Tatiana S. Oretskaya

Head of Department, Sc.D., Professor  

Department was organized in 1966. Prof. Z.A. Shabarova was the first Head of Department (1966-1999).  From 1999 to present day  Prof. T.S. Otetskaya is a Head of Department..

Department leading researchers: Sc.D. M.B.Gottikh, Ph.D. V.L.Drutsa, Ph.D.  E.A. Kubareva,  Ph.D. M.G. Ivanovskaya, Ph.D. E.A. Romanova.

The principal research efforts are focused on the design and synthesis of new oligonucleotide constructions being of interest for studying nucleic acid-protein interactions, molecular recognition, antisense and sense biotechnology. Our progress in oligonucleotide chemistry and study of chemical reactions in double-stranded DNA and specific nucleic acid-protein complexes allowed us to work successfully in the following directions. 1.    Introduction of new modifications, among them chemically reactive groups, into sugar-phosphate backbone of single- and double-stranded nucleic acids. 2.    The use of preliminarily activated DNA and RNA duplexes for probing the active centers and binding sites of enzymes, human transcription factors and transactivating enzymes of HIV-1 as well as for modulation of gene transcription or as protein “traps”. 3.    Obtaining of oligonucleotide conjugates capable to penetrate through cellular membranes and specifically inhibit translation of mRNA (antisense effect). Creation of oligonucleotide derivatives carrying non-radioactive probes, antibiotics, steroids and intercalates. 4.    Determination of structural basis of specificity and acting mechanism of DNA-recognizing enzymes: restriction endonucleases, DNA-methyltransferases, reparation enzymes, retrovirus integrases; development of novel model systems for study of these enzymes.

Participation in research projects (programs) and grant support. RFBR grants,  RFBR-CNRS grant, RFBR-DFG grant,  program “Universities of Russia”,  Russian Federation Ministry of education and science support,  INTAS Program,  the 6th Framework Program (European Commission),  and  International Network “Biochemistry of Nucleic Acids”.

Main scientific results:
1.    The effective strategy and world-class technology of modified oligonucleotide synthesis were developed. Our multi-purpose synthetic scheme allows to introduce modified residue in process of automated chemical synthesis of oligonucleotides. Chemically modified DNA strand can be widely used in development of novel model systems for study of NA-NA and NA-protein interactions.  
2.    The new and efficient methods of synthesis of modified oligonucleotides containing amino, aldehyde, carboxy, hydrazino, b-diketo, and a-diamino functions in 2’-position of carbohydrate moiety were developed. Using suggested coupling scheme the oligonucleotide conjugates with marker, reporter compounds and peptides were synthesized (Zubin E.M., Romanova E.A., Volkov E.M., Tashlitsky V.N., Korshunova G.A., Shabarova Z.A., Oretskaya T.S. Oligonucleotide-peptide conjugates as potential antisense agents. - FEBS Letters, 1999, v. 456, p. 59-62; Zatsepin T.S., Stetsenko D.A., Arzumanov A.A., Romanova E.A., Gait M.J., Oretskaya T.S. Synthesis of peptide-oligonucleotide conjugates with single and multiple peptides attached to 2'-aldehydes through tiazolidine, oxime and hydrazine linkages. – Bioconjugate Chem., 2002, v. 13, N 4, p. 822-830).  
3.    Mono-modified DNA duplexes containing new types of reactive group in sugar-phosphate moiety were synthesized through combination of automated oligonucleotide synthesis and special post-synthetic procedures. Novel DNA reagents are able to specifically cross-link to amino acids (Lys, His or Cys) in active center or binding site of NA-recognizing proteins. Such type of binding can be achieved with RNA-polymerase, human transcription factor NF-kB, trans-activating protein HIVI, reparation enzyme uracil-DNA-glycosilase, restriction-modification enzymes (Naryshkin N.A., Farrow M.A., Ivanovskaya M.G., Oretskaya T.S., Shabarova Z.A., Gait M.J. Chemical cross-linking of the human immunodeficiency virus type 1 Tat protein to synthetic models of the RNA recognition sequence TAR containing site-specific trisubstituted pyrophosphate analogues. - Biochemistry, 1997, v. 36, p. 3496-3505; Metelev V.G., Borisova O.A., Volkov E.M., Oretskaya T.S., Dolinnaya N.G. New chemically reactive dsDNAs containing single internucleotide monophosphoryldithiolinks: reactivity of 5'-mercaptooligodeoxyribonucleotides. – Nucleic Acids Res., 2001, v. 29, N 19, p. 4062-4069; Metelev V.G., Kubareva E.A., Vorob'eva O.V., Romanenkov A.S., Oretskaya T.S. Specific conjugation of DNA binding proteins to DNA templates through thiol-disulfide exchange. - FEBS Letters, 2003, v. 538, p. 48-52). The methodologies of peptide-oligonucleotide conjugate isolation and   cross-linking site determination were employed in the case of restriction endonucleases. The technique suggested can be applied to identify the NA binding site of any protein (Kubareva E. A., Thole H., Karyagina A. S., Oretskaya T. S., Pingoud A., Pingoud V. Identification of a base-specific contact between the restriction endonuclease SsoII and its recognition sequence by photocross-linking. - Nucleic Acids Res., 2000, v. 28, p. 1085-1091; Sudina A.E., Zatsepin T.S., Pingoud V., Pingoud A., Oretskaya T.S., Kubareva E.A.  Affinity modification of restriction endonucleases SsoII by 2’-aldehyde ds DNAs. -  Biochemistry (Mosc)., 2005, v. 70, p. 941-947; Pingoud V., Geyer H., Geyer R., Kubareva E., Bujnicki J.M., Pingoud A. Identification of base-specific contacts in protein–DNA complexes by photocross-linking and mass spectrometry: a case study using the restriction endonuclease SsoII. - Mol. BioSyst.,  2005, v. 1, p. 135-141).  
4.    A number of new antisense oligonucleotides and oligonucleotide-peptide conjugates were obtained. The type of conjugate structure was found to affect on the efficacy and specificity of oligonucleotide (or conjugate)-RNA interaction, stability to nuclease degradation in various biological systems, cell penetration and inhibition of gene expression (Gottikh M.B., Fedorova O.A., Baud-Demattei M-V., Giorgi-Renault S., Bertrand J-R., Shabarova Z.A., Malvy C. Alpha-beta chimeric oligonucleotides form a new stable "snail-like" structure. - J. Amer. Chem. Soc., 1996, v. 118, p. 2126-2130; Helin V., Gottikh M.B., Mishal Z., Subra F., Malvy C., Lavignon M. Cell cycle-dependent distribution and specific inhibitory effect of vectorized antisense oligonucleotides in cell culture. - Biochem.  Pharmacol., 1999, v. 58, p. 95-107).
5.    Short single stranded oligonucleotides containing modified heterocyclic bases or aromatic groups were found to be effective inhibitors of HIV I integrase. Mechanism of modified oligonucleotide - integrase interaction was precisely studied and includes the reaction between oligomer and integrase – viral DNA complex which results in quick and effective complex degradation (Brodin P., Pinskaya M.D., Volkov E.M., Romanova E.A., Leh H., Auclair C., Mouscadet J.-F., Gottikh M.B. Branched Oligonucleotide-Intercalator Conjugate Forming a Parallel Stranded Structure Inhibits HIV-1 Integrase. - FEBS Lett., 1999, v. 460, p. 270-274; Brodin P., Pinskaia M. D., Buckle M., Parsch U., Romanova E., Engels J., Gottikh M., Mouscadet J. F. Disruption of integrase/DNA complexes by short 6-oxocytosine containing oligonucleotide. -  Biochemistry, 2002, v. 41, p. 1529-1538).
6.    Using oligonucleotide-directed mutagenesis technique a series of sigma-subunit RNA-polymerase E.coli, containing amino acid replacements were obtained. These mutants can be used in determination and study of transcription initiation mechanism which is one of the key process in cell vital activity (Sergeev V.N., Chalov S.E., Drutsa V.L., Gromova E.S. A study of the Asp110-Glu112 region of EcoRII restriction endonuclease by site-directed mutagenesis. - Biochemistry (Moscow), 2000, v. 65, p. 1006-1010; Koroleva O.N., Drutsa V.L. Investigation of transcription of DNA templates with pyrophosphate internucleotide groups. – Vestnik Mosk. Univer., 2000, ser. 2 Khimiya, v.44, p.203-207).  
7. The first detailed biochemical analysis of archeal restriction enzymes has been done. It was demonstrated that a relationship exists between orthodox type II, type IIE and type IIF restriction endonucleases. These subfamilies share a not too distant common ancestor, which presumably was a homodimeric enzyme (Pingoud V., Kubareva E., Stengel G., Friedhoff P., Bujnicki J. M., Urbanke C., Sudina A., Pingoud A. Evolutionary relationship between different subgroups of restriction endonucleases. - J. Biol. Chem., 2002, v. 277, p. 14306-14314; Pingoud V., Conzelmann C., Kinzebach S, Sudina A., Metelev V., Kubareva E.,Bujnicki J.M., Lurz R., Luder G., Xu S.Y.,  Pingoud A. PspGI, a type II restriction endonuclease from the extreme thermophile Pyrococcus sp.: structural and functional studies to investigate an evolutionary relationship with several mesophilic restriction enzymes. J. Mol. Biol., 2003, v. 329, p. 913 – 929; Pingoud V., Sudina A., Geyer H., Bujnicki J.M., Lurz R., Lüder G., Morgan R., Kubareva E., Pingoud A. Specificity changes in the evolution of type II restriction endonucleases: a biochemical and bioinformatic analysis of restriction enzymes that recognize unrelated sequences. - J. Biol. Chem., 2005, v. 280, p. 4289-4298).

Main research publications:
1.    Shabarova ZA. Chemical ligation an alternative method for assembly of natural and modified DNA duplexes. Physicochemical Biology Reviews (ed. by VP Skulachev, Soviet scientific reviews). 1984; 5: 1-51. Review.
2.    Shabarova ZA. Chemical development in the design of oligonucleotide probes for binding to DNA and RNA. Biochimie. 1988; 70: 1323-34. Review.
3.   Shabarova ZA, Dolinnaya NG, Drutsa VL, Melnikova NP, Purmal AA. DNA-like duplexes with repetitions. III. Efficient template-guided chemical polymerization of d(TGGCCAAGCTp). Nucleic Acids Res. 1981; 9: 5747-61.
4.    Sokolova NI, Ashirbekova DT, Dolinnaya NG, Shabarova ZA. Chemical reactions within DNA duplexes. Cyanogenbromide as an effective oligonucleotide coupling agent. FEBS Lett. 1988; 232: 153-5.
5.    Shabarova ZA, Merenkova IN, Oretskaya TS, Sokolova NI, Skripkin EA, Alex-eyeva EV, Balakin AG, Bogdanov AA. Chemical ligation of DNA: the first non-enzymatic assembly of a biologically active gene. Nucleic Acids Res. 1991; 19: 4247-51.
6.    Koroleva ON, Drutsa VL. In vivo promoter activity of the synthetic Pribnow box. FEBS Lett. 1991; 278: 207-10.
7.    Kubareva EA, Petrauskene OV, Karyagina AS, Tashlitsky VN, Nikolskaya II, Gromova ES. Cleavage of synthetic substrates containing non-nucleotide inserts by restriction endonucleases. Change in the cleavage specificity of endonuclease SsoII. Nucleic Acids Res. 1992; 20: 4533-8.
8.    Dolinnaya NG, Blumenfeld M, Merenkova IN, Oretskaya TS, Krynetskaya NF, Ivanovskaya MG. Oligonucleotide circularization by template-directed chemical ligation. Nucleic Acids Res. 1993; 20: 5403-7.
9.    Gottikh MB, Bertrand J-R, Baud-Demattei M-V, Lescot E, Giorgi-Renault S, Sha-barova Z, Malvy C. α-β-chimeric antisense oligonucleotides: synthesis and nuclease resistance in biological media. Antisense Res. and Dev. 1994; 4: 251-8.
10.    Gottikh MB, Fedorova OA, Baud-Demattei M-V, Giorgi-Renault S, Bertrand J-R, Shabarova ZA, Malvy C. α-β-chimeric oligonucleotides form a new stable “snail-like” structure. J. Amer. Chem. Soc. 1996; 118: 2126-30.
11.    Naryshkin NA, Farrow MA, Ivanovskaya MG, Oretskaya TS, Shabarova ZA, Gait MJ. Chemical cross-linking of the human immunodeficiency virus type 1 Tat pro-tein to synthetic models of the RNA recognition sequence TAR containing site-specific trisubstituted pyrophosphate analogues. Biochemistry. 1997; 36: 3496-505.
12.    Kozlov IA, Kubareva EA, Ivanovskaya MG, Shabarova ZA. Design of new re-agents on the base of DNA duplexes for irreversible inhibition of transcription fac-tor NF-kB. Antisense and Nucleic Acid Drug Develoment. 1997; 7: 279-89.
13.    Kubareva EA, Vasilenko NL, Vorobjeva OV, Volkov EM, Oretskaya TS, Korshunova GA, Nevinsky GA. Role of DNA definite structural elements in interaction with repair enzyme uracil-DNA glycosylase. Biochemistry and Molecular Biology International. 1998; 46: 597-606.
14.    Helin V, Gottikh M, Michal Z, Subra F, Malvy C, Lavignon H. Cell cycle-dependent distribution and specific inhibitory effect of vectorized antisense oli-gonucleotides in cell culture. Biochemical Pharmacology. 1999; 58: 95-107.
15.    Zubin EM, Romanova EA, Volkov EM, Tashlitsky VN, Korshunova GA, Sha-barova ZA, Oretskaya TS. Oligonucleotide-peptide conjugates as potential an-tisense agents. FEBS Lett. 1999; 456: 59-62.
16.    Maksimenko AV, Volkov EM, Bertrand JR, Porumb H, Malvy C, Shabarova ZA, Gottikh MB. Targeting of single-stranded DNA and RNA containing adjacent pyrimidine and purine tracts by triple helix formation with circular and clamp oli-gonucleotides. Eur J Biochem. 2000; 267: 3592-603.
17.    Kubareva EA, Thole H, Karyagina AS, Oretskaya TS, Pingoud A, Pingoud V. Identification of a base-specific contact between the restriction endonuclease SsoII and its recognition sequence by photocross-linking. Nucleic Acids Res. 2000; 28: 1085-91.
18.    Metelev VG, Borisova OA, Volkov EM, Oretskaya TS, Dolinnaya NG. New chemically reactive dsDNAs containing single internucleotide monophosphoryldi-thiolinks: reactivity of 5'-mercaptooligodeoxyribonucleotides. Nucleic Acids Res. 2001; 29: 4062-9.
19.    Zatsepin TS, Stetsenko DA, Arzumanov AA, Romanova EA, Gait MJ, Oretskaya TS. Synthesis of peptide-oligonucleotide conjugates with single and multiple pep-tides attached to 2'-aldehydes through triazolidine, oxime and hydrazine linkages. Bioconjugate Chem. 2002; 13: 822-30.
20.    Kachalova AV, Stetsenko DA, Romanova EA, Tashlitsky VN, Gait MJ, Oretskaya TS. A new and efficient method for synthesis of 5'-conjugates of oligonucleotides through amide-bond formation on solid phase. Helvetica Chimica Acta. 2002; 85: 2409-16.
21.    Kubareva EA, Walter J, Karyagina AS, Vorob'eva OV, Lau PCK, Trautner T. De-termination of methylation site of DNA-methyltransferase NlaX by a hybrid     method. BioTechniques. 2002; 33: 526-531.
22.    Brodin P, Pinskaia MD, Buckle M, Parsch U, Romanova E, Engels J, Gottikh M, Mouscadet JF. Disruption of integrase/DNA complexes by short 6-oxocytosine con-taining oligonucleotide. Biochemistry. 2002; 41: 1529-38.
23.    Turutin DV, Kubareva EA, Pushkareva MA, Ullrich V, Sud'ina GF. Activation of NF-B transcription factor in human neutrophils by sulphatides and L-selectin cross-linking. FEBS Lett. 2003; 536: 241-5.
24.    Metelev VG, Kubareva EA, Vorob'eva OV, Romanenkov AS, Oretskaya TS. Spe-cific conjugation of DNA binding proteins to DNA templates through thiol-disulfide exchange. FEBS Lett. 2003; 538: 48-52.
25.    Kuznetsova S, Rykhlevskaya A, Taranenko M, Sidorkina O, Oretskaya T, Laval J. Use of crosslinking for revealing the DNA phosphate groups forming specific con-tacts with the E. coli FPg protein. Biochimie. 2003; 85: 511-9.
26.    Lesage D, Metelev V, Borisova O, Dolinnaya N, Oretskaya T, Baran-Marszak F, Taillandier E, Raphael M, Fagard R. Specific covalent binding of a NF-kB decoy hairpin oligonucleotide targeted to the p50 subunit and induction of apoptosis. FEBS Lett. 2003; 547: 115-8.
27.    Maksimenko AV, Mandrouguine VA, Gottikh MB, Bertrand J-R, Majoral J-P, Malvy C. Optimisation of dendrimer-mediated gene transfer by anionic oligomers. J. Gene Medicine. 2003; 5: 61-71.
28.    Ivanov S, Alekseev Y, Bertrand J-R, Malvy C, Gottikh MB. Formation of stable triplexes between purine RNA and pyrimidine oligodeoxyxylonucleotides. Nucleic Acids Res. 2003; 31: 4256-63.

Main last publications:
1.    Turutin DV, Kubareva EA, Pushkareva MA, Ullrich V, Sud'ina GF. Activation of NF-B transcription factor in human neutrophils by sulphatides and L-selectin cross-linking. FEBS Lett. 2003; 536: 241-5.
2.    Metelev VG, Kubareva EA, Vorob'eva OV, Romanenkov AS, Oretskaya TS. Spe-cific conjugation of DNA binding proteins to DNA templates through thiol-disulfide exchange. FEBS Lett. 2003; 538: 48-52.
3.    Zatsepin TS, Romanova EA, Stetsenko DA, Gait MJ, Oretskaya TS. Synthesis of 2’-modified oligonucleotides containing aldehyde or ethylenediamine grooups. Nucleosides Nucleotides Nucleic Acids. 2003; 22: 1383-5.
4.    Lesage D, Metelev V, Borisova O, Dolinnaya N, Oretskaya T, Baran-Marszak F, Taillandier E, Raphael M, Fagard R. Specific covalent binding of a NF-kB decoy hairpin oligonucleotide targeted to the p50 subunit and induction of apoptosis. FEBS Lett. 2003; 547: 115-8.
5.    Sumbatyan N, Maksimenko A, Gottikh M, Malvy C, Korshunova G. Synthesis and hybridization properties of oligonucleotide analogues containing ornithine back-bone modified with nucleoalanines. Nucleosides Nucleotides Nucleic Acids. 2003; 22: 1077-9.
6.    Ivanov S, Alekseev Y, Bertrand J-R, Malvy C, Gottikh MB. Formation of stable triplexes between purine RNA and pyrimidine oligodeoxyxylonucleotides. Nucleic Acids Res. 2003; 31: 4256-63.
7.    Sudina A, Volkov E, Oretskaya T, Naryshkin N, Ivanovskaya M, Kubareva E. De-tection of glycosylase, endonuclease and methyltransferase enzyme activities using immobilized oligonucleotides. IUBMB Life. 2004; 56; 139-43.
8.    Pinskaya M, Romanova E, Volkov E, Deprez E, Leh H, Brochon JC, Mouscadet JF, Gottikh M. HIV-1 integrase complexes with DNA dissociate in the presence of short oligonucleotides conjugated to acridine. Biochemistry. 2004; 43: 8735-43.
9.    Agapkina J, Smolov M, Zubin E, Mouscadet JF, Gottikh M. HIV-1 integrase can process a 3'-end crosslinked substrate. Eur J Biochem. 2004; 271: 205-11.