[Oligomerization of site-specific nicking endonuclease BspD6I at high protein concentrations].

Ability of site-specific nickase BspD6I (Nt.BspD6I) to oligomerize at concentrations > or = 0.5 microM (> or = 0.035 mg/mL) is studied. Three states of Nt.BspD6I are registered via electrophoretic studies both in the presence and in the absence of DNA. Estimation of their molecular mass allows assigning them as a monomer, a dimer and a trimer. Both dimeric and monomeric Nt.BspD6I are shown to hydrolyze its DNA substrate with the identical specificity. Calculation of the electrostatic potential distribution on the Nt.BspD6I globule surface shows that the protein molecule is a dipole. The Nt. BspD6I oligomeric forms are likely to be the result of ionic protein interactions.

[2'-Modified oligoribonucleotides, containing 1,2-diol and aldehyde groups. Synthesis and properties].

1,2-Diol-oligoribonucleotides were prepared using fully protected 2'-O-[2-(2,3-dihydroxypropyl)amino-2-oxoethyl]uridine 3'-phosphoramidite. Incorporation of the 2'-modified uridine residue into oligonucleotide chains does not significantly affect the thermal stability of RNA and RNA-DNA duplexes. Periodate oxidation of the 1,2-diol results in reactive 2'-aldehyde oligoribonucleotides. Further application of these oligonucleotides for cross-linking with bacterial ribonuclease P was investigated.

[Secondary structure of SsoII-like (cytosine-5)-DNA methyltransferases N-terminal region determined by circular dichroism spectroscopy].

(Cytosine-5)-DNA methyltransferase SsoII (M.SsoII) has a long N-terminal region (1-71 residues) preceding the sequence with conservative motifs, which are characteristic for all DNA methyltransferases of such kind. The presence of this region provides M.SsoII capability to act as a transcription regulator in SsoII restriction-modification system. To perform its regulatory function, M.SsoII binds specifically to a 15-mer inverted repeat in the promoter region of SsoII restriction-modification system genes. In the present work, properties of the protein delta(72-379)M.Ecl18kI are studied, which is a deletion mutant of the SsoII-like DNA-methyltransferase M.Ecl18kI and is homologous to M.SsoII N-terminal region. delta(72-379)M.Ecl18kI capability to bind specifically a DNA duplex containing the regulatory site is demonstrated. However, such a binding takes place only in the presence of high protein excess relative to DNA, which could indicate an altered structure in the deletion mutant in comparison with the full-length M.SsoII. Circular dichroism spectroscopy demonstrated that delta(72-379)M.Ecl18kI has a strongly pronounced secondary structure and contains 32% a-helices and 20% beta-sheets. Amino acid sequences alignment of M.SsoII N-terminal region and transcription factors of known spatial structure is made. An assumption is made how alpha-helices and beta-sheets are arranged in M.SsoII N-terminal region.

[2'-aldehyde oligonucleotides: synthesis and use for affinity modification of DNA-recognizing proteins].

Oligonucleotides with 1,2-diol grouping were prepared from 2'-O-[2-(2,3-dihydroxypropyl)amino-2-oxo-ethyl]uridine 3'-phosphoramidite. The thermal stability of modified DNA duplexes and their ability to form complexes with the p50 subunit of the NF-kappaB transcription factor and (cytosine-5)-DNA methyltransferase SsoII were studied. The periodate oxidation of the l,2-diol grouping of the oligonucleotides resulted in reactive 2'-aldehyde derivatives. The opportunity of their use for the affinity modification of DNA-recognizing proteins was studied.

[New derivatives of azobenzene for the directed modification of proteins].

Derivatives of azobenzene which contained a maleimide group in one of the benzene rings (for binding to a protein cysteine residue) and maleimide, hydroxyl, or carboxyl substitutes in another benzene ring were synthesized. The reactivity of these compounds towards a cysteine residue of a protein and their optical properties in a free state and after their attachment to the mutant forms of the SsoII restriction endonuclease were studied.

[Synthesis and characteristics of modified DNA fragments containing thymidine glycol residues].

Chemical synthesis of a series of modified oligodeoxyribonucleotides containing one or two residues of thymidine glycol (5,6-dihydro-5,6-dihydroxythymidine), the main product of oxidative DNA damage, is described. The thermal stability of DNA duplexes containing thymidine glycol residues was studied using UV spectroscopy. Introduction of even one thymidine glycol residue into the duplex structure was shown to result in its significant destabilization. Data on the interaction of DNA methyltransferases and type II restriction endonucleases with DNA ligands containing oxidized thymine were obtained for the first time. Introduction of a thymidine glycol residue into the central degenerate position of the recognition site of restriction endonuclease SsoII was found to result in an increase in the initial hydrolysis rate of the modified duplex in comparison with that of the unmodified structure. The affinity of C5-cytosine methyltransferase SsoII for the DNA duplex bearing thymidine glycol was found to be twofold higher than for the unmodified substrate. However, such a modification of the DNA ligand prevents its methylation. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.

[Hybridase cleavage of RNA. V. Complementary precision of cleavage]. / Gibridaznoe rasshcheplenie RNK. V. Komplementatsionnaia tochnost' rasshchepleniia.

The efficiency of the cleavage of RNA involved in perfect as well as imperfect hybrid duplexes composed of three components: (1) homogeneous RNA's or polyribonucleotides; (2) corresponding complementary synthetic oligodeoxyribonucleotides; (3) E. coli RNase H was investigated. The predominant RNA hydrolysis was shown to take place within the perfect hybrid duplexes formed by the target RNA and the complementary oligodeoxyribonucleotide probes. RNase H was found to cleave effectively a number of imperfect hybrid duplexes containing a central base pair mismatch.

[A model prokaryotic promotor. II. Function of designs with mutations in the "-35" region]. / Model'nyi prokarioticheskii promotor. II. Funktsionirovanie konstruktsii s mutatsiiami v oblasti "-35".

A series of DNA duplexes corresponding to the E. coli consensus promoter with random hexanucleotides in the -35 region have been synthesized and characterized. The library of recombinant plasmids with synthetic promoter-like inserts, oriented in the direction of the marker gal operon of the initial vector, have been obtained. Analysis of the library on indicator medium and S1 mapping of the in vivo transcription initiation points in several plasmids have shown that most constructions exhibit promoter properties, and the structures of their -35 regions may be varied.

[DNA duplexes containing 2'-deoxy-2'-iodoacetamidouridine as reagents for affinity modification of proteins]. / DNK-dupleksy, soderzhashchie 2'-dezoksi-2'-iodatsetamidouridin, kak reagenty dlia affinnoi modifikatsii belkov.

DNA duplexes containing the iodoacetamido group at position 2' of the ribose moiety were proposed for affinity modification of Cys in DNA-binding proteins. Reactive DNA derivatives were obtained with iodoacetic anhydride and synthetic oligodeoxyribonucleotides containing 2'-amino-2'-deoxyuridine in place of thymine at various positions. The derivatives were tested for reaction with amino acids and peptides and shown to specifically interact with Cys-containing proteins. The possibility of using the modified DNA duplexes to probe the protein SH group close to the DNA sugar-phosphate backbone in DNA-protein complexes was demonstrated with the example of subunit p50 of human transcription factor NF-kappa B.

[Covalent binding of Cys142 from SsoII methyltransferase with DNA duplexes, containing a phosphoryldisulfide internucleotide group]. / Kovalentnoe sviazyvanie Cys142 metiltransferazy SsoII s DNK-dupleksami, soderzhashchimi fosforildisulfidnuiu mezhnukleotidnuiu grupppu.

DNA duplexes containing a single phosphoryldisulfide link in place of the natural internucleotide phosphodiester bond were employed in affinity modification of Cys142 in cytosine-C5 DNA methyltransferase SsoII (M.SsoII). The possibility of duplex-M.SsoII conjugation as a result of disulfide exchange was demonstrated. The crosslinking efficiency proved to depend on the DNA primary structure, modification position, and the presence of S-adenosyl-L-homocysteine, a nonreactive analog of the methylation cofactor. The SH group of M.SsoII Cys142 was assumed to be close to the DNA sugar-phosphate backbone in the DNA-enzyme complex.

[Automated synthesis of oligodeoxyribonucleotides with terminal phosphate groups]. / Avtomticheskii sintez oligodezoksiribonukleotidov s kontsevymi fosfatnymi gruppami.

3'- and 5'-phosphorylated oligodeoxyribonucleotides have been synthesized on the "Victoria-4M" automatic synthesizer by phosphoramidite method. Two approaches have been suggested: introduction of a terminal ribo-unit as a potential source of phosphate group or the use of hydroxyl-containing polymer supports which loose the end product of the synthesis via beta-elimination reaction. Yields of oligonucleotides obtained according to both schemes proved to be almost identical. Using the first approach, oligonucleotides containing units with altered configuration of the sugar (xylo-thymidine and arabino-uridine) have been obtained.

[Synthesis of oligodeoxyribonucleotides containing 5-fluoro-2'-deoxyribocytidine]. / Sintez oligodezoksiribonukleotidov, soderzhashchikh 5-ftor-2'-dezoksiribotsitidin.

A synthesis of the phosphoroamidite derivative of 5-fluoro-2'-deoxycytidine which allows one to introduce the modified nucleoside residue into the 5'-position of the oligodeoxyribonucleotide by the standard solid phase phosphoroamidite method, has been carried out. Oligonucleotides with 5-fluoro-2'-deoxycytidine residues in various positions of the DNA strand are obtained by the combination of chemical and enzymatic syntheses.

[Chemical reactions in double-helical nucleic acids. XIV. Effectiveness of forming phosphodiester bonds between various nucleotide units]. / Khimicheskie reaktsii v dvuspiral'nykh nukleinovykh kislotakh. XIV. Effektivnost' obrazovaniia fosfodiéfirnykh sviazei mezhdu razlichnymi nukleotidnymi zven'iami.

Efficiency of the template-directed condensation of oligonucleotides depends on the nature of the nucleotide units to be joined. Fourteen out of sixteen dinucleotide combinations in double-stranded DNA were examined. Alterations of the reaction efficiency are identical for cyanogen bromide and 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide coupling reagents. Dependence of the chemical ligation efficiency on the DNA sequence-specific local conformation is discussed.

[Synthesis and properties of covalently-closed DNA duplexes containing pyrophosphate and and substituted pyrophosphate internucleotide groups]. / Sintez i svoistva kovalentno zamknutykh DNK-dupleksov, soderzhashchikh pirofosfatnuiu i zameshchennuiu pirofosfatnuiu mezhnukleotidnye gruppirovki.

A new method for the efficient synthesis of covalently closed DNA duplexes (DNA dumbbells) and the introduction of pyrophosphate and substituted pyrophosphate internucleotide groups into their structure is proposed. The method is based on chemical ligation in DNA duplexes that are formed by a polynucleotide the ends of which are brought together due to the introduction of the minihairpin structure [sequence: see text]. DNA dumbbells containing a pyrophosphate (substituted pyrophosphate) group result from the interaction as being between the 3'-terminal phosphate (methylphosphate) group of the polynucleotide and the 5'-terminal phosphate group of deoxyguanosine of the minihairpin sequence, which flanks the polynucleotide from the 5' end. 1-Ethyl-3-(3'-dimethylaminopropyl) carbodiimide was used as a condensing agent. The yield of covalently closed 42-mer DNA duplexes containing a pyrophosphate group was 98%, that of duplexes with a substituted pyrophosphate group was 25%. The reactivity of the substituted pyrophosphate group incorporated into DNA dumbbells was studied. It is shown that the group efficiently interacts with nucleophiles in an aqueous medium at pH 8.0.

[Chemical reactions in double-helical nucleic acids. XVI. Synthesis of DNA-duplexes containing regularly repeating transverse covalent cross- links]. / Khimicheskie reaktsii v dvuspiral'nykh nukleinovykh kislotakh. XVI. Sintez DNK-dupleksov, soderzhashchikh reguliarno povtoriaiushchiesia poperechnye kovalentnye sshivki.

Two self-complementary decadeoxyribonucleotides TAATGC*ATTA (where C* is a derivative of 5-methyl cytosine with a carboxy- or aminofunction attached through a spacer to the exocyclic amino group) were synthesized. Carbodiimide induced condensation of the amino and carboxyl groups in the opposite strands to give the crosslinks with a yield up to 20%. Cross-linking of two opposite strands in the duplex formed by the self-complementary aliphatic amino group-containing decanucleotide was performed with the use of glutaric aldehyde with a similar efficiency. The structure of the dimers obtained and position of the crosslinks were confirmed by the Maxam--Gilbert method. Efficiencies of the T4 DNA ligase-induced polycondensations of the double-stranded modified decanucleotides and of the cross-linked products differed significantly.

[Cleavage by restriction endonucleases MvaI and EcoRII of substrates modified in amino groups of heterocyclic bases]. / Osobennosti rasshchepleniia éndonukleazami restriktsii MvaI i EcoRII substratov, modifitsirovannykh po aminogruppam geterotsiklicheskikh osnovanii.

14-membered DNA-duplexes containing modified nucleoside residues, viz 4-N-methyldeoxycytidine (m4dC), 6-N-methyldeoxyadenosine (m6dA) or deoxyinosine (dI), in only one strand of the recognition site (CCA/TGG) of MvaI and EcoRII endonucleases were synthesized. It was shown that MvaI and EcoRII endonucleases interact with the exocyclic amino groups of the external dC residues and of the central dA residue of the recognition site exposed into the DNA major groove. These endonucleases which are isochizomers were found to possess different mechanisms of substrate cleavage. The ability of MvaI endonuclease to hydrolyze only unmodified strand of methylated duplexes allows one to make site-directed single-strand nicks in double-stranded DNA. Elimination of the 2-NH2-group located in the minor groove of DNA by substituting dI for dG had little, if any, effect on the hydrolytic activity of EcoRII and MvaI endonucleases.

[RNA synthesis using T7 phage RNA polymerase: transcription of synthetic DNA templates in solution and on polymer support]. / Sintez RNK s pomoshch'iu RNK-polimerazy faga T7: transkriptsiia sinteticheskikh DNK-matrits v rastvore i na polimernom nositele.

Transcription of synthetic DNA by T7 RNA polymerase was used to obtain oligoribonucleotides of defined sequence. The enzyme's ability to transcribe DNA immobilized on hydrazide-sepharose was revealed. DNA templates used in such synthesis can be constructed by means of enzymatic (DNA ligase) or chemical ligation (cyanogen bromide).

[Study of the mechanism of chemical ligation of DNA by cyanogen bromide]. / Izuchenie mekhanizma khimicheskogo ligirovaniia DNK pod deistviem bromtsiana.

The mechanism of chemical ligation with cyanogen bromide in the presence of an N-substituted morpholine was studied. Addition of the cyano group to the tertiary nitrogen atom of the N-substituted morpholine with the formation of a quaternary ammonium cation is shown to be the first step of the reaction; it is this cation that activates the oligonucleotide phosphate group. This method of activation can be used to obtain phosphodiester derivatives of nucleotides without DNA duplex. Optimal conditions of the chemical ligation were selected.

[Synthesis and properties of oligodeoxyribonucleotids with mono- and diphosphoryldisulfide internucleotide groupings]. / Sintez i svoistva oligodezoksiribonukleotidov s mono- i difosforildisul'fidnymi mezhnukleotidnymi gruppirovkami.

The synthesis of oligodeoxyribonucleotides bearing mono- and diphosphoryldisulfide internucleotide links was optimized. Oligonucleotide 3'-thiophosphorothioates were modified using the thiophosphoryl-disulfide exchange with preactivated 5'-deoxy-5'-mercaptooligonucleotides or 5'-phosphorothioate derivatives both with and without a complementary template. The lack of template was shown to differently affect the product ratio (homo- and heterodimers) in the reactions of mono- and diphosphoryldisulfide-containing oligonucleotides. A replacement of one natural phosphodiester bond in 15-16-mer duplexes by a mono- or diphosphoryldisulfide group causes a slight thermal destabilization of the corresponding duplex. The disulfide recombination of the resulting compounds was studied.

[Chemical reactions in double-helical nucleic acids. XV. The effect of local DNA structure on the efficacy of chemical ligation]. / Khimicheskie reaktsii v dvuspiral'nykh nukleinovykh kislotakh. XV. Vliianie lokal'noi struktury DNK na éffektivnost' khimicheskogo ligirovaniia.

A series of model DNA-duplexes with a single break in one of the chains were studied. The efficiency of the new internucleotide bond formation by means of chemical ligation was shown to depend on the DNA structure in the nick site, decreasing in the following order: pyrimidine-pyrimidine > purine-pyrimidine > pyrimidine-purine > > purine-purine. This relationship is the most pronounced in duplexes with 5'-hydroxyl group as 3'-phosphate acceptor. For duplexes, containing non-complementary base pairs in the reaction centre, the yield of ligation products also depends on the nature of the interacting nucleotides.