[Application of BODIPY-trimethylmelamine conjugate for DNA cross-linking in vitro].

Conjugate the fluorescent dye 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indatsen-8-propionic acid (BODIPY) and N2,N4,N6-trimethylmelamine was obtained. It was shown that this compound in the presence of formaldehyde generates covalent cross-links of DNA strands in vitro.

[Methoxymethyl and (p-nitrobenzyloxy)methyl groups in the synthesis of oligoribonucleotides by the phosphotriester method].

An efficient synthetic method for monomer ribonucleotide synthons containing 2'-O-methoxymethyl and 2'O-(p-nitrobenzyloxy)methyl groups used for oligonucleotide phosphotriester method with O-nucleophilic intramolecular catalysis at the stage of formation of internucleotide bond is developed. It is shown that synthons containing protecting 2'-O-(p-nitrobenzyloxy)methyl group may be used for automatic synthesis of phosphotriester oligoribonucleotides with high yields and synthons containing methoxymethyl group--to get 2'-O-modified oligonucleotides.

[Monomers containing 2'-o-alkoxymethyl groups as synthons for the synthesis of oligoribonucleotides by the phosphotriester method].

A general scheme for the synthesis of ribonucleotide monomers containing alkoxymethyl group in 2'-O-position for the solid-phase phosphotriester oligonucleotide synthesis using O-nucleophilic intramolecular catalysis has been developed. In particular, the monomers containing 2'-O-modifying 2-azidoethoxymethyl, propargyloxymethyl, or 3,4-cyclocarbonatebutoxymethyl groups has been prepared.

[N-azidomethylbenzoyl blocking group in the phosphotriester synthesis of oligonucleotides].

An effective modification of phosphotriester method for automatic synthesis of DNA and RNA fragments using O-nucleophilic intramolecular catalysis and 2-(azidometil)benzoyl group to protect amino groups of heterocyclic bases of nucleotides is described.

[Cross-linked nucleic acids: formation, structure, and biological function].

Published data on the main types of reagents capable of introducing covalent interstrand cross links into nucleic acids (NA) are summarized in the present review. The reactivity of cross-linking agents, their preferred binding sites, and methods of determining the cross-link localization in a duplex are discussed. Cell response to DNA cross linking, namely, the blocking of replication and transcription, the initiation of reparation processes, and apoptotic death of the cell, are analyzed, as well as the use of cross-linking reagents in therapy and molecular biology.

[DNA mimics on the base of pyrrolidine and hydroxyproline].

In order to improve physicochemical and biological properties of natural oligonucleotides in particular increasing their affinity for nucleic acids, the selectivity of action and biological sustainability, several types of DNA mimics were designed. The survey collected data on the synthesis and properties of the DNA mimics - peptide-nucleic acids analogues, which are derivatives of pyrrolidine and hydroxyproline. We examine some physicochemical and biological properties of negatively charged mimics of this type, containing phosphonate residues, and possessing a high affinity for DNA and RNA, selective binding with nucleic acids and stability in various biological systems. Examples of the use of these mimics as tools for molecular biological research, particularly in functional genomics are given. The prospects for their use in diagnostics and medicine are discussed.

[An azidomethyl protective group in the synthesis of oligoribonucleotides by the phosphotriester method].

A rapid and effective method of an automatic oligoribonucleotide synthesis alternative to the phosphoroamidite one was developed based on the phosphotriester approach of internucleotide bond formation under intramolecular O-nucleophilic catalysis and the use of an azidomethyl group for protection of a nucleotide 2'-hydroxyl function.

Synthesis of RNA by the rapid phosphotriester method using azido-based 2'-O-protecting groups.

The azidomethyl and 2-(azidomethyl)benzoyl as 2'-OH protecting groups are reported for preparation of oligoribonucleotides by the phosphotriester solid-phase method using O-nucleophilic intramolecular catalysis. The procedures for the synthesis of the corresponding monomer synthons were developed and the usefulness of the application of 2'-O-azidomethyl and 2'-O-2-(azidomethyl)benzoyl groups was examined in the synthesis of different RNA fragments with a chain length of 15-22 nucleotides. The azidomethyl group was found to be more preferable for effective synthesis of oligoribonucleotides. Hybridization properties of RNAs toward their complementary oligonucleotides were examined before and after the removal of 2'-O-azidomethyl groups.

Detection of the 5'-cap structure of messenger RNAs with the use of the cap-jumping approach.

An effective procedure for specific determination of the cap structure at the 5'-terminus of mRNA and for isolation of the corresponding full-length cDNA has been developed. The procedure involves covalent attachment of an oligonucleotide template extender to the 5'-cap structure of mRNA followed by RT-PCR using M-MLV SuperScript II reverse transcriptase. In the course of reverse transcription, the enzyme 'jumps over' the cap structure and includes the sequence complementary to the oligonucleotide template extender into the 3'-end of the first cDNA strand. The cap-jumping method was successfully tested using some mammalian cellular mRNAs, genomic RNAs of tobacco mosaic virus (TMV) U1 and the recently isolated crucifer-infecting tobamovirus. Moreover, cDNA products corresponding to the genomic tobamovirus RNA were obtained from total RNA extracted from tobacco plants infected by crucifer-infecting tobamovirus or tobacco mosaic virus. Using the cap-jumping method, we have shown for the first time that genomic crucifer-infecting tobamovirus (crTMV) RNA contains a 5'-cap structure. This improved method can be recommended for the construction of full-length and 5'-end enriched cDNA libraries, identification of capped RNAs and determination of their 5'-terminal sequences.

Synthesis of polyacrylamides N-substituted with PNA-like oligonucleotide mimics for molecular diagnostic applications.

Two types of oligonucleotide mimics relative to peptide nucleic acids (PNAs) were tested as probes in nucleic acid hybridisation assays based on polyacrylamide technology. One type of mimic oligomers represented a chimera constructed of PNA and phosphono-PNA (pPNA) monomers, and the other one contained pPNA residues alternating with PNA-like monomers on the base of trans -4-hydroxy-L-proline (HypNA). A chemistry providing efficient and specific covalent attachment of these DNA mimics to acrylamide polymers using a convenient approach based on the co-polymerisation of acrylamide and some reactive acrylic acid derivatives with oligomers bearing 5'- or 3'-terminal acrylamide groups has been developed. A comparative study of polyacrylamide conjugates with oligonucleotides and mimic oligomers demonstrated the suitability and high potential of PNA-pPNA and HypNA-pPNA chimeras as sequence-specific probes in capture and detection of target nucleic acid fragments to serve current forms of DNA arrays.

[Conjugates of polyacrylamide with oligonucleotides and their mimetics for diagnostic purposes]. / Kon''iugaty poliakrilamida s oligonukleotidami i ikh mimetikami dlia diagnosticheskikh tselei.

Approaches to preparing acrylamide and polyacrylamide conjugates with oligonucleotides and some peptide nucleic acid-related DNA mimics are considered. Their physicochemical properties and application to the nucleic acid analysis are discussed.

[Peptide nucleic acids and their phosphonate analogues: synthesis and hybridization characteristics]. / Peptido-nukleinovye kisloty i ikh fosfonatnye analogi: sintez i gibridizatsionnye svoistva.

The synthesis of a series of DNA mimics--peptide nucleic acids, phosphonate analogues of peptide nucleic acids, and their hybrids--is described. The preparative synthesis of the corresponding monomers and the solid phase automated synthesis of oligomers-mimics are developed. Modified phosphonate analogues of peptide nucleic acids, in particular chiral derivatives and those with additional hydroxyl groups in the side chains of the backbone as well as pyrene derivatives of peptide nucleic acids and their phosphonate analogues, are prepared. The ability of the resulting oligomers specifically to hybridize to DNA and RNA complementary chains is studied. It is shown that phosphonate analogues of peptide nucleic acids and their hybrids with peptide nucleic acids can form complexes with the DNA and RNA complementary strands, the stability of the complexes increasing in parallel with the increase in the number of peptide nucleic acid residues in the chain of the mimic. This property, along with good water solubility, provides the precondition for further evaluation of these compounds as antisense and antigene agents.

Convenient approaches to the synthesis of oligonucleotide macrocycles containing non-nucleotide linkers.

Two convenient, practical routes to the synthesis of non-nucleotide bridged cyclic oligonucleotides have been developed. The first procedure included circularization of oligonucleotides by template-directed ligation on solid phase, while the second procedure involved preparation of a circular oligomer by non-template chemical ligation of a linear precursor in solution. Using these approaches, a series of single- and double-stranded cyclic oligonucleotides with non-nucleotide bridges has been synthesized.

Synthesis and evaluation of some properties of chimeric oligomers containing PNA and phosphono-PNA residues.

In an attempt to improve physico-chemical and biological properties of peptide nucleic acids (PNAs), particularly water solubility and cellular uptake, the synthesis of chimeric oligomers consisted of PNA and phosphono-PNA analogues (pPNAs) bearing the four natural nucleobases has been accomplished. To produce these chimeras, pPNA monomers of two types containing N-(2-hydroxyethyl)phosphonoglycine, or N-(2-aminoethyl)phosphonoglycine backbone, were used in conjunction with PNA monomers representing derivatives of N-(2-aminoethyl)glycine, or N-(2-hydroxyethyl)glycine. The oligomers obtained were composed of either PNA and pPNA stretches or alternating PNA and pPNA monomers. The examination of hybridization properties of PNA-pPNA chimeras to DNA and RNA complementary strands in comparison with pure PNAs, and pPNAs as well as DNA-pPNA hybrids and DNA fragments confirmed that these chimeras form stable complexes with complementary DNA and RNA fragments. They were found to be resistant to degradation by nucleases. All these properties together with good solubility in water make PNA-pPNA hybrids promising for further evaluation as potential therapeutic agents.

Synthesis of DNA analogues with novel carboxamidomethyl phosphonamide and glycinamide internucleoside linkages.

Thymidine oligonucleotide analogues with phosphodiester bonds fully substituted by carboxamidomethyl phosphonamide, or glycinamide linkages were synthesized on a solid support, and their hybridization properties toward DNA and RNA targets were determined by Tm analysis.

[Biosynthesis of human calcitonin and mini-proinsulin in bacterial cells in the form of hybrid proteins with corresponding antisense peptides and a metal-binding peptide]. / Biosintez v bakterial'nykh kletkakh kal'tsitonina i mini-proinsulina cheloveka v vide gibridnykh belkov s sootvetstvuiushchimi antismyslovymi peptidami i metallsviazyvaiushchim peptidom.

To verify experimentally the molecular recognition theory, plasmids were constructed that provided the efficient synthesis of hybrid proteins composed of human calcitonin or miniproinsulin, the corresponding antisense peptides, and a histidine-rich metal-binding peptide. A method for isolation of the hybrid proteins by metal-chelating chromatography, cleavage, and renaturation was developed.

[An artificial gene, biosynthesis and properties of the recombinant Fc fragment of human immunoglobulin G1]. / Iskusstvennyi gen, biosintez i svoistva rekombinantnogo Fc-fragmenta immunoglobulina G1 cheloveka.

Chemicoenzymatic synthesis and cloning of a gene encoding the Fc domain of human immunoglobulin G1 were carried out. The artificial gene was expressed in Escherichia coli cells in plasmid vectors under control of a late T7 promoter. The recombinant protein isolated from the bacterial cells is capable of forming dimers and binding protein A from Staphylococcus aureus.

New efficient sulfurizing reagents for the preparation of oligodeoxyribonucleotide phosphorothioate analogues.

A set of new sulfurizing agents representing disulfides of arylsulfonic acids has been developed for the automated synthesis of phosphorothioate oligonucleotide analogues via the phosphoramidite method. These reagents, such as bis(benzenesulfonyl)disulfide, bis(p-toluenesulfonyl)disulfide, bis(p-methoxybenzensulfonyl)disulfide, and bis (p-chlorobenzenesulfonyl) disulfide, are easily prepared crystalline solid compounds. They are relatively inexpensive, easy to handle, and efficiently convert internucleotide cyanoethyl phosphite to the phosphorothioate triester within 1-2 min. The efficiency of phosphorothioate oligonucleotide synthesis with the use of these reagents is comparable to that of phosphodiester oligonucleotides.