[Bioluminescent reporters to identify gene allelic variants].

The method of single nucleotide polymorphism identification based on primer extension reaction (PEXT) with the following bioluminescent solid-phase microassay was developed. The recombinant Ca2+-regulated photoprotein obelin and coelenterazine-dependent luciferase Renilla muelleri were used as reporters. Factor V Leiden polymorphism 1691 G-->A (R506Q) of human F5 gene genotyping was used for investigation. Genomic DNA was amplified by PCR using primers, flanking polymorphic site of 140 base pairs. PCR products were used as a template for two PEXT reaction using two primers with 3'-end nucleotides, complementary either normal or mutant alleles. At complementarity of template and allelic-typical primer its extension with DNA-polymerase takes place. The products carried biotin due to availability ofbiotinylated dUTP in the reactions mixture. The assay was carried out using obelin-streptavidin chemical conjugates. Optimal PEXT-reaction conditions providing high reliability of SNP genotyping were found. A new approach to determine both alleles in one well was developed applying two bioluminescent reporters. Availability of the proposed approach was shown in the study of clinical DNA samples.

[Cloning of genes, purification and properties investigation of recombinant DNA ligases from the thermophilic archaeon Pyrococcus abyssi and Methanobacterium thermoautotrophicum].

The genes encoding of DNA ligases from the thermophilic archaeon Pyrococcus abyssi (PabDNA ligase) and Methanobacterium thermoautotrophicum (MthDNA ligase) were cloned and expressed in Escherichia coli. The activity of purified enzymes was studied by ligation of two oligonucleotides, one of which had preformed hairpin structure. In the used system the maximal output of reaction products for both DNA ligases was observed near 70 degrees C that is explained by substrate thermostability. At stoichiometric ratio of enzymes and substrate the output of a product reaches of plateau at 70-75% of theoretical ones. Investigated DNA ligases showed different thermostability. The half-time life of PabDNA ligase was about 60 min at 90 degrees C. MthDNA ligase was completely inactivated at this temperature during 10 min. Recombinant DNA ligases from P. abyssi and M. thermoautotrophicum possessed high stability during a storage at 4 degrees C.

[Oligonucleotide derivatives in the nucleic acid hybridization analysis. I. Covalent immobilization of oligonucleotide probes onto the nylon].

The features of UV-induced immobilization of oligonucleotides on a nylon membranes and the effectiveness of enzymatic labeling of immobilized probes at heterophase detection of nucleic acids are studied. Short terminal oligothymidilate (up to 10 nt) sequences are suggested to attach to the probe via a flexible ethylene glycol based linker. The presence of such fragment enhances the intensity of immobilization and reduces UV-dependent degradation of the targeted (sequence-specific) part of the probe by reducing the dose needed for the immobilization of DNA. The optimum dose of UV-irradiation is determined to be ~0.4 J/cm(2) at the wavelength 254 nm. This dose provides high level of hybridization signal for immobilized probes with various nucleotide composition of the sequence specific moiety. The amide groups of the polyamide are shown to play the key role in the photoinduced immobilization of nucleic acids, whereas the primary amino groups in the structure of PA is not the center responsible for the covalent binding of DNA by UV-irradiation, as previously believed. Various additives in the soaking solution during the membrane of UV-dependent immobilization of probes are shown to influence its effectiveness. The use of alternative to UV-irradiation system of radical generation are shown to provide the immobilization of oligonucleotides onto the nylon membrane.

[Bent dsDNA with defined geometric characteristics in terms of complexes of bridged oligonucleotides].

An opportunity of designing nontypical double-stranded DNA structures containing nonnatural inserts in a regular nucleotide DNA sequence has been investigated. The looped nucleotide inserts on the basis of adenylates and thymidilates, and nonnucleotide inserts on the basis of phosphodiesters of diethyleneglycol, 1,10-decanediol, and 3-hydroxy-2-hudroxymethyltetrahydrofuran were introduced into the backbone of a 32-mer native DNA duplex. These inserts formed the internal loops in the modified double-stranded DNA fragments which were shown to lead to bending of the linear duplex structure by 16 to 83 degrees. The dependencies of the bend angle of dsDNA on the composition and the length of the looped regions were determined. It was established that the bend of the irregular region of dsDNA depended on the electrostatic interaction of the phosphate residues. The tension in the complex structure could be reduced by the introduction of additional nucleotide units opposite the loop, which led to some relaxation of the bent helix. The resulting parameters of the bend values were shown to be in a good agreement with the published data obtained by NMR spectroscopy. It was demonstrated that the variation of the nature or the length of the insert allowed one to regulate the level of the local perturbation of the duplex structure and, thereby, influence both the bend level of the double helix and the destabilization of the modified complex.

[A highly sensitive and rapid method for the detection of DNA fragments using the photoprotein obelin as a reporter].

The recombinant Ca2+-activated photoprotein obelin was used as a reporter protein in a solid-phase bioluminescent hybridization DNA assay. Oligonucleotide probes were immobilized on the surface of a fine-grained polymer or microbiological plates of different types. A 30-mer oligonucleotide or its derivative with a biotin residue on the 3'-terminus, as well as a denatured double-stranded PCR fragment of the hepatitis C virus with the sequence of the 30-mer oligonucleotide, was used as a DNA template. The probe in the hybridization complex was labeled by elongation of the chain using Taq DNA polymerase in the presence of biotinylated deoxyuridine triphosphate. The results of the bioluminescent assay were compared with the results of colorimetric analysis obtained with alkaline phosphatase as a reporter protein. It was shown that the use of the bioluminescent obelin label substantially accelerates the DNA detection procedure, ensures a high sensitivity of the assay (no less than 10(-15) mol of DNA template), and enables quantitative determination of the amount of DNA template in the tested sample.

[Enhancement of hybridization analysis efficacy by means of controlled fragmentation of analyzed DNA].

The influence of fragmentation of analyzed DNA amplicon on the efficacy of specific sequence detection by means of heterophase hybridization analysis was investigated. The detection of DNA sequence was carried out colorimetrically after introduction of biotin label into the oligonucleotide probe immobilized on a solid support upon its limited elongation in the complex with the analyzed DNA using Taq polymerase. Two simple and reproducible approaches to DNA analyte fragmentation were suggested. They are based on the formation apurinic/apyrimidinic sites in DNA followed by their degradation upon the thermal treatment. Apurinization of DNA was achieved with a mild acidic treatment. The apyrimidinic sites were formed when DNA fragment containing dTMP and dUMP residues in various ratios was treated with uracil-DNA-glycosylase (UDG). DNA analytes pretreated by one of these approaches can be used without additional purification for hybridization analysis of DNA with the use of Taq polymerase. The efficacy of hybridization analysis is shown to be higher in the case of the fragmented DNA in comparison with the native DNA amplicon. The use of fragmented DNA analytes allows utilizing bridged oligonucleotides as highly selective probes with reduced hybridization properties.

[Determination of Herpes simplex virus DNA with the use of solid-phase methods for the detection of PCR products].

To determine DNA of herpes simplex virus (HSV), types 1 and 2, the polymerase chain reaction (PCR) method was developed with the subsequent detection of amplification products by means of electrophoresis or the molecular hybridization of nucleic acids (MHNA). Two variants of MHNA have been compared: hybridization in the solution of a biotinylated probe with digoxigenin-labeled PCR with the subsequent sorption of hybridization complexes onto streptavidin-covered plates and solid-phase hybridization of digoxigenin-labeled PCR with a biotinylated probe. Effective hybridization was observed after the denaturation of targets at 95 degrees C in the solution of 50 mM NaOH, but not in neutral solutions. To increase the level of sensitivity of hybridization in solution, the exact selection of the amount of the probe was shown to be necessary, for both its excess and deficiency essentially decreased the method sensitivity. A decrease in the ionic power of hybridization solutions from 6 h SSC to 1 h SSC led to greater specificity of hybridization without a decrease in the method sensitivity. A rise in the temperature of hybridization and subsequent washing to 45 degrees C decreased the sensitivity of the method. The limit of the sensitivity PCR with electrophoretic detection was 30 HSV genome equivalents, and 10 genome equivalents in the presence MHNA in the solution and on the solid phase.

[Cleavage of RNA in hybrid duplexes by E. coli ribonuclease H. II. Substrate properties of nucleotides containing non-nucleotide linkers]. / Rasshcheplenie RNK v sostave gibridnykh dupleksov ribonukleazoi H E. coli. II. Substratnye svoistva oligonukleotidov, soderzhashchikh nenukleotidnye vstavki.

The 20-mer bridged oligodeoxynucleotides containing short oligomers joined by the hexamethylenediol and hexaethylene glycol linkers were shown to form complementary DNA/DNA and RNA/DNA complexes whose thermostability depends on the length and number of the nonnucleotide linkers. Hybrid complexes of the bridged oligonucleotides proved to be substrates for the E. coli ribonuclease H. The presence of one-three nonnucleotide linkers in a 20-mer decreased the hydrolysis efficacy only 1.2-1.4-fold. It is the composition of the RNA cleavage products that was influenced the most significantly by the nonnucleotide linkers. RNase H simultaneously hydrolyzed the RNA 3'-ends of each hybrid duplex involving a bridged oligonucleotide. The presence of an inverted 3'-3'-phosphodiester bond at the 3'-end of the oligodeoxyribonucleotide only slightly affected the RNase H activity.

[Nucleic acids interactions with short oligonucleotide derivatives. II. Tandem of short oligonucleotides as highly sensitive system for identification of single base substitutions in target DNA]. / Vzaimodeistvie proizvodnykh korotkikh oligonukleotidov s nykleinovymi kislotami. II. Tandem korotkikh oligonukleotidov--vysokochuvstvitel'naia sistema dlia obnaruzheniia odnobukvennoi zameny v DNK-misheni.

A new approach for modification of target DNAs with tandems of derivatives of short oligonucleotides was suggested that allows highly selective modification of perfect duplexes only. At physiological temperatures, the efficiency of DNA modification by a dodecanucleotide alkylating agent was demonstrated to be the same for both perfect and mismatch-containing duplexes, whereas the tetranucleotide reagent in the presence of two flanking effectors alkylated with high selectivity the target DNA in the perfect duplex only.

[Interaction of short nucleotide derivatives with nucleic acids. IV. Modification of DNA by an alkylating tetranucleotide reagents in the presence of effectors in perfect and imperfect complexes]. / Vzaimodeistvie proizvodnykh korotkikh oligonukleotidov s nukleinovymi kislotami. IV. Modifikatsiia DNK alkiliruiushchim proizvodnym tetranucleotida v prisutstvii éffektorov v sostave pravil'nykh i nepravil'nykh kompleksov.

It was demonstrated that any mismatches in a complex formed by an ssDNA target and a tetranucleotide at 25 or 37 degrees C can be discriminated by alkylating the DNA with a tetranucleotide carrying a 4-[N-methyl-N-(2-chloroethyl)]aminobenzylethylamine residue at the 5'-terminal phosphate in the presence of a pair of flanking effectors, octanucleotide di-N-(2-hydroxyethyl)-phenazinium derivatives. The discrimination factor (ratio of the extent of the target modification in the perfect and mismatch-containing complexes) for a single mismatch in the tetranucleotide binding site at 25 degrees C varied between 4 and 500 depending on the type of mismatch and its location in the complex and exceeded 400 at 37 degrees C for all the investigated mismatches. The DNA target modification by the alkylating derivative of the 3'-estrone ester of tetranucleotide pCAGX (mean = C, T, A or G) was selective in the presence of a pair of hydrophobic effectors, octanucleotide 5'-cholesteryl-3'-phenazinium derivatives. The discrimination factors for 3'-terminal mismatches T.G, A.G, and G.G were 1,8,400, and 400, respectively.

[Interaction of derivatives of short oligonucleotides with nucleic acids. I. Effect of various types of effectors on alkylation of DNA-targets]. / Vzaimodeistvie proizvodnykh korotkikh oligonukleotidov s nukleinovymi kislotami. I. Vliianie razlichnykh tipov éffektorov na alkilirovanie DNK-mishenei.

It was shown that the tandem of the derivatives of short oligonucleotides efficiently and site specifically interacts with target 20 base deoxyribonucleotide (M). It was demonstrated that the very low hybridization ability of tetranucleotide (D) and its 3'-cholesterol and 3'-estrone esters (D-ChS and D-EsS, respectively) increases significantly in the presence of the effectors: octanucleotides (E1 and E2), and their 5',3'-diphenazinium (Phn-E1-Phn and Phn-E2-Phn) and 5'-cholesteryl-3'-phenazinium (ChS-E1-Phn and ChS-E2-Phn) derivatives, which flank them on the target strand. The influence of the effectors on the interaction of the target M with tetranucleotide D or its alkylating derivatives (RCl-D) increases in a series E1 + E2 < ChS-E1-Phn + ChS-E2-Phn < Phn-E1-Phn + Phn-E2-Phn. For the steroid derivatives, D-ChS and D-EsS, and the reagents based on them (RCl-D-ChS and RCl-D-EsS), this series is E1 + E2 < Phn-E1-Phn + Phn-E2-Phn < ChS-E1-Phn + ChS-E2-Phn. The modification level of the target M with derivatives RCl-D-EsS in the presence of ChS-E1-Phn and ChS-E2-Phn reaches 40% even at 37 degrees C under conditions close to physiological. The possibility of using 5'-cholesteryl-3'-phenazinium-containing oligonucleotides as effectors of the interaction of target DNA with the derivatives of short oligonucleotides was demonstrated.