DEPARTMENT OF NUCLEIC ACIDS CHEMISTRY
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.