[Determination of melatonin in biological fluids].

The review considers main approaches to solving the problem in the quantification of melatonin and describes physicochemical and biological methods for melatonin determination in biological fluids (saliva, urine, plasma) and tissues. The impetus to writing this paper came from the considerably growing interest of the scientific community in melatonin and the in-depth studies of its physiological role, which had revealed a diversity of its previously unknown most important functions and confirmed its presence in many compartments of the body. Due to the specificity of its molecular structure and low concentration, melatonin remains invisible for many conventional analyses. The way out may be combination detection that involves the benefits of a few procedures and that has emerged at the interface of various sciences.

[Point contacts of T7 RNA polymerase in the promotor complex, as determined with phosphate-activated oligonucleotide derivatives]. / Testirovanie tochechnykh kontaktov v promotornom komplekse RNK-polimerazy bakteriofaga T7 s pomoshch'iu fosfat-aktivirovannykh proizvodnykh oligonukleotidov.

The contacts between phosphate groups of promoter DNA an Lys or His of T7 RNA polmerase (Pol) in the Pol-promoter complex were studied with single- and double- stranded oligonucleotides, which corresponded to the T7 promoter consensus and contained activated phosphate groups at position +1, +2, or -14 relative to the transcription start. To obtain reactive groups, terminal phosphates were modified with N-oxybenzotriazole (HOBT), and internucleotide phosphates were repalced with a trisubstituted pyrophosphate (TSP). The resulting derivatives produced covalent complexes with T7 Pol. Covalent bonding involved His in the case of TSP at position +1 or HOBT at position +1 or -14, and Lys in the case of TSB at position -14.

[Synthesis and properties of mixed ribodeoxyribooligonucleotide duplexes containing an internucleotide trisubstituted pyrophosphate bond]. / Sintez i svoistva smeshannykh ribodezoksiribooligonukleotidnykh dupleksov, soderzhashchikh mezhnukleotidnuiu trizameshchennuiu pirofosfatnuiu sviaz'.

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.

[A new approach to the problem of immobilizing oligonucleotides on carboxyl-containing nylon membranes for nucleic acid hybridization]. / Novyi podkhod k probleme immobilizatsii oligonukleotidov na karboksilsoderzhashchikh neilonovykh membranakh dlia gibridizatsii s nukleinovymi kislotami.

A simple and efficient method of the covalent immobilization of oligonucleotides on carboxyl-containing nylon membranes was proposed. The method is based on the reaction between a postsynthetically introduced aminoalkyl group of an oligonucleotide and membrane carboxyl catalyzed by a water soluble N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide. Judging from the mechanism of carboxyl group activation by the carbodiimide, optimal conditions of the immobilization were selected, which made it possible to increase the immobilization efficiency and carrier capacity as compared with those described previously. Using a 23-unit membrane-immobilized oligonucleotide as an example, the oligonucleotides immobilized were shown to be capable of hybridizing with complementary sequences.

[Synthesis and properties of oligonucleotide derivatives containing chemical active ureido groups]. / Sintez i svoistva novykh proizvodnykh oligonukleotidov, soderzhashchikh khimicheski aktivnye ureidnye gruppirovki.

The 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide (EDC)-induced modification of the carboxyl group in a synthetic oligodeoxyribonucleotide was studied. Treatment of a carboxyl-containing oligonucleotide with EDC in water or aqueous buffer solutions leads to the rapid formation (with a 80-90% yield) of the corresponding ureido derivative, which can easily be isolated by PAGE. These derivatives are stable in neutral and weakly acid aqueous solutions, whereas under weakly basic conditions they efficiently (50-90%) acylate amino groups. Reagents of this type can be used for affinity modification of enzymes and other proteins and for preparing conjugates of oligonucleotides with other compounds.

[A new method of covalent immobilization of oligodeoxyribonucleotides on nylon membranes for hybridization with nucleic acids]. / Novyi metod kovalentnoi immobilizatsii oligodezoksiribonukleotidov na neilonovykh membranakh dlia gibridizatsii s nukleinovymi kislotami.

A new method of covalent immobilization of oligodeoxyribonucleotides on nylon membranes which contain surface amino groups was developed. The method consists in condensation between the amino group of the membrane and the carboxyl group of modified oligonucleotide by means of 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide. The carboxyl group was introduced into the oligonucleotide by means of postsynthetic attachment of peptide (reduced glutathione) at the terminal phosphate group of the oligonucleotide, using the N-hydroxybenzotriazole method of phosphate activation. Membranes containing a covalently immobilized 23-membered oligonucleotide were tested in hybridization with complementary oligonucleotide, and with single-stranded DNA of bacteriophage M13 which has a complementary sequence. The method of covalent immobilization is very simple and convenient. The membranes with covalently immobilized oligonucleotides may be used not only in hybridization analysis, but also for purification of nucleic acids and proteins which recognize nucleotide sequences and in sense biotechnology.

[A new method of nonradioactive labelling of oligonucleotides and their use as allele-specific probes for detecting mutations causing beta-thalassemia]. / Novyi metod neradioaktivnogo mecheniia oligonukleotidov i ispol'zovaniie ikh v kachestve allel'-spetsificheskikh zondov dlia viavleniia mutatsii vyzyvaiushchikh beta-talassemiiu.

We have developed simple and efficient methods for synthesis of biotin and horseradish peroxidase (HRP)-labeled oligonucleotides. Biotinylated oligonucleotides were obtained in quantitative yields, and oligonucleotide conjugates with HRP in 60-80% yields. Allele-specific oligonucleotide probes for the diagnostics of IVS 1-110 mutation in the beta-globin gene causing beta-thalassemia were thus obtained. Temperature conditions for the non-radioactive ASO hybridization with the amplified segment of the human beta-globin gene and wash conditions were selected. HRP-labelled probes were used in hybridization without preliminary separation after synthesis. To decrease nonspecific enzyme binding we have elaborated special conditions for membrane blocking. Detection of the biotinylated probe was carried out with the help of a streptavidin--HRP conjugate. O-Dianisidine was used as a chromogenic substrate. We have demonstrated the usefulness of this method in the analysis of amplified samples of DNA obtained from blood of patients homozygous in the mutant gene, and heterozygous carriers.

[Affinity modification of EcoRII restriction endonuclease by a DNA-duplex containing a monosubstituted pyrophosphate internucleotide bond]. / Affinnaia modifikatsiia éndonukleazy restriktsii EcoRII DNA-dupleksom, soderzhashchim monozameshchennuiu pirofosfatnuiu mezhnukleotidnuiu sviaz'.

Oligonucleotide duplex with an active monosubstituted pyrophosphate bond within the recognition site of the EcoRII restriction endonuclease was cross-linked to this enzyme with a yield of 10-15%. The cross-linking specificity was proved by the absence of the cross-linking to a DNA duplex with the same modification but without the EcoRII recognition site as well as by unmodified EcoRII substrate's inhibition of the cross-linking.

[Design of a universal biotin-containing oligonucleotide diagnostic kit for detecting viroid plant diseases]. / Konstruirovanie universal'nogo biotinsoderzhashchego oligonukleotidnogo diagnostikuma dlia vyiavleniia viroidnykh zabolevanii rastenii.

A non-radioactive diagnosticum for plant viroid diseases has been designed, based on hybridization of a biotin-labeled 26-member oligonucleotide probe with the viroid RNA site identical for potato spindle tuber viroid and chrysanthemum stunt viroid. The biotin label has been introduced into the synthetic oligonucleotide probe by the high-yield acylation of the oligonucleotide aminoalkylamide with the biotin imidazolide or N-hydroxysuccinimide ester. Hybridization techniques have been elaborated for nucleic acids isolated from plant sap. The hybrids obtained have been detected with streptavidin and biotinylated alkaline phosphatase or with the covalent conjugate of streptavidin and alkaline phosphatase, the sensitivity being as low as 1 ng. The methodology used can be applied for revealing viroids and for large scale and quick investigation of plant cell cultures.

[Determination of potato spindle tumor viroid and chrysanthenum stund viroid using biotinylated olideoxyribonucleotides]. / Opredelenie viroidov veretenovidnosti klubnei kartofelia i karlikovosti krizantem s ispol'zovaniem biotinilirovannogo oligodezoksiribonukleotida.

A 26 base long oligodeoxyribonucleotide complementary to a common RNA sequence of potato spindle tuber viroid (PSTV) and chrysantemum stunt viroid (CSV) was synthesized. The 3'-end biotinylated one was used for the detection of PSTV and CSV RNA immobilized on nitrocellulose filters by nucleic acid hybridization. Visualization of hybridization results was performed by two ways, either by streptavidin-alkaline phosphatase conjugate or streptavidine and biotinylated alkaline phosphatase. It was possible to detect 0.65 ng of purified CSV and PSTV RNA. The suggested system of viroid diseases detection can be used by agricultural and horticultural enterprises.

[Chemical reactions in double helical nucleic acids. XIII. Directed introduction of acylphosphate internucleotide bonds into the DNA-duplex structure]. / Khimicheskie reaktsii v dvuspiral'nykh nukleinovykh kislotakh. XIII. Napravlennoe vvedenie atsilfosfatnoi mezhnukleotidnoi sviazi v strukturu DNA-dupleksov.

DNA duplex, containing an acylphosphate internucleotide bond in a predetermined position of the sugar-phosphate backbone, was synthesized. The synthesis was carried out by condensing on the complementary matrix two heptanucleotides, one of which possessed at the 3'-end a glycine residue, connected with the oligonucleotide by the phosphoramide bond, whereas the 5'-end phosphate group of the other was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC). The yield of the oligonucleotide with an acylphosphate bond was 24%. The stability and chemical properties of the synthesized compound were studied in comparison with analogous oligonucleotide containing a substituted pyrophosphate internucleotide bond. The former was shown to be an effective acylating agent in the aqueous medium in contrast to the latter which is a phosphorylating agent.

[Design of new oligonucleotide derivatives resistant to cell nucleases]. / Konstruirovanie novykh proizvodnykh oligonukleotidov, ustoichivykh k deistviiu kletochnykh nukleaz.

A new type of modified antisense oligodeoxyribonucleotides is suggested, containing an intercalating drug (e.g., daunomycin (Dnm-NH2)) at one end of the oligonuleotide, and a compound, improving the oligonucleotide transport into the cell, e.g., polymyxin B1 (PmH) at its other end. Schemes for joining these antibiotics to the terminal groups of oligonucleotides have been developed. From undecanucleotide pTGTAAAACGACp (I); the corresponding derivative (Pm)pTGTAAAACGACp (NH-Dnm) (II) was obtained with 20% yield. It was found that joining of daunomycin significantly lowers the rate of the oligonucleotide's exonuclease degradation, whereas joining of polymyxin blocks the degradation completely. Derivative (II) is much more stable to hydrolysis by cell enzymes that the unmodified oligonucleotide (I): upon incubation with E. coli cells (I) is quantitatively hydrolyzed with in 40 minutes while (II) is hydrolyzed by 30% with in 24 h. Derivative (II) is also better adsorbed by E. coli cells. This sort of derivative of antisense oligonucleotides may prove to be efficient inhibitors of the gene expression.

[Chemical reactions in double-stranded nucleic acids. IX. Directed introduction of substituted pyrophosphate bonds into DNA structure]. / Khimicheskie reaktsii v dvuspiral'nykh nukleinovykh kislotakh. IX. Napravlennoe vvedenie zameshchennykh pirofosfatnykh sviazei v strukturu DNK.

An effective synthesis of oligodeoxyribonucleotides containing a substituted pyrophosphate bond in the definite position of the sugar-phosphate backbone has been developed by template-directed condensation of two heptanucleotides. One of them containing 5'-phosphate group to be activated and 3'-phosphate group of the other being substituted with ethoxy-, buthylamino-, morpholino- or ethyl glycinate residues. Water-soluble carbodiimide (EDAC) proved to be more efficient in the phosphate group activation than N-hydroxybenzotriazole ester (yields of substituted pyrophosphates 35-80 and 10-15% respectively). The substituted pyrophosphate bong is quite stable in neutral aqueous solution. Mild conditions of selective cleavage of this bond yielding the initial oligonucleotides were found.

[Testing of G----A mutation in position 110 of a minor intron of beta-globin genes in patients with thalassemia in Azerbaijan]. / Testirovanie mutatsii G----A v 110-m polozhenii malogo introna beta-globinovogo gena u bol'nykh talassemiei iz Azerbaidzhana.

A point mutation G-A in the 110 position of the beta-globin gene small intron has been revealed by cloning and sequencing from the material of a homozygote beta-thalassemia patient in Azerbaijan. In the present study two allele-specific oligonucleotide probes for testing the mutation have been synthesized. Assessment frequency of the mutation among the beta-thalassemia patients in Azerbaijan has been performed with the use of the amplified beta-globin gene fragments obtained by using the thermostable DNA-polymerase from T. thermophilus with the subsequent dot-hybridization in gel of the amplified material with the oligonucleotide probes. The possibility to test the mutation by hybridization of the oligonucleotide probes with the donors and beta-thalassemia patients restricted genomic DNA has been analyzed. Only one of 50 thalassemia alleles of beta-globin genes under study has been shown to possess the mutation mentioned.

[Chemical reactions in double-stranded nucleic acids. V. Directed introduction into the DNA sugar-phosphate backbone of aliphatic diamine or glycol residues]. / Khimicheskie reaktsii v dvuspiral'nykh nukleinovykh kislotakh. V. Napravlennoe vvedenie v sakharofosfatnyi ostov DNK ostatkov alifaticheskikh diaminov ili glikolei.

The chemical ligation method was used for directed introduction of aliphatic diamines or glycols into sugar-phosphate backbone of DNA. Via condensation of heptanucleotide derivatives at the terminal phosphate (aminoalkylamides or hydroxyalkyl esters) with the adjacent heptanucleotide on the corresponding template, 14 bp DNA duplexes containing residues of ethylene-, propylene- or hexamethylenediamine, as well as residues of ethylene- or propyleneglycol in one of its strands, were synthesised. In a similar way duplexes were obtained in which residues of the above diamines or glycols are substituted for a mononucleotide in one of the complementary strands. Examplified by synthesis of DNA duplexes containing ethylenediamine or ethyleneglycol residues, three methods of the phosphate group activation using carbodiimide, imidazolide and N-hydroxybenzotriazole ester were tested; the last method gave the highest yields and purity of the products. Yields of duplexes without nucleotide omissions were 50, 36 and 7% for aminoethyl, aminopropyl and aminohexylamides, and 20 and 17% for hydroxyethyl and hydroxypropyl esters, respectively, whereas duplexes with nucleotide omissions were synthesised with lower yields. Each of the modified DNA duplexes thus obtained contains a recognition site of EcoRII, SsoII or MvaI restriction nucleases, thus being a potential substrate of these enzymes.

[Chemical reactions in double-stranded nucleic acids. VI. N-hydroxybenzotriazole esters of oligonucleotides--reagents for synthesis of DNA-duplexes with modified sugar-phosphate backbone]. / Khimicheskie reaktsii v dvuspiral'nykh nukleinovykh kislotakh. VI. N-oksibenzotriazolovye éfiry oligonukleotidov--reagenty dlia sinteza DNK-dupleksov s modifitsirovannym sakharofosfatnym ostovom.

With the aid of a new type of phosphorylating agents, N-hydroxybenzotriazole phosphodiesters of oligonucleotides, template-directed synthesis of DNA duplexes was carried out with ribonucleotide, aliphatic diamine or amino acid residues at a predetermined position of the sugar-phosphate backbone. Introduction of a ribonucleotide into an oligonucleotide strand gives rise to a mixture of 2'-5' and 3'-5' isomers, whose ratio depends on the condensation conditions. Residues of amino acids (lysine, serine, tyrosine) or aliphatic diamines were substituted for one or two mononucleotides in the duplex. Phosphoamide bonds with the participation of an aliphatic diamine or lysine are formed most efficiently in the presence of N-methylimidazole and if the reacting groups are in the close proximity to each other. Phosphodiester bond synthesis with the participation of hydroxy groups of serine or tyrosine proceeds less effectively. Oligonucleotide N-hydroxybenzotriazole esters exceeds previously used phosphoimidazolides in efficiency of the chemical ligation. An amino acid residue incorporated into the duplex may be used in construction of new compounds by joining the carboxyl with various groups.