V. R. Chechetkin

Size-dependence of three-periodicity and long-range correlations in DNA sequences

Abstract 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.

Discrimination Between Perfect and Mismatched Duplexes with Oligonucleotide Gel Microchips: Role of Thermodynamic and Kinetic Effects During Hybridization

Abstract 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.

Kinetics of Hybridization on Surface Oligonucleotide Microchips: Theory, Experiment, and Comparison with Hybridization on Gel-Based Microchips

Abstract 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.

Sequencing by hybridization with the generic 6-mer oligonucleotide microarray : an advanced scheme for data processing.

Abstract DNA sequencing by hybridization was carried out with a microarray of all 46 = 4,096 hexadeoxyribonucleotides (the generic microchip). The oligonucleotides immobilized in 100 × 100 × 20µm 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.

Kinetics of hybridization on the oligonucleotide microchips with gel pads.

Abstract 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.

Three-Quasiperiodicity, Mutual Correlations, Ordering and Long-Range Modulations in Genomic Nucleotide Sequences for Viruses

Using Fourier transform, we investigate the structural characteristics of genomes for several viruses. The mutual correlations and ordering of the different nucleotides in genomes are compared versus their counterparts from the random sequences with the same nucleotide composition. In order to assess the stastical significance of correlations and ordering we introduce the expression for structural entropy of a sequence and derive the corresponding analytical criteria. The method specially aimed at the investigation of the long-range correlations is described as well and the relationship between the long-range correlations and the modulations of three-quasiperiodicity is discussed.

Conserved sequences in the current strains of HIV-1 subtype A in Russia are effectively targeted by artificial RNAi in vitro.

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.

Hydrogel-based protein and oligonucleotide microchips on metal-coated surfaces: enhancement of fluorescence and optimization of immunoassay.

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.

Genome-wide study of correlations between genomic features and their relationship with the regulation of gene expression

The broad class of tasks in genetics and epigenetics can be reduced to the study of various features that are distributed over the genome (genome tracks). The rapid and efficient processing of the huge amount of data stored in the genome-scale databases cannot be achieved without the software packages based on the analytical criteria. However, strong inhomogeneity of genome tracks hampers the development of relevant statistics. We developed the criteria for the assessment of genome track inhomogeneity and correlations between two genome tracks. We also developed a software package, Genome Track Analyzer, based on this theory. The theory and software were tested on simulated data and were applied to the study of correlations between CpG islands and transcription start sites in the Homo sapiens genome, between profiles of protein-binding sites in chromosomes of Drosophila melanogaster, and between DNA double-strand breaks and histone marks in the H. sapiens genome. Significant correlations between transcription start sites on the forward and the reverse strands were observed in genomes of D. melanogaster, Caenorhabditis elegans, Mus musculus, H. sapiens, and Danio rerio. The observed correlations may be related to the regulation of gene expression in eukaryotes. Genome Track Analyzer is freely available at http://ancorr.eimb.ru/.

Coexistence of different base periodicities in prokaryotic genomes as related to DNA curvature, supercoiling, and transcription

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.