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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.

The functional groups of the DNA methylation site that are involved in the DNA interaction with methyltransferase SsoII at the recognition stage were identified. The contacts in the enzyme-substrate complex were analyzed in the presence of S-adenosyl-L-homocysteine using the interference footprinting assay with formic acid, hydrazine, dimethyl sulfate, or N-ethyl-N-nitrosourea as a modifying reagent. It was shown that the replacement of the central A.T by the G.C pair in the methylation site did not affect the enzyme-DNA interaction, whereas the use of a substrate with one chain methylated (monomethylated substrate) instead of the unmethylated substrate dramatically changes the DNA contacts. The binding constants of unmethylated and monomethylated substrates with methyltransferase SsoII in the presence of S-adenosyl-L-homocysteine were calculated.

A method for directional introduction of oleylamine residues to any position of oligodeoxyribonucleotides during their automated synthesis was developed. The presence of oleylamine residues in 3'- or 5'-terminal nucleotides was shown to have no effect on the thermodynamic stability of DNA duplexes formed by such oligonucleotides and the complementary sequences. The rate of the snake venom phosphodiesterase hydrolysis of oligonucleotides containing oleylamine residues in the 3'-terminal units was shown to be markedly lower than that of natural oligonucleotides.

A rapid and effective method of testing of a repair enzyme, uracil-DNA-glycosylase, was proposed. As a substrate, a deoxyuridine-containing 5'-32P-labeled deoxyoligonucleotide covalently attached to a polystyrene support (Tenta Gel S-NH2) was used. The ammonia cleavage of the apyrimidine site formed in the enzymic reaction followed by the transition of the labeled oligonucleotide fragment from the solid phase into solution allowed the detection of the enzymic activity.

A method for preparing a modified derivative of 2'-amino-2'-deoxy-arabino-adenosine ready for directed insertion into an oligonucleotide chain during solid-phase synthesis was elaborated. A series of the title oligonucleotides (6-25-mers) containing a 2'-amino-2'-deoxy-arabino-adenosine fragment were prepared. A high reactivity of the 2'-amino group during the acylation with carboxylic acid anhydrides was demonstrated. It was shown that the insertion of the modified fragments into oligonucleotides did not inhibit the formation of the DNA duplex.

Fragments of double-stranded RNAs and mixed double-stranded ribo/deoxyribooligonucleotides containing an internucleotide trisubstituted pyrophosphate bond in a predetermined position of the sugar-phosphate backbone were synthesized. The modified internucleotide bond was created between synthetic oligonucleotides by chemical ligation with carbodiimide. The chemical ligation conditions were optimized for a series of duplexes that differed in the content and mutual arrangement of ribo- and deoxyribonucleotide blocks. The yield of oligonucleotides containing the trisubstituted internucleotide pyrophosphate bond was 90% in an all-deoxy duplex and 15-68% in ribooligonucleotide-containing duplexes. The modified duplexes thus obtained are relatively stable in an aqueous solution at pH 5.5-7.5, whereas the modified internucleotide bond is cleaved by amines to form phosphamide derivatives of the corresponding oligonucleotides. Automated solid-phase synthesis of oligonucleotide 3'-ethylphosphates is described.

Synthesis of cross-linked modified DNA duplexes is described. The structure of the duplexes was confirmed by digestion with the AluI restriction endonuclease. Thermostability and resistance to enzymatic hydrolysis of the cross-linked duplexes were studied.

A method for the synthesis of DNA duplex with covalently linked strands was elaborated, and the thermal and hydrolytic stability of the duplex was studied. The strands were connected via an amide bond between carboxyl and aliphatic amino groups in the presence of water-soluble carbodiimide. For this purpose, a series of modified 5- to 26-mer oligonucleotides with primary amino or carboxyl group were prepared, and their properties were investigated.

Techniques for preparing catalytically active corrin complexes of cobalt with monofunctional e-carboxyl- and e-amino groups in a macroligand are suggested. Two methods of the covalent attachment of such Co-corrin complexes to the terminal 3'- and 5'-phosphate groups of oligodeoxyribonucleotides were studied: the introduction of the complex to the oligonucleotide after oligonucleotide synthesis in aqueous solution and in the course of automated solid-phase oligonucleotide synthesis. Introducing of the Co-corrin complex during solid-phase synthesis was more efficient. It was demonstrated that the oligonucleotide probe thus obtained was able to act as a nuclease in the presence of ascorbic acid, the location of the clearage sites being determined by the addressing oligonucleotide.

Effective methods of multiple incorporations of nucleotides with the inverted configuration of C2' hydroxyl group have been developed. The presence of tU and tC as 3'-terminal oligonucleotide modifications is shown to increase their resistance to snake venom phosphodiesterase. The obtained compounds are promising for the use in antisense biotechnology.

Mixed oligomers, representing oligonucleotides connected with long non-nucleotide spacers, have been synthesized using phosphoramidite chemistry. The oligonucleotide moieties of the mixed oligomers fully or partially correspond in the structure to the consensus elements of -35 (TTGACA) and -10 (TATAATG) regions of prokaryotic promoters. The non-nucleotide spacers, approximating in size 17-membered DNA fragments, were synthesized using phosphoramidite derivatives of polyethylene glycol (PEG600), tetraethylene glycol or dodecanediol. It is shown that the oligonucleotide moieties of the mixed oligomers can form "normal" DNA like antiparallel complementary complexes, being the substrates of T4 DNA ligase. To obtain the DNA-like polymers with alternating natural and non-natural regions or cyclic structures, the enzymatic ligation of different complexes of the oligomers synthesized was studied.

An effective method for the oligonucleotide cyclization using BrCN-induced chemical ligation was developed. The novel idea to incorporate non-nucleotide inserts makes the process of cyclizations independent of the inner secondary structure of the linear precursor. An set of assays were developed to confirm the cyclic structure of the compounds obtained.

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.

The synthesis, by means of the standard phosphoramidite chemistry, of modified oligodeoxynucleotides (5-20 residues) containing 2'-amino-2'-deoxypyrimidine nucleosides has been carried out, and their ability to form duplexes with complementary DNA has been investigated. A high reactivity of such compounds in N-acylation was shown.

The DNA ligase-induced assembly of synthetic oligodeoxyribonucleotides on polymer supports was used to obtain immobilized DNA, containing the T7 RNA polymerase promoter and coding for a 14-membered oligoribonucleotide. The obtained template can carry out RNA synthesis in a flowing-type reactor. Sepharose 4B and Toyopearl HW-55 were used as supports.

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).

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.