Binding of netropsin to DNA and synthetic polynucleotides

The interaction of netropsin with DNA and synthetic polydeoxyribonucleotides was studied by absorption spectrophotometry and circular dichroism. The results are consistent with a model in which a netropsin molecule occupies five base pairs in binding and carries three reaction sites each capable of interacting with one AT base pair. We associate these reaction sites with the antibiotic peptide groups which probably interact with AT base pairs by a hydrogen bonding mechanism.     

Gel-based oligonucleotide microarray approach to analyze protein–ssDNA binding specificity

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}N₁N₂N₃N₄N₅N₆{N}-3′ consisted of a fixed hexamer motif N₁N₂N₃N₄N₅N₆ 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°C to 50°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.     

Gel-Based Microchips: History and Prospects

The review describes the history of formation and development of the microchip technology and its role in the human genome project in Russia. The main accent was done on the three-dimensional gel-based microchips developed at the Center of Biological Microchips headed by A.D. Mirzabekov since 1988. The gel-based chips of the last generation, IMAGE chips (Immobilized Micro Array of Gel Elements), have a number of advantages over the previous models. The microchips are manufactured by photoinitiated copolymerization of gel components and immobilized molecules (DNA, proteins, and ligands). This ensures an even distribution of the immobilized probe throughout the microchip gel element with a high yield (about 50% for oligonucleotides). The use of methacrylamide as a main component of the polymerization mixture resulted in a substantial increase of gel porosity without affecting its mechanical properties and stability; this allowed one to work with the DNA fragments of up to 500 nt in length, as well as with quite large protein molecules. At present, the gel-based microchips are widely applied to solve different problems. The generic microchips containing a complete set of possible hexanucleotides are used to reveal the DNA motifs binding with different proteins and to study the DNA–protein interactions. The oligonucleotide microchips are a cheap and reliable diagnostic tool designed for mass application. Biochips have been developed for identification of the tuberculosis pathogen and its antibiotic-resistant forms; of orthopoxviruses, including the smallpox virus; of the anthrax pathogen; and chromosomal rearrangements in leukemia patients. The protein microchips can be adapted for further use in proteo-mics. Bacterial and yeast cells were also immobilized in the gel, maintaining their viability, which opens a wide potential for creating biosensors on the basis of microchips.     

An oligonucleotide microarray for multiplex real-time PCR identification of HIV-1, HBV, and HCV

We describe a novel microarray-based approach for simultaneous identification and quantification of human immunodeficiency virus type 1 (HIV-1) and hepatitis B and C viruses (HBV and HCV) in donor plasma specimens. The method is based on multiplex real-time RT-PCR performed within the microarray hydrogel pads. Double-stranded amplification products are simultaneously detected using nonspecific SYBR Green I dye due to the reaction run in separate pads bearing 5'-immobilized specific primers. Both the sensitivity and specificity of the assay, based on 132 blood specimens analyzed, were 100% (56, 26, and 8 specimens were seropositive to HBV HCV and HIV-1, respectively; 22 were positive to both HIV-1 and HCV and 2 positive to all three viruses; 18 samples were pathogen-negative). The dynamic range of the quantitative analysis covered a six-order interval ranging from 100 to 106 genome equivalents per assay. The 95% detection limits were 14 gEq for HIV-1, 10 gEq (1.7 IU) for HBV, and 15 gEq (7.5 IU) for HCV per assay. The proposed approach is considered to be versatile and could be adapted for simultaneous identification and quantification of numerous genetic targets.     

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.     

2'-O-Methyl Oligoribonucleotide Analogs Used to Change the Temperature Characteristics of Immobilized Probes and to Enhance the Specificity of Hybridization

Biophysics - The specificity of interactions between oligonucleotide probes immobilized in a biological microchip (biochip) and target DNA depends on a number of factors, with the main roles being...     

Investigation of frequency of occurrence of core Y haplogroups in residents of Moscow and St. Petersburg

The collection of DNA from 239 Moscow residents and 62 St. Petersburg residents has been investigated by means of a biochip for genotyping haplogroup markers of the Y chromosome, including M130 (C), M145 (DE), P257 (G), M69 (II), U179 (I), M304 (J), M185 (L), M231 (N), M175 (O), P224 (R), L146 (R1a), and M343 (R1b). The distribution of haplogroup frequency in populations native to Moscow and St. Petersburg has been obtained. Three subsamples that vary in the duration of residence (one, two, or three generations) were compared. The increase in J, G, and R1b frequencies may be related to immigration from the Caucasus and other regions.     

Biochip analysis of BRCA1/2 and CHEK2 common mutations in ovarian cancer and primary multiple tumors involving the ovaries (Russian population)

Ovarian cancer (OC) is among the leading causes of cancer-related mortality in women. A high risk of OC (lifetime estimates ranging 10–60%) is determined by BRCA1/2 mutations. The 1100delC variant of CHEK2 is associated with predisposition to breast cancer (BC) in women. With the known spectrum and frequencies of mutations of these genes, it is possible to identify a risk group in a population. Using biochip technology, the frequencies of eight BRCA1/2 and CHEK2 mutations (185delAG, 300T>G, 4153delA, 4158A>G, and 5382insC of BRCA1; 695insT and 6174delT of BRCA2; and 1100delC of CHEK2) were studied in Russian women with OC, including 68 patients with organ-specific OC and 19 with primary multiple tumors (PMTs) involving the ovaries. Four BRCA1 mutations were observed: 185delAG, 300T>G, 4153delA, and 5382insC. The last one was most common in OC, accounting for 87.5% of all cases with mutant BRCA1, and occurred at a frequency of 50.0% in PMT. BRCA2 and CHEK2 mutations were not found in the two groups.     

Effects of external transport on discrimination between perfect and mismatch duplexes on gel-based oligonucleotide microchips

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.