Biomedical subjects

D V Prokopenko

Publications and source records attributed to D V Prokopenko.

8 recordsLinked to original sources

PubMed2005

Biosensing and monitoring of cell populations using the hydrogel bacterial microchip.

Advanced development of the hydrogel bacterial microchip (HBMChip) technique is proposed. The microchip represents an array of hemispherical gel elements 0.3-60 nl in volume attached to hydrophobic glass surface and containing live immobilized microbial cells. Separate gel elements contain each up to 10(5) cells and retain them inside even while the cells are dividing. Porous structure of the gel provides easy access of nutrients and tested substances to the immobilized cells. Optical signals from the cells are easily measurable and allow monitoring of intracellular metabolism using vital fluorescent stains or engineered constructs encoding bioluminescent or fluorescent reporters. Two possible application modes of the HBMChip have been investigated, i.e. the observation of bacteria and biosensing. The dynamics of nucleic acids synthesis in growing E. coli cells has been analyzed using vital fluorescent stain SYTO 9. A special function has been suggested for evaluation of the cell growth parameters. Biosensing properties of the HBMChip have been illustrated by quantitative analysis of antibiotics and the detection of sodium meta-arsenite.

Anti-Bacterial Agents↗

PubMed2003

Analysis of binding specificity of disulfide bonded dimeric lambda-Cro V55C protein with generic hexamer oligonucleotide microchip.

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.

Bacteriophage lambda↗

PubMed2002

Specificity of mammalian Y-box binding protein p50 in interaction with ss and ds DNA analyzed with generic oligonucleotide microchip.

p50 protein is a member of the Y-box binding transcription factor family and is a counterpart of YB-1 protein. The generic microchip was used to analyze the sequence specificity of p50 binding to single (ss) and double-stranded (ds) oligodeoxyribonucleotides. The generic microchip contained 4,096 single-stranded octadeoxyribonucleotides in which all possible core 6-mers (4(6)=4,096) were flanked at their 3' and 5'-ends with degenerated nucleotides. The oligonucleotides were chemically immobilized within polyacrylamide gel pads fixed on a glass slide. The binding of p50 to the generic microchip was shown to be the most specific to ss GGGG motif and then to ss CACC and CATC motifs. GC-rich ds oligonucleotides of the generic microchip, and particularly those containing GGTG/CACC, GATG/CATC, and GTGG/CCAC heterogeneous motifs, were most efficiently destabilized due to interaction with p50. Gel-shift electrophoresis has shown that the protein exhibits much higher binding specificity to 24-mer oligoA-TGGGGG-oligoA containing G-rich 6-mer, in comparison with 24-mer oligoA-AAATAT-oligoA carrying A,T-rich 6-mer in full correspondence with the data obtained with the microchip. Studies of DNA-binding proteins using gel-immobilized ss and ds DNA fragments provide a unique possibility to detect low-affinity complexes of these proteins with short sequence motifs and assess the role of these motifs in sequence-specific interactions with long recognition sites.

Animals↗

PubMed2001

Massive parallel analysis of the binding specificity of histone-like protein HU to single- and double-stranded DNA with generic oligodeoxyribonucleotide microchips.

A generic hexadeoxyribonucleotide microchip has been applied to test the DNA-binding properties of HU histone-like bacterial protein, which is known to have a low sequence specificity. All 4096 hexamers flanked within 8mers by degenerate bases at both the 3'- and 5'-ends were immobilized within the 100 x 100 x 20 mm polyacrylamide gel pads of the microchip. Single-stranded immobilized oligonucleotides were converted in some experiments to the double-stranded form by hybridization with a specified mixture of 8mers. The DNA interaction with HU was characterized by three type of measurements: (i) binding of FITC-labeled HU to microchip oligonucleotides; (ii) melting curves of complexes of labeled HU with single-stranded microchip oligonucleotides; (iii) the effect of HU binding on melting curves of microchip double-stranded DNA labeled with another fluorescent dye, Texas Red. Large numbers of measurements of these parameters were carried out in parallel for all or many generic microchip elements in real time with a multi-wavelength fluorescence microscope. Statistical analysis of these data suggests some preference for HU binding to G/C-rich single-stranded oligonucleotides. HU complexes with double-stranded microchip 8mers can be divided into two groups in which HU binding either increased the melting temperature (T(m)) of duplexes or decreased it. The stabilized duplexes showed some preference for presence of the sequence motifs AAG, AGA and AAGA. In the second type of complex, enriched with A/T base pairs, the destabilization effect was higher for longer stretches of A/T duplexes. Binding of HU to labeled duplexes in the second type of complex caused some decrease in fluorescence. This decrease also correlates with the higher A/T content and lower T(m). The results demonstrate that generic microchips could be an efficient approach in analysis of sequence specificity of proteins.

Bacterial Proteins↗

PubMed2001

Parallel multiplex thermodynamic analysis of coaxial base stacking in DNA duplexes by oligodeoxyribonucleotide microchips.

Parallel thermodynamic analysis of the coaxial stacking effect of two bases localized in one strand of DNA duplexes has been performed. Oligonucleotides were immobilized in an array of three-dimensional polyacrylamide gel pads of microchips (MAGIChips'). The stacking effect was studied for all combinations of two bases and assessed by measuring the increase in melting temperature and in the free energy of duplexes formed by 5mers stacked to microchip-immobilized 10mers. For any given interface, the effect was studied for perfectly paired bases, as well as terminal mismatches, single base overlaps, single and double gaps, and modified terminal bases. Thermodynamic parameters of contiguous stacking determined by using microchips closely correlated with data obtained in solution. The extension of immobilized oligonucleotides with 5,6-dihydroxyuridine, a urea derivative of deoxyribose, or by phosphate, decreased the stacking effect moderately, while extension with FITC or Texas Red virtually eliminated stacking. The extension of the immobilized oligonucleotides with either acridine or 5-nitroindole increased stacking to mispaired bases and in some GC-rich interfaces. The measurements of stacking parameters were performed in different melting buffers. Although melting temperatures of AT- and GC-rich oligonucleotides in 5 M tetramethylammonium chloride were equalized, the energy of stacking interaction was significantly diminished.

Acridines↗

PubMed2000

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

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.

Algorithms↗

PubMedDate not supplied

[Analysis of perfect and mismatched DNA duplexes by a generic hexanucleotide microchip].

The thermodynamic analysis was done for the duplexes formed by fluorescently labeled oligonucleotide targets on a genetic hexanucleotide microchip. All 4096 different hexanucleotide chains were immobilized as probes in individual gel pads of the microchip. To strengthen the hybridization, each hexamer was extended at both ends by one nucleotide from the equimolar mixture of all four nucleotides to serve as nonselective linkers. It has been shown that the melting curves for oligonucleotide duplexes formed on the microchip and in a solution are quite similar. The influence of ionic surrounding has been studied in terms of the hybridization efficiency and discrimination between the mismatched and perfect duplexes. Different approaches have been tested to compensate the dependence of duplex stability on the GC content. It has been demonstrated that the use of chaotropic agents, addition of nonlabeled GC-rich competitor oligonucleotides, as well as creation of a temperature gradient along the microchip reproducing the distribution of melting temperatures, efficiently level out the AT/GC differences. The use of tetramethylammonium chloride for the same purpose was accompanied by weakening to some extent the discrimination between the mismatched duplexes and the perfect ones.

Base Composition↗

PubMedDate not supplied

[Alginate gel microchip for real-time monitoring of intracellular processes in bacterial and yeast cells].

A method of alginate-based hydrogel cell microchip manufacturing is proposed. The development of mild conditions for cell immobilization in microvolumes of non-toxic alginate gel allows extending the range of microorganisms used. Different approaches to cell analysis using microchip have been approved in pilot studies. By the example of Escherichia coli, Bordetella bronchiseptica and Saccharomyces cerevisiae it is shown that cell microchip can be successfully applied for monitoring of nucleic acid and protein synthesis in growing cells simultaneously using two fluorescent dyes. The influence of chloramphenicol on the nucleic acids and protein synthesis in five bacterial strains has been studied on the microchip. The microchip was also applied for the analysis of inducible fluorescent protein EGFP synthesis in E. coli cells, the correlation between the level of EGFP synthesis and concentration of the inductor in the medium has been established.

Alginates↗