[The second generation universal oligonucleotide microarray for subtyping of influenza virus A].

The microchip for influenza A subtyping was developed, functioning on a principle "one spot--one subtype". Each spot contains the set of oligonucleotide probes, specific for a particular subtype of hemagglutinin, neuraminidase or matrix gene. Reliability of the proposed chip version is the same as for earlier created in our group full-size microchip for separate hemagglutinin and neuraminidase subtyping. To visualize the image, analyzed DNA can be labeled by either fluorescent dye or biotin with the further fixation in system streptavidin-gold nanoparticles and image development by silver precipitation. In the second case common version of scanner can be used for the image analysis, that essentially simplifies procedure of influenza A subtyping.

[Oligonucleotide microarray for subtyping of influenza virus A neuraminidase].

Microarray for influenza A neuraminidase subtyping was presented. Selection of oligoprobes proceeded in two steps. First step included selection of peptides specific for each subtype of neuraminidase. At the second step oligoprobes were calculated using found peptides structures with the subsequent additional selection of the most specific and representative probes. From 19 to 24 probes were used for determination of each subtype of neuraminidase. Microchip testing for 19 samples with the most widespread types (N1 and N2) specifies in unequivocal definition 18 of them and only one isolate has not been identified.

[Sulfonium derivatives of thioxanthenone, a new class of photodetritylating agents for microarray oligonucleotide synthesis].

The usability of a new class of photo acids, namely, sulfonium hexaphosphates based on thioxanthenone, for the removal of the dimethoxytrityl protective group in the process of oligonucleotide synthesis has been studied in order to search for new detritylating agents for microarray oligodeoxyribonucleotide synthesis. 2,4-Diethyl-9-oxo-10-(4-heptyloxyphenyl)-9H-thioxanthenium hexafluorophosphate has been successfully used for the solid-phase synthesis of (dT)(10).

[Subtyping of influenza virus A hemagglutinine with hybridization microarray].

An oligonucleotide microarray for influenza A hemagglutinine subtyping was presented. The number of probes for determination of each subtype hemagglutinine (H1-H13, H15, H16, pandemic flu H1N1)varied from 13 to 28. When testing of the microarray using 40 type A influenza virus isolates the hemagglutinin subtypes were unambiguously determined for 36 specimens.

[Oligonucleotide conjugates with minor groove ligands as probes for hybridization microarray chips].

A possibility of using oligonucleotide conjugates with minor groove ligands as probes for hybridization microarray chips was studied. The oligonucleotide conjugates contain a hairpin ligand (MGB) composed of two tripyrrolcarboxamide residues with an aminocaproic acid residue as a linker and bound to the oligonucleotide duplex AT tract in a site-specific manner. We used as (5'-3') probes GACAAGAp, GACAAAAp, GACAAGA-MGB, and GACAAAA-MGB. The oligonucleotides labeled with Cy3 cyanine dye, Cy3-ACTAATTTTGTC and Cy3-ACTAATCTTGTC, were used as targets. The maximal MGB effect on the fluorescence level of microarray chip spots, which caused its fourfold increase as compared with the initial unmodified duplex, was observed for the duplex containing only AT pairs in the ligand binding site. The presence of A-C and G-T mutations in the binding site (imperfect duplexes) or a C-G pair (perfect duplex) affects the change in fluorescence level to a considerably lesser degree.

[TaqMan probes based on oligonucleotide-hairpin minor groove ligand conjugates].

Hybridization of TaqMan probes derived from oligonucleotides containing fluorophores (fluorescein, FAM, or tetramethylrhodamine, (Tamra)), fluorescence quenchers (BHQ1 or BHQ2), and a conjugated hairpin ligand (MGB) composed of two tripyrrolcarboxamide residues connected through an aminobutyric acid residue were proposed for discrimination of point mutations using the real time PCR technique. Identification of point A/C substitution was shown to be highly specific for hepatitis C virus subtypes 1a and 1b with two variants of the probe (5'-3'): ATTGAGCGGGTTTAp-BHQ2-MGB for subtype 1a and FAM-ATTGAGCGGGTTGAp-BHQ1-MGB for subtype 1b. Perfect duplexes (A.T and G.C pairs) increase fluorescence in the process of amplification, whereas imperfect duplexes (A.G and T.C pairs) induce no fluorescence changes. This phenomenon enables simultaneous genotyping of hepatitis C virus subtypes 1a and 1b.

[An oligonucleotide microarray for detection and discrimination of orthopoxviruses based on oligonucleotide sequences of two viral genes].

An oligonucleotide microarray for detection and identification of orthopoxviruses was developed. Genus specific and orthopoxvirus species-specific regions of the genes encoding chemokine binding and alpha/beta-interferon binding proteins were used as a target. The developed microarray allows the variola, monkeypox, cowpox, vaccinia, camel-pox and ectromelia (mousepox) viruses to be distinguished with a high degree of reliability.

Interaction of endonuclease ecoRI with short specific and nonspecific oligonucleotides.

The interaction of EcoRI with different oligodeoxyribonucleotides (ODNs) was analyzed using the method of the slow step-by-step simplification in their complexity. Orthophosphate (KI = 31 mM), 2-deoxyribose 5-phosphate (KI = 4.6 mM) and different dNMPs (KI = 2.1-2.5 mM) were shown to be the minimal ligands of the enzyme. The lengthening of a nonspecific d(pN)n (n = 1-6) by one nucleotide unit resulted in the increase of their affinity by a factor of approximately 2.0. Weak nonspecific electrostatic contacts of EcoRI with internucleotide phosphate groups of ODNs can account for about 5 orders of magnitude in the ligand affinity, whereas the contribution of specific interactions between EcoRI and d(pN)n is no more than 2 orders of magnitude of a total ODN's affinity.

Formation of the D-loop structure complexes between DNA and oligonucleotides and affinity modification of DNA by chemically reactive derivatives of oligonucleotides lead to the recombination.

INTRODUCTION: Affinity modification of DNA by chemically reactive derivatives of complementary oligonucleotides (ODNs) and antisense ODNs has shown an application for the inhibition of gene expression and the growth of viruses and parasites in high organisms. Unfortunately, the rapid advancement of antisense therapeutic approaches is not parallel to the investigation of possible consequences of antisense and gene-directed ODNs on genetic material of the cells being treated. Here we tried for the first time to estimate a possible genetic impact of antisense ODNs and their chemically reactive derivatives on the cells using bacteria and the plasmid DNA. MATERIAL AND METHODS: Recombination of direct repeats, induced by the formation of reversible complexes of plasmid DNA with complementary ODNs and after covalent binding of the alkylating derivative of the ODNs with DNA, has been investigated. For this purpose, a polylinker sequence flanked by 165 bp direct repeats was inserted within the tet gene of pBR 327. This plasmid was used to construct DNA containing AT- and GC-rich sequences placed in the central region of the polylinker. RESULTS: Transformation of E. coli cells with the plasmids (and with mixtures of the plasmids with d(pN)17 complementary to the AT- and GC-rich sequences) did not produce deletions. After modification of plasmids with alkylating derivatives of d(pN)17, the deletion of the polylinker DNA region (recombination) revealed the restoration of the tet gene function. The same effect was found at the cell transformations with the D-loop complex of the plasmids with ODNs, but the frequency of the transformants was about 1.5-2 times lower. The data obtained demonstrate that the complexes of DNA with complementary ODNs and the modification of the plasmids by reactive ODN derivatives result in induction of the recombination process and in loss of genetic material.

Single-stranded oligodeoxyribonucleotides are substrates of Fpg protein from Escherichia coli.

The interaction of Escherichia coli Fpg protein, which catalyzes excision of several damaged purine bases including 8-oxoguanine (oxoG) from DNA with a set of single- (ss) and double-stranded (ds) 23-mer oligodeoxyribonucleotides (ODNs) containing 8-oxoguanine(s) at various positions, has been investigated. The affinities of different ss ODNs (KM = 0.55-1.3 microM) were shown to be 12-170 times less than those for corresponding ds ODNs (KM = 6-60 nM). Depending on the position of the oxoG within the ODNs, relative initial rates of conversion of ss substrates may be less than, comparable, or greater than those for ds ODNs. The enzyme can remove 5'-terminal oxoG from ODNs only if the 5'-end is phosphorylated. Fpg does not release oxoG residues from the ultimate and penultimate 3'-terminal positions. Duplexes containing two adjacent oxoG are poor substrates for the glycosylase.

HIV-1 reverse transcriptase is capable of elongating derivatives of sequence specific noncomplementary oligodeoxynucleotides.

We have carried out a comparison of KM and Vmax values for various primers in the polymerization reaction catalyzed by the HIV-1 RT. The affinity of RT for complementary d(pT)6 containing two different 5'-end pyranone derivatives was 2-3 orders of magnitude higher (KM = 3-15 nM) than that of d(pT)6 (KM = 12.6 mM). Oligodeoxynucleotides (ODNs) noncomplementary to poly(A) template were not elongated by RT. However, derivatives of d(CAGGTG) containing the 5'-terminal chromone and coumarin related groups were efficient primers showing KM (30-300 nM) and Vmax (75-93%) values comparable with that for d(pT)10 (800 nM; 100%). The [d(CAGGTG)]ddT ODN derivatives were effective inhibitors of RT. The primer function of derivatives of noncomplementary ODNs appears to be due to the additional interactions of their 5'-terminal groups with the enzyme tRNA-binding site.

Structural constraints in the HIV-1 reverse transcriptase-primer/template complex for the initiation of DNA synthesis from primer tRNALys3.

The topography and functional implications of the complex formed in vitro between human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and its primer tRNALys3 were studied in this work. On the basis of previous results showing the high affinity both of the native primer, tRNALys3, as well as that of mismatched short oligonucleotide primers for HIV-1 RT, we synthesized chimeric primers containing tRNALys3 linked to U and T residues of different lengths. We found that the affinity of the oligonucleotide primers for HIV-1 RT is dramatically increased when linked to primer tRNA. Our results also show that in the tRNA.RT complex, before annealing tRNALys3 to the retroviral RNA genome, the 3'-terminal nucleotide of tRNALys3 is positioned at a distance of one nucleotide unit away from the template in the active polymerization site of the enzyme.

[Photomodification of DNA with a perfluorylazide-derived oligonucleotide]. / Fotomodifikatsiia DNK perftorarilazidoproizvodnym oligonukleotida.

The kinetics of photomodification of oligodeoxyribonucleotide pd(GTGTGA) with a derivative of the complementary oligodeoxyribonucleotide pd(CACACA) bearing a 3-(n-azidotetrafluorobenzoylamino)propylamine residue (ArN3) at the terminal phosphate group was studied at 20 degrees C. It was found that the target's G3 residue is preferentially modified. Along with the transformation of the arylazide moiety, degradation of the oligonucleotide fragment of the reagent occurred with a partial loss of affinity. With the use of the reagent labeled at the 5'-end, sites of photomodification were found. From the dependence of the modification level on the reagent concentration at the initial time of irradiation, the association constant (Kx = (1.40 +/- 0.24) x 10(5) M-1) was determined. From the dependence of the modification level on the concentration of the pre-irradiated reagent, the constant of the transformed reagent-target association in solution (Kr = (2.49 +/- 0.30) x 10(4) M-1) was determined. From the time-dependence of the modification level [PZ]/P0, the rate constant for the limiting step of photomodification (k0 = (5.31 +/- 0.28) x 10(-4) S-1) and the modification efficiency of the target in the complex with the reagent (gamma = 1) were found.

Recognition and conversion of single- and double-stranded oligonucleotide substrates by 8-oxoguanine-DNA glycosylase from Escherichia coli.

The substrate specificity of 8-oxoguanine-DNA glycosylase was studied. The data showed for the first time that the enzyme can cleave single-stranded deoxyoligonucleotides containing an 8-oxoG link. The values of K(m) and Vmax for a range of single-stranded and double-stranded oligonucleotides (23 bp) containing 8-oxoG at different positions of one chain. These parameters consistently depended on the position of 8-oxoG in single-stranded or double-stranded substrates. A possible mechanism responsible for substrate recognition by 8-oxoguanine-DNA glycosylase is described.