A V Chudinov

Publications (25)21.57 Total impact

• Article: Targeting duplex DNA with chimeric α,β-triplex-forming oligonucleotides.

  N A Kolganova, A K Shchyolkina, A V Chudinov, A S Zasedatelev, V L Florentiev, E N Timofeev
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  ABSTRACT: Triplex-directed DNA recognition is strictly limited by polypurine sequences. In an attempt to address this problem with synthetic biology tools, we designed a panel of short chimeric α,β-triplex-forming oligonucleotides (TFOs) and studied their interaction with fluorescently labelled duplex hairpins using various techniques. The hybridization of hairpin with an array of chimeric probes suggests that recognition of double-stranded DNA follows complicated rules combining reversed Hoogsteen and non-canonical homologous hydrogen bonding. In the presence of magnesium ions, chimeric TFOs are able to form highly stable α,β-triplexes, as indicated by native gel-electrophoresis, on-array thermal denaturation and fluorescence-quenching experiments. CD spectra of chimeric triplexes exhibited features typically observed for anti-parallel purine triplexes with a GA or GT third strand. The high potential of chimeric α,β-TFOs in targeting double-stranded DNA was demonstrated in the EcoRI endonuclease protection assay. In this paper, we report, for the first time, the recognition of base pair inversions in a duplex by chimeric TFOs containing α-thymidine and α-deoxyguanosine.
  Nucleic Acids Research 05/2012; 40(16):8175-85. · 8.03 Impact Factor
• Article: Detection of KRAS mutations in tumor cells using biochips

  M. A. Emelyanova, F. A. Amossenko, A. V. Chudinov, S. A. Surzhikov, T. P. Kazubskaya, L. N. Lubchenko, T. V. Nasedkina
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  ABSTRACT: Somatic mutations in the KRAS gene are important markers of some types of tumors, for example, pancreatic cancer, and may be useful in early diagnostics. A biochip has been developed which allows deter-mining most frequent mutations in 12, 13, and 61 codons of the KRAS gene. To increase the sensitivity of the method and to enable the analysis of minor fractions of tumor cells in clinical samples, the method of blocking wild type sequence PCR amplification by LNA-oligonucleotides has been used. The product of LNA-clamp PCR was further hybridized with oligonucleotide probes, immobilized on the biochip. The biochip was tested with 42 clinical DNA samples from patients with pancreatic cancer, mostly duct adenocarcinomas. As reference methods, RFLP analysis and sequencing were used. The developed approach allows detecting somatic mutations in the KRAS gene if the portion of tumor cells with mutation is at least 1% of the whole cell population. Keywordssomatic mutations–tumor– KRAS gene–biochips
  Molecular Biology 04/2012; 45(5):797-803. · 0.66 Impact Factor
• Source

  Available from: Denis O Fesenko

  Article: Preparing of single-stranded DNA in single-stage PCR with low-melt excess primer for hybridization on biochips

  D. O. Fesenko, A. E. Kornienko, A. V. Chudinov, T. V. Nasedkina
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  ABSTRACT: A method for fluorescently labeled single-stranded DNA (ssDNA) production during single-stage polymerase chain reaction (PCR) for subsequent hybridization on a biochip was described. This approach, whose efficiency was confirmed in the case of DARC gene, is considered as an alternative to two-stage nested PCR, consisting of two separate reactions: symmetric and asymmetric. Implementation of PCR in a single stage was achieved due to the use of a truncated excess primer in the second stage that does not anneal on the matrix during the cycles of symmetric stage of PCR and that enters the reaction after decrease of the annealing temperature in asymmetric stage. As a result, high efficiency of genotyping by means of hybridization on biochips is maintained. The suggested approach will allow us to reduce the time, working hours, and risk of contamination when researching biochips. Keywordsproduction of ssDNA–biochips–single-stage nested PCR
  Molecular Biology 04/2012; 45(2):237-240. · 0.66 Impact Factor
• Article: Water-soluble cyanine dyes for biological microchip technology

  V. E. Kuznetsova, T. A. Luk’yanova, V. A. Vasiliskov, O. V. Kharitonova, A. V. Chudinov, A. S. Zasedatelev
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  ABSTRACT: Novel indodicarbocyanine dyes were obtained and their spectroscopic characteristics were determined. For equal concentrations of the dyes, the relative fluorescence efficiency was measured at the excitation wavelengths λ = 635 and 655 nm and the emission wavelengths λ = 670 and 690 nm, respectively.
  Russian Chemical Bulletin 04/2012; 56(12):2438-2442. · 0.38 Impact Factor
• Article: Novel asymmetric indodicarbocyanine dyes

  V. E. Kuznetsova, A. V. Davydov, V. A. Vasiliskov, A. A. Stomakhin, A. V. Chudinov, A. S. Zasedatelev
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  ABSTRACT: Novel indodicarbocyanine dyes functionalized at position 5 of the indolenine system were obtained and characterized by spectroscopic methods.
  Russian Chemical Bulletin 04/2012; 56(11):2263-2267. · 0.38 Impact Factor
• Article: [An association study of polymorphisms in HTR2A, BDNF and SLC6A4 genes with paranoid schizophrenia and suicidal behavior.]

  D Iu Galaktionova, O A Gra, I I Nizamutdinov, V E Shershov, V E Kuznetsova, A V Chudinov, A E Gareeva, D F Zakirov, E K Khusnutdinova, Iu P Lysov, T V Nasedkina
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  ABSTRACT: We have developed a biochip for the analysis of candidate genes for schizophrenia. Using this biochip, allele and genotype frequencies for the polymorphisms of HTR2A, BDNF and SLC6A4 genes in 198 patients with schizophrenia and 192 healthy individuals have been obtained. The allele T of the HTR2A polymorphism rs6314 was identified as protective against the development of paranoid schizophrenia (p=0,014). An analysis of gene-gene interactions using the Multifactor-Dimensionality Reduction (MDR) algorithm has shown a statistically significant association of combined genotypes rs6311 G/-, rs6313 C/-, rs6314 C/C, rs7997012 G/- with the disease (p=0.019). Also it has been shown that the G/G genotype of the polymorphism rs6311 (p=0.013) and the C/C genotype of the polymorphism rs6313 (p=0.008) in the HTR2A gene are associated with the suicide attempt in schizophrenic patients. Correspondingly, an A allele, А/- genotypes of the polymorphism rs6311 G>A and a T allele, T/- genotypes of the polymorphism rs6313 C>T were found to be less frequent in schizophrenic patients with a history of suicide attempt than in schizophrenic patients without a history of suicide attempt, thus suggesting their protective role in the development of suicidal behavior. The results confirm the hypothesis that the HTR2A plays an important role in the etiology of schizophrenia and suicidal behavior.
  Zhurnal nevrologii i psikhiatrii imeni S.S. Korsakova / Ministerstvo zdravookhraneniia i meditsinskoi promyshlennosti Rossiiskoi Federatsii, Vserossiiskoe obshchestvo nevrologov [i] Vserossiiskoe obshchestvo psikhiatrov 01/2012; 112(10):39-44. · 0.12 Impact Factor
• Source

  Available from: Vladimir R Chechetkin

  Article: Kinetic effects on signal normalization in oligonucleotide microchips with labeled immobilized probes.

  S V Pan'kov, V R Chechetkin, O G Somova, O V Antonova, O V Moiseeva, D V Prokopenko, R A Yurasov, D A Gryadunov, A V Chudinov
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  ABSTRACT: 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 tuberculosis.
  Journal of biomolecular structure & dynamics 11/2009; 27(2):235-44. · 4.99 Impact Factor
• Article: Development of a Biochip for Analyzing Polymorphism of the Biotransformation Genes

  A. S. Glotov, T. V. Nasedkina, T. E. Ivaschenko, R. A. Urasov, S. A. Surzhikov, S. V. Pan’kov, A. V. Chudinov, V. S. Baranov, A. S. Zasedatelev
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  ABSTRACT: Large-scale population studies, diagnosis of genetic predisposition to a broad range of multifactorial diseases, and screening of polymorphic loci associated with individual drug resistance need efficient, accurate, and rapid techniques for identifying many mutations. One of the most promising techniques is hybridization on an oligonucleotide microarray (biochip). The efficiency of this method in assessing genetic polymorphism was demonstrated using an example of mutations in CYP1A1, CYP2D6, GSTM1, GSTT1, NAT2, CYP2C9, CYP2C19, and MTHFR. The biochip constructed provides a convenient tool for pharmacogenetic research.
  Molecular Biology 04/2005; 39(3):357-365. · 0.66 Impact Factor
• Source

  Available from: Denis O Fesenko

  Article: Alginate gel biochip for real-time monitoring of intracellular processes in bacterial and yeast cells

  D. O. Fesenko, T. V. Nasedkina, A. V. Chudinov, D. V. Prokopenko, R. A. Yurasov, A. S. Zasedatelev
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  ABSTRACT: A method was developed for producing cell biochips on the basis of calcium alginate. Cell immobilization in microvolumes of nontoxic alginate gel under mild conditions extended the range of testable micro-organisms. The possibility of studying the intracellular processes with alginate gel biochips was demonstrated in model experiments with Escherichia coli, Bordetella bronchiseptica, and Saccharomyces cerevisiae. Cell biochips proved to be suitable for simultaneous monitoring of nucleic acid and protein syntheses with two fluorescent dyes. The effect of chloramphenicol on nucleic acid synthesis was studied with five bacterial strains. Inducible synthesis of the green fluorescence protein (EGFP) in E. coli cells was monitored with the use of biochips. The level of EGFP synthesis correlated with the inductor concentration in the medium.
  Molecular Biology 12/2004; 39(1):84-89. · 0.66 Impact Factor
• Article: Hydrogel drop microchips with immobilized DNA: properties and methods for large-scale production.

  A Yu Rubina, S V Pan'kov, E I Dementieva, D N Pen'kov, A V Butygin, V A Vasiliskov, A V Chudinov, A L Mikheikin, V M Mikhailovich, A D Mirzabekov
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  ABSTRACT: Although gel-based microchips offer significant advantages over two-dimensional arrays, their use has been impeded by the lack of an efficient manufacturing procedure. Here we describe two simple, fast, and reproducible methods of fabrication of DNA gel drop microchips. In the first, copolymerization method, unsaturated groups are chemically attached to immobilized molecules, which are then mixed with gel-forming monomers. In the second, simpler polymerization-mediated immobilization method, aminated DNA without prior modification is added to a polymerization mixture. Droplets of polymerization mixtures are spotted by a robot onto glass slides and the slides are illuminated with UV light to induce copolymerization of DNA with gel-forming monomers. This results in immobilization of DNA within the whole volume of semispherical gel drops. The first method can be better controlled while the second one is less expensive, faster, and better suited to large-scale production. The microchips manufactured by both methods are similar in properties. Gel elements of the chip are porous enough to allow penetration of DNA up to 500 nucleotides long and its hybridization with immobilized oligonucleotides. As shown with confocal microscope studies, DNA is hybridized uniformly in the whole volume of gel drops. The gels are mechanically and thermally stable and withstand 20 subsequent hybridizations or 30-40 PCR cycles without decrease in hybridization signal. A method for quality control of the chips by staining with fluorescence dye is proposed. Applications of hydrogel microchips in research and clinical diagnostics are summarized.
  Analytical Biochemistry 03/2004; 325(1):92-106. · 3.00 Impact Factor
• Article: Synthesis of a Water-Soluble Ytterbium Porphyrin–Bovine Serum Albumin Conjugate

  A. V. Chudinov, V. D. Rumyantseva, A. V. Lobanov, G. K. Chudinova, A. A. Stomakhin, A. F. Mironov
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  ABSTRACT: The ytterbium complex of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin was synthesized as an IR-fluorescent label and covalently bound to bovine serum albumin. The resulting conjugate fluoresces at 985 nm and is of interest for use in IR-fluorescent tumor diagnostic, immunoassay, and energy transfer studies.
  Russian Journal of Bioorganic Chemistry 12/2003; 30(1):89-93. · 0.64 Impact Factor
• Article: A Fluorescent Dye with Low Specificity to DNA Nucleotide Sequences: Quantitative Assessment of Oligonucleotides Immobilized in Microchip Gel Pads

  A. L. Mikheikin, A. V. Chudinov, A. I. Yaroshchuk, A. Yu. Rubina, S. V. Pan'kov, A. S. Krylov, A. S. Zasedatelev, A. D. Mirzabekov
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  ABSTRACT: To assess the DNA amount in samples (e.g., in biological microchip gel pads) by means of fluorescent dyes, one should use the dyes whose fluorescence weakly depends on DNA composition and structure. With the ImD-310 dye created for this purpose, we have analyzed the staining of single- and double-stranded oligo- and polynucleotides of different nucleotide composition, length, and concentration both in solution and being immobilized in biological microchip gel pads. It turned out that ImD-310 has no pronounced specificity to the single- and double-stranded nucleotide sequences, while the intensity of fluorescence for the dye complexes with d(A)8, d(T)8, d(C)8, and d(G)8 at high temperatures (50C) differs by less than 25%. A linear correlation has been established between the intensity of fluorescence and the amount of oligonucleotides immobilized on a biological microchip. The plots of the intensity of fluorescence against the concentration of NaCl and the temperature were obtained. By using a generic microchip containing all 4096 hexamer oligonucleotides, it has been determined that the dye has no distinct specificity to any certain motifs of the nucleotide sequence. Thus, ImD-310 may serve as an efficient fluorescent probe to quickly estimate the amount of oligonucleotides immobilized in a microchip, in an electrophoretic gel, etc.
  Molecular Biology 10/2003; 37(6):902-911. · 0.66 Impact Factor
• Article: [Identification of Mycobacterium tuberculosis strains and a simultaneous identification of their drug resistance by the hybridization method on oligonucleotide microchips].

  D A Griadunov, V M Mikhaĭlovich, S A Lapa, N I Rudinskiĭ, V E Barskiĭ, A V Chudinov, A S Zasedatelev, A D Mirzabekov
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  ABSTRACT: A method of multiplex polymerase chain reaction (PCR) with subsequent hyoridization on oligonucleotide microchips was worked out to identify the Mycobacterium tuberculosis complex and to determine simultaneously the bacterial sensitivity to 2 first-line drugs, i.e. rifampin and isoniazid. The method provides for detecting above 95% of rifampin-resistant and around 80% of isoniazid-resistant strains within 1 day.
  Molekuliarnaia genetika, mikrobiologiia i virusologiia 02/2003;
• Article: [Synthesis of conjugates of bovine serum albumin with water-soluble ytterbium porphyrins].

  A V Chudinov, V D Rumiantseva, A V Lobanov, G K Chudinova, A A Stomakhin, A F Mironov
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  ABSTRACT: The ytterbium complex of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin was synthesized as an IR-fluorescent label and covalently bound to bovine serum albumin. The resulting conjugate fluoresces at 985 nm and is of interest for use in IR-fluorescent tumor diagnostic, immunoassay, and energy transfer studies. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.
  Bioorganicheskaia khimiia 30(1):99-104.
• Article: [New indodicarbocyanine dyes for the biological microchip technology].

  V E Kuznetsova, V A Vasiliskov, O V Antonova, V M Mikhaílovich, A S Zasedatelev, A V Chudinov
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  ABSTRACT: New indodicarbocyanine dyes with the carboxybutyl group in position 3 of the indolenine fragment bearing methyl and sulfonic groups in positions 5 and 7 of the cycle were synthesized in order to find the most effective fluorescent labels for the biological microchip technology. The position of absorption and fluorescence maxima, the total charge of the dye molecule, and water solubility depend on the location and the total amount of methyl and sulfonic groups. The spectral characteristics of the dyes synthesized were determined. The relative fluorescence efficiencies of the dyes at equal concentrations were measured at excitation wavelengths of 635 and 655 nm and emission wavelengths of 670 and 690 nm, respectively.
  Bioorganicheskaia khimiia 34(1):141-4.
• Article: [Boradiazaindacene dyes for technology of biological microchips].

  E E Belobritskaia, M V Neunylova, V A Vasiliskov, V D Rumiantseva, A V Chudinov, A S Zasedatelev
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  ABSTRACT: A number of boradiazaindacene dyes containing a carboxyl group separated from a fluorophore by two methylene units were synthesized. The compounds have narrow spectral bands with absorption maxima at 480-530 nm and fluorescence maxima at 500-550 nm. Succinimide esters of these compounds and the corresponding fluorescent-labeled olgionucleotides were also prepared. Boradiazaindacene dyes can be used as fluorescent labels for oligonucleotides for analysis of melting curves of duplexes on microchips either by themselves or in combination with Texas Red. They can also be applied for labeling primers for polymerase chain reaction.
  Bioorganicheskaia khimiia 33(6):664-6.
• Article: A new pair of donor-acceptor markers for immunoassay: a porphyrin-cyanine dye. Energy transfer in solutions and Langmuir films.

  G K Chudinova, I A Nagovitsyn, A V Chudinov, V V Savransky, A M Prokhorov
  Doklady Biochemistry and Biophysics 386:268-70. · 0.33 Impact Factor
• Source

  Available from: Sergey Tokalov

  Article: [Fluorescence of meso-tetrakis(4-(carboxy)phenyl)porphine covalently bound to oligonucleotides d(CG)5 and d(TA)5].

  I A Besschetnova, A V Chudinov, D N Kaliuzhnyĭ, A K Shchelkina, O F Borisova, S V Tokalov, V E Kuznetsova, A V Lobanov, V D Rumiantseva, V E Barskiĭ, A D Mirzabekov
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  ABSTRACT: The amino-reactive derivative of tetraphenylporphine meso-tetrakis[4-(carboxy)phenyl]porphine (TCPP) was synthesized, which is characterized by a high molar absorption coefficient (epsilon 416 = 36,500 M-1.cm-1). TCPP was covalently attached to oligonucleotides d(CG)5 [d(CG)5-TCPP] and d(TA)5 [d(TA)5-TCPP]. The spectral characteristics of these complexes were studied in 0.01 M phosphate buffer, pH 7 at 23 degrees C. UV-visible absorption spectra of these complexes have a clearly pronounced Soret band at (414 +/- 1) nm for d(CG)5-TCPP and at (412 +/- 1) nm for d(TA)5-TCPP. The fluorescence spectra of these complexes have maxima at (648 +/- 2) nm for d(CG)5-TCPP and at (658 +/- 2) nm for d(TA)5-TCPP. In this study we also determined fluorescence quantum yields q and fluorescence lifetimes tau [q = 0.099 +/- 0.011, tau = (9.0 +/- 0.3) ns for d(CG)5-TCPP and q = 0.080 +/- 0.011, tau = (8.7 +/- 0.3) ns for d(TA)5-TCPP]. A temperature rise from 5 to 50 degrees C produced only slight (within 23%) emission changes in both samples studied. Taking into account: a) high fluorescence yields (q), b) weak dependence of q on temperature, c) weak q dependence of q on the oligonucleotide type, we conclude that TCPP may be used as a sensitive fluorescence label in DNA studies.
  Biofizika 47(2):259-67. · 0.43 Impact Factor
• Article: [Biochip development for polymorphism analysis in biotransformation system genes].

  a S Glotov, T V Nasedkina, T E Ivashchenko, R A Iurasov, S A Surzhikov, S V Pan'kov, A V Chudinov, V S Baranov, A S Zasedatelev
  [show abstract] [hide abstract]
  ABSTRACT: Large-scale population researches, diagnostics of genetic predisposition to multifactorial diseases, screening of the polymorphic loci associated with individual sensitivity to pharmaceutical preparations, require the development of effective, exact and rapid methods of analysis for detection of many mutations simultaneously. One of the most perspective methods to solve these problems is a method of allele-specific hybridization with biochips. Taking the analysis of mutations in genes CYP1A1, CYP2D6, GSTM1, GSTT1, NAT2, CYP2C9, CYP2C19 and MTHFR as an example we showed the efficiency of using the approach for identification of individual genetic polymorphism. We believe that the biochips can be also a convenient tool in pharmacogenetics researches.
  Molekuliarnaia biologiia 39(3):403-12.
• Article: [Dye with low specificity to nucleotide sequences of DNA: use for assessing the quantity of oligonucleotides, immobilized in cells of biological microchips].

  A L Mikheĭkin, A V Chudinov, A I Iaroshchuk, A Iu Rubina, S V Pan'kov, A S Krylov, A S Zasedatelev, A D Mirzabekov
  [show abstract] [hide abstract]
  ABSTRACT: To assess the DNA amount in samples (e.g., in biological microchip gel pads) by means of fluorescent dyes, one should use the dyes whose fluorescence weakly depends on DNA composition and structure. With the ImD-310 dye created for this purpose, we have analyzed the staining of single- and double-stranded oligo- and polynucleotides of different nucleotide composition, length, and concentration both in solution and being immobilized in biological microchip gel pads. It turned out that ImD-310 has no pronounced specificity to the single- and double-stranded nucleotide sequences, while the intensity of fluorescence for the dye complexes with d(A)8, d(T)8, d(C)8, and d(G)8 at high temperatures (50 degrees C) differs by less than 25%. A linear correlation has been established between the intensity of fluorescence and the amount of oligonucleotides immobilized on a biological microchip. The plots of the intensity of fluorescence against the concentration of NaCl and the temperature were obtained. By using a generic microchip containing all 4096 hexamer oligonucleotides, it has been determined that the dye has no distinct specificity to any certain motifs of the nucleotide sequence. Thus, ImD-310 may serve as an efficient fluorescent probe to quickly estimate the amount of oligonucleotides immobilized in a microchip, in an electrophoretic gel, etc.
  Molekuliarnaia biologiia 37(6):1061-70.