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The most cited biological Institute in Russia *

Department of nucleic acids chemistry

The division was founded in 1966 and up to 1999 it was headed by Prof. Z.A. Shabarova. Since 1999 the division has been headed by Prof. T.S. Oretskaya. The main researchers in the devision are: Prof. M.B. Gottikh, Prof. E.A. Kubareva, PhD V.L. Drutza, PhD E.A. Romanova, PhD M.G. Ivanovskaya.

Research areas

  1. Synthesis and development of new types of modified DNA and RNA fragments including those containing modified chemically active groups in any position of the sugar-phosphate backbone. The use of synthetic NA reagents capable of reacting spontaneously with nucleophylic amino acids in the NA - protein complex in order to define the structural basics for specificity in the complex as well as mechanisms of action of regulatory proteins and gene expression modulation.

  2. The development of the fotoregulation of the DNA-binding enzymes activity by changing the configuration of azobenzene derivatives. This approach changes the local structure within the protein or else limits the active center accessibility for the substrate.

  3. The study of the influence of point lesions in the DNA on: the double helix structure, the binding with functional proteins and enzymes, on biological methylation of nucleic acids.

  4. The determination of molecular structure of the key proteins in the DNA mismatch repair system (MMR) and the study of the regulation mechanisms that guide the complex activity.

  5. The development of general approaches towards the analysis of the “molecular anatomy” and the functioning of various RNA-protein complexes. The study of the role of a new type of regulators – short untranslated RNAs (6S RNAs). These RNAs are capable of a transcription initiation by the means of a direct binding to bacterial RNA polymerase. The study of supramolecular RNA-protein complexes and of a newly discovered RNAse P analog PRORP1 from Arabidopsis thaliana. PRORP1 is involved in tRNA processing in mitochondria and chloroplasts in plants.

  6. The study of the HIV-1 integrase that is one the main HIV enzymes. The study involves the analysis of the integrase interaction with viral and cellular DNA, the characteristics of the structural peculiarities of the viral DNA that correspond to the binding specificity with integrase, the study of the kinetics of the integrase catalyzed reactions. The study of various new HIV-1 integrase inhibitors and their mechanism of action. The development of a system for directed DNA integration by the means of a direct modification of HIV-1 integrase as well as of an integrase from prototype foamy virus so that these enzymes would specifically recognize a certain sequence within human genome.

Research results

(i) The design and synthesis of oligonucleotides with chemically active groups for preparation of NA fragments derivatives and affine modification of proteins were developed

A strategy involving flexible variation of type and position of modification in the sugar-phosphate DNA backbone was developed for obtaining nucleic acids fragments with designated features. A universal scheme of incorporating modified units into oligonucleotides by combining methods of automatic chemical synthesis and postsynthetic modification was developed. Several substitutions were incorporated in a specified position in the DNA fragment. A DNA - reagents was constructed for an affine modification of lysine, cysteine and arginine residues in proteins. Modified oligonucleotides were proven active with wide range of proteins and enzymes. Finally, a set of oligonucleotide derivatives was synthesized with various hydrazides, hydrazines and carbazates including compounds with reporter groups (biotin), markers (acridine, pyrene), cross-linking reagents (terephtalic acid dihydrazide), lypophilic groups and peptide molecules [Zatsepin T.S. et al. Bioconjug.Chem. 2002; Zatsepin T.S. et al. Tetrahedron Lett., 2006; Zatsepin T.S. et al. Nucleosides, Nucleotides & Nucl.Acids, 2007; Dolinnaya N.G. et al. Curr. Org. Chem.2009; Khomyakova E.A. et al. Nucleosides, Nucleotides & Nucl. Acids, 2011].

(ii) New DNA reagents were characterized as instruments in studying the molecular structure of nucleic acid - protein complexes

New DNA reagents were characterized as instruments in studying the molecular structure of nucleic acid - protein complexes on prestationary state and of supramolar biopolimer complexes. Several cell targets were used: NF-kB transcription factor, restriction-modification enzymes, point DNA lesions reparation enzymes, integration enzymes (HIV-1 integrase). It was shown that protein affine modification allows to study fine interactions between proteins and nucleic acids and the sensitivity of this method can be compared to such techniques as site-directed mutagenesis and X-ray crystallography  [Metelev V. et al. IUBMB Life, 2006; Romanenkov A.S. Biochem.-Moscow, 2006; Kosinski J. et al. Proteins, 2007; Fedotova E.A. et al. Biochem.-Moscow, 2009; Ryazanova A.Y. et al. Analyst, 2011; Ryazanova A.Y. et al. Nucleosides, Nucleotides & Nucl. Acids, 2011; Khomyakova Е.А. et al. Russ.J. of Bioorg.Chem. 2010].

(iii) The structural and functional characteristics of complexes between DNA and a bifunctional enzyme C5-cytosine DNA-methyltransferase SsoII (M.SsoII) were studied

The structural and functional characteristics of complexes between DNA and a bifunctional enzyme C5-cytosine DNA-methyltransferase SsoII (M.SsoII). This enzyme not only methylates the cytosine residue but also binds to the regulatory site in the promoter area of the SsoII restriction-modification system which suppresses the transcription of enzyme’s own gene and stimulates the transcription of the restriction endonuclease gene [Romanenkov A.S. et al. Biochem.-Moscow, 2006; Fedotova E.A. et al. Biochem.-Moscow, 2009]. The kinetics of interaction between M.SsoII and several DNA duplexes were studied and the highest affinity was observed for the complex formed by M.SsoII and the DNA duplex containing the methylation site [Ryazanova A.Y. et al. Analyst, 2011]. The use of bifunctional chemical reagents allowed to show that protein region responsible for methylation is bound to the site within the DNA duplex flanking the regulatory sequence [Ryazanova A.Y. et al. Nucleosides, Nucleotides & Nucl. Acids, 2011].

(iv) The protein activity regulation strategy was designed by using external signal

The activity external regulation strategy was designed in order to control the functioning of site-specific endonucleases. The so called “molecular spring” and “molecular gates” approaches have been applied to a specifically constructed forms of PvuII and SsoII restriction endonucleases which allowed to show that the exposure to light of different wave lengths changes the activity of DNA-hydrolyzing enzymes in case certain derivatives of azobenzene were used as a photo switch reagent [Schierling B. et al. 2010, PNAS; Hien L.T. 2009, Rus. J. of Bioorg. Chem.; Hien L.T. et al. 2011, Bioconjugate Chem.].

(v) Physico-chemical and biological properties of thymidine glycol containing DNA were investigated

Physico-chemical and biological properties of thymidine glycol containing DNA were investigated that is one of the most widespread DNA damages. Thymidine glycol containing DNA duplexes were used for studying the influence of oxidative damage on the thermodynamic properties of double helix as well as on its structure and its interaction with DNA-recognizing proteins [Yang F. et al. 2009, Analyst]. Synthetic DNA molecules containing thymidine glycol residues were used to study the ability of specialized DNA-polymerases to “overcome” oxidized bases during replication [Belousova E.A. et al. 2010, Biochemistry].

(vi) The features of protein - DNA recognition in the DNA mismatch repair system were determined

The features of protein - DNA recognition in the mismatch repair system were determined. MutS and MutL are the key proteins in the bacterial DNA mismatch repair system (MMR). The peculiarities in the complex between MutL, MutS and the DNA were studied using cross-linking approach coupled to FRET technique. A novel model of a MutS-MutL-DNA complex has been suggested that explains a subsequent accomplishment of reparation by these enzymes.

(vii) DNA methyltransferases: methylation of the damaged DNA, inhibition, mechanism were investigated

The influence of a widely spread pollution agent benzo[a]pyrene as well as 8-oxoguanine and 6-methylguanine on DNA methylation by bacterial and mammalian (murine Dnmt3a) DNA methyltransferases (MTases) has been studied. It has been shown that these DNA lesions primarily affect the catalytic step of the reaction and to a much lesser degree they affect the enzyme binding to the damaged DNA. Methylation efficiency depends on the structure of the damage and its localization within the double helix. The inhibition of the MTases has been examined, novel inhibitors have been proposed and mechanism of methylation has been studied.

The data obtained will give insight into the relationship between DNA damage, epigenetic modifications and development of different diseases as well as will make an important contribution to the development of anticancer therapy through the MTase inhibition [Baskunov V.B. et al. Biochem., 2005; Subach O.M. et al., Biochem., 2006; Subach O.M. et al. FEBS J., 2007; Kirsanova O.V. et al. Biochem.-Moscow, 2009; Maltseva D.V. et al. Biochem. год; Maltsevа D. V.et al. Biochem.-Moscow, 2010; Darii M.V. et al BBA., 2010; Cherepanova N.A.et al., J Enzyme Inhib. Med. Chem., 2011; Lukashevich O.V. et al. Biochem. 2011].

(viii) A new technique of detecting non-canonical base pairs in DNA was developed

A new technique of detecting non-canonical base pairs in DNA was developed. The technique is based on molecular recognition of complementary base pairs in a tandem system formed by metal-binding centers coordination bonds localized at terminal positions in the oligonucleotide tandem. A model system showed that the oligonucleotide hybridization on a DNA matrix can only happen in the presence of nickel ions, while a point mutation located opposite to the short oligonucletides connection site impedes the DNA duplex formation [Hien L.T. et al. Med.Chem. Lett. 2009]. A direct express detection technique for outlooping thymine residues in DNA duplexes has been developed. The technique is based on duplex treatment by potassium permanganate and specific point mutation recognition by a chemical reagent [Logvina N.A. Biochem.-Moscow, 2011; Yakubovskaya M.G. et al. Biochimie, 2010].

(ix) The study of the retroviral integration mechanism and the search for HIV-1 integrase inhibitors were carried out

A detailed kinetic study of enzymatic reactions has for the first time shown that the HIV-1 integrase is a single turn-over enzyme. A set of modified analogs of viral DNA was used to determine the DNA sites that directly interact with designated lysine residues in the integrase. A new model of integrase interaction with viral DNA has been proposed. This model was further used for new integration inhibitors development including those based on short modified oligonucleotides. The studies conducted show that oligonucleotides are allosteric inhibitors of integrase: they integrate within the integrase - DNA complex and provoke its fast dissociation. Some oligonucleotide inhibitors can suppress viral replication in infected cells.

A system for the directed integration of DNA into the human genome has been developed. It is based on both HIV integrase and an integrase from prototype foamy virus. A novel synthetic protein consisting of six “zinc-finger” domains and designed specifically for binding a designated region within human genome has been added to both integrases. These hybrid proteins were proven capable of directing integration to a specific site in an in vitro system. [Smolov M. et al. FEBS J., 2006; Agapkina J. et al.  JBC   2006; Michel F. et al. EMBO J., 2009; Grigorov B. et al. NAR, 2011; Korolev S. et al. Nucleosides, Nucleotides & Nucl. Acids, 2011; Agapkina J. et al. ACS Med. Chem. Lett., 2011]

(x) The structure and function of E.coli RNA polymerase were studied

Studies were conducted on the mechanism of transcriptional interference in a DNA system containing closely located promoter regions. A set of new mutant variants of E. coli RNA-polymerase σ-subunits containing point substitutions and deletions in their N-terminal region has been obtained and thoroughly investigated. It has been shown that the mutated σ-subunits can be used for directed regulation of promoter activity in an artificial multipromoter system. A specific influence of several amino acids and short peptides on various transcription stages has been shown in vitro. A study using atomic force microscopy has for the first time shown that the E. coli RNA-polymerase σ-subunit can form highly organized amyloid-like aggregates [Koroleva O.N. et al. J. Biosci., 2011; Koroleva O.N. et al. Nucleosides, Nucleotides & Nucl. Acids, 2011; Dubrovin E.V. et al. Nanomedicine: Nanotechn., Biol. and Med.,2012].

Educational activities

Educational activities in forms of lectures, practical trainings and seminars are provided for students and post-graduates of Moscow State University particularly from Faculty of Chemistry and Faculty of Bioengineering and Bioinformatics.

During the last 3 years Department researchers supervised 10 PhD theses and 12 graduate works that were successfully defended.

Lecture courses

Professor Gottikh M.B – course “Chemistry of Nucleic Acids” for 4-th year students of Faculty of Chemistry.

Professor Oretskaya T.S. – course “Chemistry of mono- and dicarbohydrates” for 3-th year students of Faculty of Chemistry

Employees

Assistant employees

Students

Selected papers Recent papers
  1. Agapkina J., Zatsepin T., Knyazhanskaya E., Mouscadet J.F., Gottikh M. (2011) Structure-Activity Relationship Studies of HIV-1 Integrase Oligonucleotide Inhibitors. ACS Medicinal Chemistry Letters, 2 (7): 532-537.

  2. Grigorov B., Bocquin A., Gabus C., Avilov S., Mely Y., Agopian A., Divita G., Gottikh M., Witvrouw M., Darlix J.L. (2011) Identification of a methylated oligoribonucleotide as a potent inhibitor of HIV-1 reverse transcription complex. Nucleic Acids Research, 39 (13): 5586-5596.

  3. Khomyakova E.A., Zubin E.M., Smirnov I.P., Pozmogova G.E., Stetsenko D.A., Oretskaya T.S. (2011) DNA Or RNA Oligonucleotide 2 '-Hydrazides for Chemoselective Click-Type Ligation with Carbonyl Compounds. Nucleosides, Nucleotides & Nucleic Acids, 30 (7): 577-584.

  4. Hien L.T., Zatsepin T.S., Schierling B., Volkov E.M., Wende W., Pingoud A., Kubareva E.A., Oretskaya T.S. (2011) Restriction Endonuclease Ssoll with Photoregulated Activity-a "Molecular Gate" Approach. Bioconjugate Chemistry, 22 (7): 1366-1373.

  5. Ryazanova A.Y., Winkler I., Friedhoff P., Viryasov M.B., Oretskaya T.S., Kubareval E.A. (2011) Crosslinking of (Cytosine-5)-DNA Methyltransferase SsoII and Its Complexes with Specific Dna Duplexes Provides An Insight Into Their Structures. Nucleosides, Nucleotides & Nucleic Acids, 30 (7): 632-650.

  6. Koroleva O.N., Busby S.J.W., Drutsa V.L. (2011) Effects of substitutions at position 180 in the Escherichia coli RNA polymerase sigma(70) subunit. Journal of Biosciences, 36 (1): 43-54.

  7. Lukashevich O.V., Baskunov V.B., Darii M.V., Kolbanovskiy A., Baykov A.A., Gromova E.S. (2011) Dnmt3a-CD Is Less Susceptible to Bulky Benzo[a]pyrene Diol Epoxide-Derived DNA Lesions Than Prokaryotic DNA Methyltransferases. Biochemistry, 50 (5): 875-881.

  8. Logvina N.A., Yakubovskaya M.G., Dolinnaya N.G. (2011) Rapid photometric detection of thymine residues partially flipped out of double helix as a method for direct scanning of point mutations and apurinic DNA sites. Biochemistry (Moscow), 76 (2): 245-252. >>

  9. Ryazanova A.Y., Kubareva E.A., Grman I., Lavrova N.V., Ryazanova E.M., Oretskaya T.S., Hianik T. (2011) The study of the interaction of (cytosine-5)-DNA methyltransferase SsoII with DNA by acoustic method. Analyst, 136 (6): 1227-1233.

  10. Yakubovskaya M.G., Belyakova A.A., Gasanova V.K., Belitsky G.A., Dolinnaya N.G. (2010) Comparative reactivity of mismatched and unpaired bases in relation to their type and surroundings. Chemical cleavage of DNA mismatches in mutation detection analysis. Biochimie, 92 (7): 762-771. >>

  11. Belousova E.A., Maga G., Fan Y., Kubareva E.A., Romanova E.A., Lebedeva N.A., Oretskaya T.S., Lavrik O.I. (2010) DNA Polymerases beta and lambda Bypass Thymine Glycol in Gapped DNA Structures. Biochemistry, 49 (22): 4695-4704.

  12. Schierling B., Noel A.J., Wende W., Hien L.T., Volkov E., Kubareva E., Oretskaya T., Kokkinidis M., Rompp A., Spengler B., Pingoud A. (2010) Controlling the enzymatic activity of a restriction enzyme by light. Proceedings of the National Academy of Sciences of the United States of America, 107 (4): 1361-1366.

  13. Hien L.T., Oretskaya T.S., Zatsepin T.S. (2009) Metal ion CHElate-aSSisted LIGAtion (CHESS LIGA) for SNP detection on microarrays. Medicinal Chemistry Letters, 19 (15): 4018-4021.

  14. Dolinnaya N.G., Zubin E.M., Kubareva E.A., Zatsepin T.S., Oretskaya T.S. (2009) Design and Synthesis of 2 '-Functionalised Oligonucleotides. Their Application for Covalent Trapping the Protein-DNA Complexes. Current Organic Chemistry, 13 (11): 1029-1049.

  15. Michel F., Crucifix C., Granger F., Eiler S., Mouscadet J.F., Korolev S., Agapkina J., Ziganshin R., Gottikh M., Nazabal A., Emiliani S., Benarous R., Moras D., Schultz P., Ruff M. (2009) Structural basis for HIV-1 DNA integration in the human genome, role of the LEDGF/P75 cofactor. EMBO Journal, 28 (7): 980-991.

  16. Subach O.M., Maltseva D.V., Shastry A., Kolbanovskiy A., Klimasauskas S., Geacintov N.E., Gromova E.S. (2007) The stereochemistry of benzo[a]pyrene-2'-deoxyguanosine adducts affects DNA methylation by SssI and HhaI DNA methyltransferases. FEBS J., 274 (8): 2121-2134. >>

  17. Kazanova E.V., Zubin E.M., Kachalova A.V., Volkov E.M., Oretskaya T.S., Stetsenko D.A., Gottikh M.B. (2007) A convenient solid-phase method for the synthesis of novel oligonucleotide-folate conjugates. Nucleosides, Nucleotides & Nucleic Acids, 26 (10): 1273-1276.

  18. Zatsepin T.S., Oretskaya T.S., Gait M.J., Stetsenko D.A. (2007) Preparation of 2 '-hydrazino oligonucleotides and their reaction with aldehydes and 1,3-diketones. Nucleosides, Nucleotides & Nucleic Acids, 26 (6): 795-798.

  19. Metelev V., Romanenkov A., Kubareva E., Zubin E., Polouchine N., Zatsepin T., Molochkov N., Oretskaya T. (2006) Structure-based cross-linking of NF-kappa B p50 homodimer and decoy bearing a novel 2 '-disulfide trapping site. IUBMB Life, 58 (11): 654-658.

  20. Zatsepin T.S., Gait M.J., Oretskaya T.S., Stetsenko D.A. (2006) Synthesis of 2′-hydrazine oligonucleotides and their efficient conjugation with aldehydes and 1,3-diketones. Tetrahedron Letters, 47 (31): 5515-5518. >>

  21. Agapkina J., Smolov M., Barbe S., Zubin E., Zatsepin T., Deprez E., le Bret M., Mouscadet J.F., Gottikh M. (2006) Probing of HIV-1 integrase/DNA interactions using novel analogs of viral DNA. Journal of Biological Chemistry, 281 (17): 11530-11540.

  22. Smolov M., Gottikh M., Tashlitskii V., Korolev S., Demidyuk I., Brochon J.C., Mouscadet J.F., Deprez E. (2006) Kinetic study of the HIV-1 DNA 3 '-end processing - Single-turnover property of integrase. FEBS Journal, 273 (6): 1137-1151.