusters in the regulation of many human genes. However, the genes that reside within the rDNA-contacting regions have not been identified. The purpose of this study was to detect and characterize the regions where rDNA clusters make frequent contacts and to identify and categorize genes located in these regions. We analyzed the regions that contact rDNA using 4C data and show that these regions are enriched with genes specifying transcription factors and non-coding RNAs involved in differentiation and development. The rDNA-contacting genes are involved in neuronal development and are associated with different cancers. Heat shock treatment led to dramatic changes in the pattern of rDNA-contacting sites, especially in the regions possessing long stretches of H3K27ac marks. Whole-genome analysis of rDNA-contacting sites revealed specific epigenetic marks and the transcription sites of 20&ndash, 100 nt non-coding RNAs in these regions. The rDNA-contacting genes jointly regulate many genes that are involved in the control of transcription by RNA polymerase II and the development of neurons. Our data suggest a role for rDNA clusters in the differentiation of human cells and carcinogenesis.

Highly active antiretroviral therapy has greatly reduced the morbidity and mortality of AIDS. However, many of the antiretroviral drugs are toxic with long-term use, and all currently used anti-HIV agents generate drug-resistant mutants. Therefore, there is a great need for new approaches to AIDS therapy. RNAi is a powerful means of inhibiting HIV-1 production in human cells. We propose to use RNAi for gene therapy of HIV/AIDS. Previously we identified a number of new biologically active siRNAs targeting several moderately conserved regions in HIV-1 transcripts. Here we analyze the heterogeneity of nucleotide sequences in three RNAi targets in sequences encoding the reverse transcriptase and integrase domains of current isolates of HIV-1 subtype A in Russia. These data were used to generate genetic constructs expressing short hairpin RNAs 28-30-bp in length that could be processed in cells into siRNAs. After transfection of the constructs we observed siRNAs that efficiently attacked the selected targets. We expect that targeting several viral genes important for HIV-1 reproduction will help overcome the problem of viral adaptation and will prevent the appearance of RNAi escape mutants in current virus strains, an important feature of gene therapy of HIV/AIDS.

Colorectal cancer (CRC) is the third most common malignancy in industrialized countries. Despite the advances in diagnostics and development of new drugs, the 5-year survival remains only 60-65%. Our approach to early diagnostics of CRC is based on the determination of serological signatures with an array of hemispherical hydrogel cells containing immobilized proteins and oligosaccharides (glycochip). The compounds immobilized on the glycochip include tumor-associated glycans (SiaTn, Tn, TF, Le(C) , Le(Y) , SiaLe(A) , and Manβ1-4GlcNAcβ) and antibodies against human immunoglobulins IgG, IgA, and IgM. The glycochip detects antibodies against tumor-associated glycans in patients' sera. The simultaneous measurement of the levels of immunoglobulins enhances the diagnostic impact of the signatures. In this work, we found previously unreported increase in antibodies against oligosaccharide Manβ1-4GlcNAcβ in patients with CRC. In parallel with these experiments, we determined the levels of oncomarkers carcinoembryonic antigen (CEA), cancer antigen (CA) 19-9, CA 125, CA 15-3, human chorionic gonadotropin (HCG), and alpha-fetoprotein (AFP) using another gel-based biochip with immobilized antibodies (oncochip) developed earlier in our laboratory. In total, 69 samples from healthy donors, 33 from patients with colorectal carcinoma, and 27 from patients with inflammatory bowel diseases were studied. The use of combined signatures of antiglycan antibodies and oncomarkers provides much better predictive value than the conventional measurement of oncomarkers CEA and CA 19-9. Positive predictive value of CRC diagnoses using together glycochip and oncochip reached 95% with the sensitivity and specificity 88% and 98%, respectively. Thus, the combination of antibody profiling with detection of conventional oncomarkers proved to be a promising tool in diagnostics of CRC.

The development of gene-therapy technology using RNAi for AIDS/HIV-1 treatment is a prospective alternative to traditional anti-retroviral therapy. RNAi targets could be selected in HIV-1 transcripts and in CCR5 mRNA. Previously, we experimentally selected a number of efficient siRNAs that target HIV-1 RNAs. The viral genome mutates frequently, and RNAi strength is very sensitive, even for a single mismatches. That is why it is important to study nucleotide sequences of targets in clinical isolates of HIV-1. In the present study, we analyzed mutations in 6 of about 300-bp regions containing RNAi targets from HIV-1 subtype A isolates in Russia. Estimates of the mean frequencies of mutations in the targets were obtained and the frequencies of mutations in the different codon positions were compared. The frequencies of mutations in the vicinity of the targets and directly within the targets were also compared and have been shown to be approximately the same. The frequencies of indels in the chosen regions have been assessed. Their frequencies have proved to be two to three orders of magnitude less compared to that for mutations.

The discrete double Fourier transform (DDFT) was developed to search for large-scale multi-periodic patterns in the presence of noise and is based on detection of the equidistant series of harmonics generated by the periodic patterns in the discrete Fourier transform (DFT) spectra. As DDFT retains all generic features of the Fourier transform, the corresponding pattern correlation function (PCF) related to DDFT can be introduced similarly to the data correlation function (DCF) related to DFT on the basis of the Wiener–Khinchin relationship. Peaks in PCF indicate the number of periodic patterns in a dataset under analysis and have direct correspondence with the counterpart peaks in the DFT spectrum. The close correspondence between positions of the peaks in the PCF and DFT spectra strongly enhances statistical significance of detected periodicities. Similar PCFs can also be defined for the cepstrum transform. The combined DFT–DCF and DDFT–PCF technique was applied to the detection of cycles in geomagnetic activity using disturbance storm-time (Dst) index. In addition to the known 27-day, semiannual and 11-year cycles of geomagnetic activity, we have also found the annual cycle of activity. The results were compared with those obtained by the cepstrum transform. A multiple cross-check makes the combined technique much more efficient and robust in comparison with the detection based on a unique particular method.

The assembly and maturation of viruses with icosahedral capsids must be coordinated with icosahedral symmetry. The icosahedral symmetry imposes also the restrictions on the cooperative specific interactions between genomic RNA/DNA and coat proteins that should be reflected in quasi-regular segmentation of viral genomic sequences. Combining discrete direct and double Fourier transforms, we studied the quasi-regular large-scale segmentation in genomic sequences of different ssRNA, ssDNA, and dsDNA viruses. The particular representatives included satellite tobacco mosaic virus and the strains of satellite tobacco necrosis virus, STNV-C, STNV-1, STNV-2, Escherichia phages MS2, phiX174, alpha3, and HK97, and Simian virus 40. In all their genomes, we found the significant quasi-regular segmentation of genomic sequences related to the virion assembly and the genome packaging within icosahedral capsid. We also found good correspondence between our results and available cryo-electron microscopy data on capsid structures and genome packaging in these viruses. Fourier analysis of genomic sequences provides the additional insight into mechanisms of hierarchical genome packaging and may be used for verification of the concepts of 3-fold or 5-fold intermediates in virion assembly. The results of sequence analysis should be taken into account at the choice of models and data interpretation. They also may be helpful for the development of antiviral drugs., Comment: 55 pages, 14 figures

The efficient development of antiviral drugs, including efficient antiviral small interfering RNAs (siRNAs), requires continuous monitoring of the strict correspondence between a drug and the related highly variable viral DNA/RNA target(s). Deep sequencing is able to provide an assessment of both the general target conservation and the frequency of particular mutations in the different target sites. The aim of this study was to develop a reliable bioinformatic pipeline for the analysis of millions of short, deep sequencing reads corresponding to selected highly variable viral sequences that are drug target(s). The suggested bioinformatic pipeline combines the available programs and the ad hoc scripts based on an original algorithm of the search for the conserved targets in the deep sequencing data. We also present the statistical criteria for the threshold of reliable mutation detection and for the assessment of variations between corresponding data sets. These criteria are robust against the possible sequencing errors in the reads. As an example, the bioinformatic pipeline is applied to the study of the conservation of RNA interference (RNAi) targets in human immunodeficiency virus 1 (HIV-1) subtype A. The developed pipeline is freely available to download at the website http://virmut.eimb.ru/. Brief comments and comparisons between VirMut and other pipelines are also presented.

Gene therapy for AIDS based on RNA interference (RNAi) is currently looked upon as a promising alternative to conventional antiretroviral chemotherapy. The high variability of HIV-1 is the main challenge in developing new approaches to AIDS therapy. To date, about 18 million HIV-1 infected individuals receive antiretroviral therapy worldwide. As of 2017, about 44% of individuals with AIDS received antiretroviral therapy in Russia. Since the RNAs used for efficient RNAi and the corresponding targets in the viral transcript should be perfectly complementary to each other, it is necessary to continuously monitor the nucleotide sequences of clinical HIV-1 isolates obtained from blood and cells of naïve patients and patients receiving antiretroviral therapy. Comprehensive analysis of the mutation frequencies in the viral genome is only possible with deep sequencing approaches. The present paper reports on an analysis of the mutation frequencies in six 100 bp genome regions in clinical HIV-1 isolates obtained from blood plasma of four Russian AIDS patients who have been receiving antiretroviral therapy for several years. These regions contain efficient RNAi targets. The average frequencies of all possible transversions and transitions within the RNAi targets and in their proximity have been estimated. It has been demonstrated that reverse transcriptase inhibition decreases the frequency of a number of reverse mutations. It has been found that mutations in RNAi targets are rarer (5-75 times lower than the mutation frequency for different nucleotide substitutions) than in the adjacent sequences. Our findings speak in favor of these conservative targets for developing new approaches to gene therapy of AIDS.

HIV-1 is one of the most variable viruses. The development of gene therapy technology using RNAi for AIDS/HIV-1 treatment is a potential alternative for traditional anti-retroviral therapy. Anti-HIV-1 siRNA should aim to exploit the most conserved viral targets. Using the deep sequencing of potential RNAi targets in 100-nt HIV-1 genome fragments from the clinical HIV-1 subtype A isolates in Russia, we found that the frequencies of all possible transversions and transitions in certain RNAi targets are 3-38 times lower than in adjacent sequences. Therefore, these targets are conserved. We propose the development of these RNAi targets for AIDS/HIV-1 treatment. Deep sequencing also enables the detection of the characteristic mutational bias of RT during the replication of viral RNA.

DNA double-strand breaks (DSBs) are associated with different physiological and pathological processes in different organisms. To understand the role of DSBs in multiple cellular mechanisms, a robust method for genome-wide mapping of chromosomal breaks at one-nucleotide resolution is required. Many years ago, we detected large DNA fragments migrating from DNA-agarose plugs in pulsed-field gels, which we named ‘forum domains’ [1,2]. Recently, we developed a method for genome-wide mapping of DSBs that produces these 50–150 kb DNA domains using microarrays or 454 sequencing (Tchurikov et al., 2011; 2013). Now we have used Illumina sequencing to map DSBs in repetitive rDNA units in human HEK293T cells. Here we describe in detail the experimental design and bioinformatics analysis of the data deposited in the Gene Expression Omnibus with accession number GSE49302 and associated with the study published in the Journal of Molecular Cell Biology (Tchurikov et al., 2014).

A prototype of test-system for simultaneous quantitative assay of nine tumor markers in blood serum was developed. The main constituent of the test-system is OM-9 biochip containing immobilized antibodies against nine oncomarkers: α-fetoprotein (AFP), carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG), cancer antigen 15-3 (CA 15-3), cancer antigen 125 (CA 125), cancer antigen 19-9 (CA 19-9), prostate-specific antigen, total (PSAtot) and free (PSAfree) forms, neuron-specific enolase (NSE). The biochip-based assay procedure for carrying out simultaneous quantitative determination of nine tumor markers in patient's blood serum: two-steps sandwich-immunoassay, was proposed. The main analytical characteristics of the method were obtained. The results permit to consider the prototype of the test-system as a promising instrument for clinical application. The test-system prototype was tested using blood serum samples of oncological patients (252 samples) and healthy donors (185 samples). Increased concentrations of one or more tumor markers above the normal level were found in 76.6% cases of oncological patients and only in 6% cases of healthy donors. For colorectal cancer patients group, application of modern statistical methods of data-processing in medical researchers, i.e. ROC-analysis and logistic regression, indicted that the simultaneous assay of nine tumor markers on biochips showed much more diagnostic significance (area under the ROC-curve (AUC) reached 0.84) than traditional assay of 2 tumor markers, CEA and CA 19-9 (AUC = 0.59). The developed biochip-based test-system can be recommended both for the estimation of people's health, e.g., for standard medical examination, and for tracking of tumoral process in postsurgical period or after specific tumor treatment.

We analyzed the periodic patterns in E. coli promoters and compared the distributions of the corresponding patterns in promoters and in the complete genome to elucidate their function. Except the three-base periodicity, coincident with that in the coding regions and growing stronger in the region downstream from the transcriptions start (TS), all other salient periodicities are peaked upstream of TS. We found that helical periodicities with the lengths about B-helix pitch ~10.2-10.5 bp and A-helix pitch ~10.8-11.1 bp coexist in the genomic sequences. We mapped the distributions of stretches with A-, B-, and Z- like DNA periodicities onto E.coli genome. All three periodicities tend to concentrate within non-coding regions when their intensity becomes stronger and prevail in the promoter sequences. The comparison with available experimental data indicates that promoters with the most pronounced periodicities may be related to the supercoiling-sensitive genes., 23 pages, 6 figures, 2 tables

In this study, we analyzed the microbiomes of uncultivated (virgin) and cultivated (cropland) soils from the southern part of Russia using DNA shotgun deep sequencing and metagenomic studies. The processed reads were mapped to the nucleotide sequences of bacteria, fungi, archaea, and viruses extracted from the non redundant database of NCBI. Our data indicate that Archaea and Fungi are more abundant in the virgin soil. The analysis of the top ten most abundant taxa from phylum to species levels revealed only slightly statistically significant differences between the microbial communities of the soils. In contrast, among the less abundant organisms at all taxa levels, we detected considerable differences between the virgin soil and the cropland microbiomes. The higher biodiversity of the virgin soil was confirmed by functional annotation and abundance analysis using the eggNOG and KEGG databases. Collectively, our data strongly indicate that the biodiversity of virgin soil is much higher, suggesting that intensive farming reduces the microbial biodiversity and causes specific changes in the pattern of less abundant microbiome taxa.

Among various factors affecting operation of oligonucleotide microchips, the variations in concentration and in homogeneous distribution of immobilized probes over the cells are one of the most important. The labeling of immobilized probes ensures the complete current monitoring on the probe distribution and is reliable and convenient. Using hydrogel-based oligonucleotide microchips, the applicability of Cy3-labeled immobilized probes for quality control and signal normalization after hybridization with Cy5-labeled target DNA was investigated. This study showed that proper signal normalization should be different in thermodynamic conditions and in transient regime with hybridization far from saturation. This kinetic effect holds for both hydrogel-based and surface oligonucleotide microchips. Besides proving basic features, the technique was assessed on a sampling batch of 50 microchips developed for identifying mutations responsible for rifampicin and isoniazid resistance of Mycobacterium tubercu...

ISSN 1607-6729, Doklady Biochemistry and Biophysics, 2009, Vol. 427, pp. 171–174. © Pleiades Publishing, Ltd., 2009.Original Russian Text © Zh.I. Zubtsova, M.A. Filippova, E.N. Savvateeva, D.A. Zubtsov, V.R. Chechetkin, E.V. Grishin, A.S. Zasedatelev, A.Yu. Rubina, 2009, published inDoklady Akademii Nauk, 2009, Vol. 427, No. 1, pp. 118–121.

Protein microchips are designed for high-throughput evaluation of the concentrations and activities of various proteins. The rapid advance in microchip technology and a wide variety of existing techniques pose the problem of unified approach to the assessment and comparison of different platforms. Here we compare the characteristics of protein microchips developed for quantitative immunoassay with those of antibodies immobilized on glass surfaces and in hemispherical gel pads. Spotting concentrations of antibodies used for manufacturing of microchips of both types and concentrations of antigen in analyte solution were identical. We compared the efficiency of antibody immobilization, the intensity of fluorescence signals for both direct and sandwich-type immunoassays, and the reaction-diffusion kinetics of the formation of antibody-antigen complexes for surface and gel-based microchips. Our results demonstrate higher capacity and sensitivity for the hydrogel-based protein microchips, while fluorescence saturation kinetics for the two types of microarrays was comparable.

The development of gene-therapy technology using RNAi for AIDS/HIV-1 treatment is a prospective alternative to traditional anti-retroviral therapy. RNAi targets could be selected in HIV-1 transcripts and in CCR5 mRNA. Previously, we experimentally selected a number of efficient siRNAs that target HIV-1 RNAs. The viral genome mutates frequently, and RNAi strength is very sensitive, even for a single mismatches. That is why it is important to study nucleotide sequences of targets in clinical isolates of HIV-1. In the present study, we analyzed mutations in 6 of about 300-bp regions containing RNAi targets from HIV-1 subtype A isolates in Russia. Estimates of the mean frequencies of mutations in the targets were obtained and the frequencies of mutations in the different codon positions were compared. The frequencies of mutations in the vicinity of the targets and directly within the targets were also compared and have been shown to be approximately the same. The frequencies of indels in the chosen regions have been assessed. Their frequencies have proved to be two to three orders of magnitude less compared to that for mutations.

A prototype of test-system for simultaneous quantitative assay of nine tumor markers in blood serum was developed. The main constituent of the test-system is OM-9 biochip containing immobilized antibodies against nine oncomarkers: α-fetoprotein (AFP), carcinoembryonic antigen (CEA), human chorionic gonadotropin (HCG), cancer antigen 15-3 (CA 15-3), cancer antigen 125 (CA 125), cancer antigen 19-9 (CA 19-9), prostate-specific antigen, total (PSAtot) and free (PSAfree) forms, neuron-specific enolase (NSE). The biochip-based assay procedure for carrying out simultaneous quantitative determination of nine tumor markers in patient's blood serum: two-steps sandwich-immunoassay, was proposed. The main analytical characteristics of the method were obtained. The results permit to consider the prototype of the test-system as a promising instrument for clinical application. The test-system prototype was tested using blood serum samples of oncological patients (252 samples) and healthy donors (185 samples). Increased concentrations of one or more tumor markers above the normal level were found in 76.6% cases of oncological patients and only in 6% cases of healthy donors. For colorectal cancer patients group, application of modern statistical methods of data-processing in medical researchers, i.e. ROC-analysis and logistic regression, indicted that the simultaneous assay of nine tumor markers on biochips showed much more diagnostic significance (area under the ROC-curve (AUC) reached 0.84) than traditional assay of 2 tumor markers, CEA and CA 19-9 (AUC = 0.59). The developed biochip-based test-system can be recommended both for the estimation of people's health, e.g., for standard medical examination, and for tracking of tumoral process in postsurgical period or after specific tumor treatment.

Protein hydrogel-based microchips are being developed for high-throughput evaluation of the concentrations and activities of various proteins. To shorten the time of analysis, the reaction-diffusion kinetics on gel microchips should be accelerated. Here we present the results of the experimental and theoretical analysis of the reaction-diffusion kinetics enforced by mixing with peristaltic pump. The experiments were carried out on gel-based protein microchips with immobilized antibodies under the conditions utilized for on-chip immunoassay. The dependence of fluorescence signals at saturation and corresponding saturation times on the concentrations of immobilized antibodies and antigen in solution proved to be in good agreement with theoretical predictions. It is shown that the enhancement of transport with peristaltic pump results in more than five-fold acceleration of binding kinetics. Our results suggest useful criteria for the optimal conditions for assays on gel microchips to balance high sensitivity and rapid fluorescence saturation kinetics.

We perform spectral entropy and GC content analyses in the beta-esterase gene cluster, including the Est-6 gene and the psiEst-6 putative pseudogene, in seven species of the Drosophila melanogaster species subgroup. psiEst-6 combines features of functional and nonfunctional genes. The spectral entropies show distinctly lower structural ordering for psiEst-6 than for Est-6 in all species studied. Our observations agree with previous results for D. melanogaster and provide additional support to our hypothesis that after the duplication event Est-6 retained the esterase-coding function and its role during copulation, while psiEst-6 lost that function but now operates in conjunction with Est-6 as an intergene. Entropy accumulation is not a completely random process for either gene. Structural entropy is nucleotide dependent. The relative normalized deviations for structural entropy are higher for G than for C nucleotides. The entropy values are similar for Est-6 and psiEst-6 in the case of A and T but are lower for Est-6 in the case of G and C. The GC content in synonymous positions is uniformly higher in Est-6 than in psiEst-6, which agrees with the reduced GC content generally observed in pseudogenes and nonfunctional sequences. The observed differences in entropy and GC content reflect an evolutionary shift associated with the process of pseudogenization and subsequent functional divergence of psiEst-6 and Est-6 after the duplication event.

The efficiency of discrimination between perfect and mismatched duplexes during hybridization on microchips depends on the concentrations of target DNA in solution and immobilized probes, buffer composition, and temperature of hybridization and is determined by both thermodynamic relationships and hybridization kinetics. In this work, optimal conditions of discrimination were studied using hybridization of fluorescently labeled target DNA with custom-made gel-based oligonucleotide microchips. The higher the concentration of immobilized probes and the higher the association constant, the higher the concentration of the formed duplexes and the stronger the corresponding fluorescence signal, but, simultaneously, the longer the time needed to reach equilibrium. Since mismatched duplexes hybridize faster than their perfect counterparts, perfect-to-mismatch signal ratio is lower in transient regime, and short hybridization times may hamper the detection of mutations. The saturation time can be shortened by decreasing the probe concentration or augmenting the gel porosity. This improves the detection of mutations in transient regime. It is shown that the decrease in the initial concentration of oligonucleotide probes by an order of magnitude causes only 1.5-2.5-fold decrease of fluorescence signals after hybridization of perfect duplexes for 3-12 h. At the same time, these conditions improve the discrimination between perfect and mismatched duplexes more than two-fold. A similar improvement may be obtained using an optimized dissociation procedure.

Binding specificity of mutant V55C disulfide bonded dimeric lambda-Cro protein (CroVC) to double-stranded DNA (dsDNA) was studied using generic hexamer oligonucleotide microchip. The curves of dissociation of hybridized DNA in the presence and absence of CroVC were converted into the effective discriminant constants to assess the relevant thermodynamic equilibrium binding constants for dsDNA-protein complexes. Then, tiling of longer oligonucleotides with shorter oligomers was used to search for sequence motifs with the highest binding specificity similarly to sequencing by hybridization. The comparison of the deduced sequences with the known natural operator half-sites demonstrated the principal ability to discern and reconstruct the major parts of 7-mer motifs corresponding to the strongest binding of CroVC subunits. Our results show the applicability of generic microchips to the analysis of binding specificity in the case of multi-subunit DNA-binding proteins.

The kinetics of hybridization on the oligonucleotide microchip with gel pads is studied both theoretically and experimentally. The monitoring of kinetics was performed with the measurements of fluorescence intensity produced by the labeled target oligonucleotides. As is shown, the hybridization time depends on the stability of the formed duplexes, the concentrations of target and probe oligonucleotides, and the diffusion of target oligonucleotides in solution and gel pad. The initial stage of hybridization is determined by the flow of target oligonucleotides from solution, then, followed by the diffusive propagation with approximately constant concentration of oligonucleotides at the boundary of gel pad and, finally, by the exponential saturation. The theoretical predictions of hybridization kinetics reveal a good correspondence with the experimental results and may be used for the choice of the optimal hybridization conditions. The possible applications of kinetic hybridization curves to the discrimination problems and assessment of diffusion coefficients in gel pads are briefly discussed. Finally, we discuss the relationships between the binding kinetics and the general functioning of biomolecular microchips.

We have analyzed nucleotide polymorphism within a 5.3-kb region encompassing the functional Est-6 gene and the ψEst-6 putative pseudogene in 28 strains of Drosophila melanogaster and one of D. simulans. Two divergent sequence types were detected, which are not perfectly associated with Est-6 allozyme variation. The level of variation (π) is very close in the 5′-flanking region (0.0059) and Est-6 gene (0.0057), but significantly higher in the intergenic region (0.0141) and putative pseudogene (0.0122). The variation in the 3′-flanking region is intermediate (0.0083). These observations may reflect different levels of purifying selection in the different regions. Strong linkage disequilibrium occurs within the region studied, with the largest values revealed in the putative pseudogene and 3′-flanking region. Moreover, recombination is restricted within ψEst-6. Gene conversion is detected both within and (to a lesser extent) between Est-6 and ψEst-6. The data indicate that ψEst-6 exhibits some characteristics that are typical of nonfunctional genes, while other characteristics are typically attributed to functional genes; the same situation has been observed in other pseudogenes (including Drosophila). The results of structural entropy analysis demonstrate higher structural ordering in Est-6 than in ψEst-6, in accordance with expectations if ψEst-6 is indeed a pseudogene. Taking into account that the function of ψEst-6 is not known (but could exist) and following the terminology of J. Brosius and S. J. Gould, we suggest that the term “potogene” may be appropriate for ψEst-6, indicating that it is a potential gene that may have acquired some distinctive but unknown function.

DNA sequencing by hybridization was carried out with a microarray of all 4(6) = 4,096 hexadeoxyribonucleotides (the generic microchip). The oligonucleotides immobilized in 100 x 100 x 20-microm polyacrylamide gel pads of the generic microchip were hybridized with fluorescently labeled ssDNA, providing perfect and mismatched duplexes. Melting curves were measured in parallel for all microchip duplexes with a fluorescence microscope equipped with CCD camera. This allowed us to discriminate the perfect duplexes formed by the oligonucleotides, which are complementary to the target DNA. The DNA sequence was reconstructed by overlapping the complementary oligonucleotide probes. We developed a data processing scheme to heighten the discrimination of perfect duplexes from mismatched ones. The procedure was united with a reconstruction of the DNA sequence. The scheme includes the proper definition of a discriminant signal, preprocessing, and the variational principle for the sequence indicator function. The effectiveness of the procedure was confirmed by sequencing, proofreading, and nucleotide polymorphism (mutation) analysis of 13 DNA fragments from 31 to 70 nucleotides long.

The hidden periodicities in various genomes can be identified via a Fourier transform of genomic DNA sequences. The typical search strategies are described and the corresponding statistical criteria are presented. The methods are applied to the structural analysis of genome for bacteriophage PHIX174. The results indicate the important role of the coherent structural modifications along the several main periods in the formation of the genome. Some possible mechanisms for the features observed are discussed.

The investigation of size-dependence for three-periodic Fourier harmonics may resolve between the model with the long-range correlations for different nucleotides and the representation of the genome in terms of a mosaic picture with uncorrelated structural units. The study indicates superstructural organization in genomic DNA sequences. The possible physical mechanisms for formation of such a structure are discussed.

The spectral representation gives an effective approach to the analysis of statistical characteristics of symbolic sequences. We derive the corresponding criteria for the random case. The criteria ensure the dichotomic classification (random-non-random) for relatively short sequences of about several thousand symbols. The theory is applied to inflation models, symbolic dynamics and DNA sequences.

An approach to circuit renaturation-hybridization of dsDNA on oligonucleotide microchips is described. A close circuit cycling device has been developed, and the feasibility of the proposed technique was demonstrated on two platforms. First, a commercial microchip for detection of rifampicin resistance in Mycobacterium tuberculosis was used. Hybridization of a 126 nt long single-stranded DNA (ssDNA) fragment of the rpoB gene according to manufacturer's protocol has been compared to hybridization of the same double-stranded DNA (dsDNA) fragment using the developed approach. Hybridization signals obtained by both methods were comparable in intensity and correlated closely. Second, a 22 nt long hairpin-forming oligonucleotide was designed and hybridized with a custom microchip containing probes complementary to both strands of the oligonucleotide. Conventional hybridization of this oligonucleotide did not yield any significant signals. Cleavage of the hairpin loop resulted in the formation of a 9 bp long intermolecular duplex. Hybridization of the duplex using the suggested technique yielded strong signals. The proposed approach allows analyzing target DNA in double-stranded form bypassing the preparation of single-stranded targets. Moreover, both complementary chains could be analyzed simultaneously, providing a reliable internal control. Being combined with fragmentation this method opens new possibilities in analyzing ssDNA with complex secondary structure.

Manufacturing of hydrogel-based microchips on metal-coated substrates significantly enhances fluorescent signals upon binding of labeled target molecules. This observation holds true for both oligonucleotide and protein microchips. When Cy5 is used as fluorophore, this enhancement is 8–10-fold in hemispherical gel elements and 4–5-fold in flattened gel pads, as compared with similar microchips manufactured on uncoated glass slides. The effect also depends on the hydrophobicity of metal-coated substrate and on the presence of a layer of liquid over the gel pads. The extent of enhancement is insensitive to the nature of formed complexes and immobilized probes and remains linear within a wide range of fluorescence intensities. Manufacturing of gel-based protein microarrays on metal-coated substrates improves their sensitivity using the same incubation time for immunoassay. Sandwich immunoassay using these microchips allows shortening the incubation time without loss of sensitivity. Unlike microchips with probes immobilized directly on a surface, for which the plasmon mechanism is considered responsible for metal-enhanced fluorescence, the enhancement effect observed using hydrogel-based microchips on metal-coated substrates might be explained within the framework of geometric optics.

The standard genetic code is known to be robust to translation errors and point mutations. We studied how small modifications of the standard code affect its robustness. The robustness was assessed in terms of a proper stability function, the negative variations of which correspond to a more robust code. The fraction of more robust codes obtained under small modifications appeared to be unexpectedly high, about 0.1-0.4 depending on the choice of stability function and code modifications, yet significantly lower than the corresponding fraction in the random codes (about a half). In this sense the standard code ought to be considered distinctly non-random in accordance with previous observations. The distribution of the negative variations of stability function revealed very abrupt drop beyond one standard deviation, much sharper than for Gaussian distribution or for the random codes with the same number of codons in the sets coding for amino acids or stop-codons. This behavior holds for both the standard code as a whole and its binary NRN-NYN, NWN-NSN, and NMN-NKN blocks. Previously, it has been proved that such binary block structure is necessary for the robustness of a code and is inherent to the standard genetic code. The modifications of the standard code corresponding to more robust coding may be related to the different variants of the code. These effects may also contribute to the rates of replacements of amino acids. The observed features demonstrate the joint impact of random factors and natural selection during evolution of the genetic code.

It has been shown that recombination-dissociation processes influence the relaxation correction to the concentration dependence of the conductivity of electrolytes. A theoretical treatment has been carried out in the framework of the continual stochastic formalism of birth-and-death processes with a long-range interaction. Evaluations of the observed effects have been given and the possibility of their experimental measurement has been discussed. The question of the concentration dependence of the Hall effect has also been considered.

Gel-based oligonucleotide microarray approach was developed for quantitative profiling of binding affinity of a protein to single-stranded DNA (ssDNA). To demonstrate additional capabilities of this method, we analyzed the binding specificity of ribonuclease (RNase) binase from Bacillus intermedius (EC 3.1.27.3) to ssDNA using generic hexamer oligodeoxyribonucleotide microchip. Single-stranded octamer oligonucleotides were immobilized within 3D hemispherical gel pads. The octanucleotides in individual pads 5'-{N}N(1)N(2)N(3)N(4)N(5)N(6){N}-3' consisted of a fixed hexamer motif N(1)N(2)N(3)N(4)N(5)N(6) in the middle and variable parts {N} at the ends, where {N} represent A, C, G and T in equal proportions. The chip has 4096 pads with a complete set of hexamer sequences. The affinity was determined by measuring dissociation of the RNase-ssDNA complexes with the temperature increasing from 0 degrees C to 50 degrees C in quasi-equilibrium conditions. RNase binase showed the highest sequence-specificity of binding to motifs 5'-NNG(A/T/C)GNN-3' with the order of preference: GAG > GTG > GCG. High specificity towards G(A/T/C)G triplets was also confirmed by measuring fluorescent anisotropy of complexes of binase with selected oligodeoxyribonucleotides in solution. The affinity of RNase binase to other 3-nt sequences was also ranked. These results demonstrate the applicability of the method and provide the ground for further investigations of nonenzymatic functions of RNases.

We discuss intermittency effects in the distribution of scalar passive impurities within fully developed hydrodynamic turbulence. It is shown that the observable stronger intermittency effects in the distribution of passive impurities with respect to that for the energy dissipation rate can naturally be explained in the framework of composite random cascade models. We discuss doubly random bounded and unbounded log-normal models, the doubly randomβ-model, and the two-scale Cantor set approximation. Then the problem of mutual correlations is discussed. The various results are compared with experiments.

We discuss intermittency effects in fully developed hydrodynamic turbulence. It is shown that the application of the bounded log-normal distribution to the fluctuations of the local energy dissipation rate resolves some basic difficulties related to Kolmogorov's third hypothesis and gives a good agreement with experiment. The nonlinear interaction of the large-scale and inertial-range turbulent pulsations of the velocities may explain the observable characteristics of the intermittency. We give also a detailed comparison of the results obtained with the use of the bounded log-normal distribution with that obtained in the framework of the homogeneous and random /~-models, a two-scale Cantor set approximation, and the original unbounded log-normal distribution suggested by Kolmogorov and Obukhov.

The theory based on the minimum propagating zone (MPZ) criterion overestimates the critical heat of quench in the partially stabilized superconducting magnets. This difference is especially important for the high values of Stekly parameter. We discuss the variational principle for calculation of the critical heat of quench and compare our results with that of based on the MPZ theory and obtained by the direct dynamic numerical simulations. It is shown that variational calculations can effectively be used for the determination of the critical heat. At the end, we discuss the Lyapunov approach to the stability problem.

Using hydrogel-based oligonucleotide microchips developed previously for the choice of drugs during leukemia treatment and the other diseases, it is shown that the acceleration of external transport by mixing buffer solution with peristaltic pump not only enhances the observable fluorescence signals, but also improves significantly the discrimination between perfect and mismatch duplexes at the intermediate stage of hybridization on the oligonucleotide microchips. The discrimination efficiency for a given hybridization time grows monotonously with the frequency of flow pulsations. The mixing with frequency 10 Hz accelerates the hybridization rate approximately thrice and improves the discrimination efficiency 1.5-2.5 times higher for overnight hybridization. To study these effects, we have developed the special peristaltic pump mixing solution in a hybridization chamber of 35 mul in volume (area approximately 1 x 1 cm(2) and height 0.3 mm). We present also the brief theoretical summary for the interpretation and assessment of the observed experimental features.

The optimal design of oligonucleotide microchips and efficient discrimination between perfect and mismatch duplexes strongly depend on the external transport of target DNA to the cells with immobilized probes as well as on respective association and dissociation rates at the duplex formation. In this paper we present the relevant theory for hybridization of DNA fragments with oligonucleotide probes immobilized in the cells on flat substrate. With minor modifications, our theory also is applicable to reaction-diffusion hybridization kinetics for the probes immobilized on the surface of microbeads immersed in hybridization solution. The main theoretical predictions are verified with control experiments. Besides that, we compared the characteristics of the surface and gel-based oligonucleotide microchips. The comparison was performed for the chips printed with the same pin robot, for the signals measured with the same devices and processed by the same technique, and for the same hybridization conditions. The sets of probe oligonucleotides and the concentrations of probes in respective solutions used for immobilization on each platform were identical as well. We found that, despite the slower hybridization kinetics, the fluorescence signals and mutation discrimination efficiency appeared to be higher for the gel-based microchips with respect to their surface counterparts even for the relatively short hybridization time about 0.5-1 hour. Both the divergence between signals for perfects and the difference in mutation discrimination efficiency for the counterpart platforms rapidly grow with incubation time. In particular, for hybridization during 3 h the signals for gel-based microchips surpassed their surface counterparts in 5-20 times, while the ratios of signals for perfect-mismatch pairs for gel microchips exceeded the corresponding ratios for surface microchips in 2-4 times. These effects may be attributed to the better immobilization efficiency and to the higher thermodynamic association constants for duplex formation within gel pads.

Hidden periodicities play a very important role in the regulatory and structural functioning of genomic DNA strands. Primarily, it concerns the fundamental three-periodicity inherent to protein coding regions in all taxonomic groups, two-periodicity in introns of eukaryots as well as periodicities related to helix and chromatin pitches, while the other periodicities appear to be species specific. Rather roughly (and without sharp boundary) the underlying periodicities may be divided by two groups. In the first case the periodicities are due to particular nucleotides (or very short oligomers) quasi-regularly positioned in a seemingly random background. This type of regularity can be identified via either standard frequency analysis or more elaborate Fourier methods. For the second group a periodicity is related to the quasi-random replacements in initially complete repeating motifs (situation typical, e.g., for modifications of satellites). In the last case the statistical reconstruction of underlying repeats is a much less trivial task. The authors show that this problem can successfully be solved with multi-symbol extension of energy-minimizing neural networks (EMNN). The reconstruction of underlying motifs may shed additional light on the evolutionary and functional modifications in various genomes.