Biomedical subjects
Publications and source records attributed to D V Pyshnyĭ.
Oligo(2'-tetrahydropyranylribonucleotides) and their analogues containing a 3'-3'-internucleotide bond at the 3'-terminus are nuclease-resistant and possess rather high affinity toward RNA, the main target in the antisense approach.
Properties and three-dimensional structure of the tandem DNA duplex pTGGAGCTG.(pCAGC+(PhnL)pTCCA) in aqueous solution, where L is an amino linker and Phn is an N-(2-hydroxyethyl)phenazinium residue, were studied spectrophotometrically and by two-dimensional 1H NMR spectroscopy (COSY and NOESY). When a tandem complex involving a Phn residue-bearing oligonucleotide is formed, the dye aromatic system intercalates into the double helix at the nick site and takes part in two stacking interactions: a strong one (3.5-4 A) with the T5-A12 base pair of its own duplex moiety and a weak one (4-5 A) with the C4-G13 pair of the adjoining duplex (mainly with the C4 base). This arrangement of the dye residue, providing a cross-interaction of the phenazinium moiety with the base pairs of the adjacent duplex structures, results in the stabilization of the whole tandem complex.
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.
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The modification of a target DNA by alkylating oligonucleotide derivatives possessing various capacities for complex formation was studied. The binding properties of oligonucleotides were changed either by increasing their length (tetra-, octa-, and dodecamers) or by introducing a point substitution and/or an N-(2-hydroxyethylphenazinium) residue. It was found that conformational changes occurring in the structure of the target.reagent complex upon elevating the reaction temperature affect the efficiency and site-specificity of the alkylation. In the case of complete saturation of the target with the reagent, an increase in the hybridization ability of the reagent reduced the efficiency of the target modification. It was found that the modification by the tetranucleotide reagent (in the presence of an effector adjacent to the 3' end) occurs exclusively at an intracomplex target base. In the case of the dodecamer, which forms a stable, highly cooperative complex with the target, several bases of the target undergo alkylation, and an increase in temperature changes the site-specificity of alkylation. In this process, the redistribution of the target modification sites toward stronger nucleophilic centers enhances alkylation at temperatures near the melting temperature of the target.dodecanucleotide complex despite a decrease in the extent of target association.
A tetranucleotide was highly specifically and quantitatively ligated with a pair of flanking octanucleotides carrying both radioactive and nonradioactive reporter groups. The sequence of the ligation of oligonucleotide components in a tandem on a complementary template was studied. The first stage was found to be the enzyme-catalyzed activation of the phosphate group of octanucleotide, a tandem component that possesses a higher hybridization capacity than the tetramer. It is shown that the introduction of terminal reporter groups into octanucleotides does not decrease the efficiency of their tandem ligation.
The high ligation specificity of a tetranucleotide with a pair of flanking octanucleotides on DNA template by the action of T4 phage DNA ligase is shown. In a tetranucleotide-DNA template complex containing a mismatch, almost no ligation products are formed. The ligation of a tandem octanucleotide-tetranucleotide-octanucleotide makes it possible to identify accurately any single nucleotide substitution in a tetranucleotide binding site.
The influence of effectors [octanucleotides and their 3',5'-di-N-(2-hydroxyethyl)phenazinium derivatives] on the modification of a target DNA by alkylating oligonucleotide derivatives forming duplexes of different stability with the target ws studied. It is shown that, being in tandem complexes immediately adjacent to the reactive group of an oligonucleotide reagent possessing a high hybridization capacity, the effector, on the one hand, enhances the stability of the reagent target duplex, and on the other hand, changes the site-specificity of alkylation and decreases the efficiency of the target modification at temperatures that provide a high extent of the target association with the reagent. Conversely, in the case of oligonucleotide reagents forming weak complexes with the target, effectors enhance both the stability of the target.reagent duplex and the extent of the target throughout the temperature range tested. The data indicate that the varying influence of effectors on the target modification by reagents with different hybridization capacities is due to conformational features of the target reagent duplexed regions. Increasing the rigidity of the target.reagent duplex reduces the efficiency of the target modification in tandem complexes.
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.
High efficiency was demonstrated for the photomodification of a DNA target by a 5'-p-azidotet-rafluorobenzoyl reagent based on a tetranucleotide and its 3'-phosphoestrone ester in the presence of a pair of flanking effectors. These effectors are oligonucleotide derivatives with N-(2-hydroxyethyl)phenazinium groups or those connected to cholesterol residues at the terminal phosphates.
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.
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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.
A new approach to the identification of point mutations by allele-specific PCR was proposed. The mutation R408W of the human phenylalanine hydroxylase gene was used as a model. A high specificity of the approach was achieved by the use of primers partially complementary to the genomic DNA. Polyethylene glycol covalently attached to one of the allele-specific primers provides for the differential identification of the PCR products due to a change in electrophoretic mobility.
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A new approach was proposed for detecting amplified DNA fragments by hybridization with a highly selective oligonucleotide probe obtained by ligation of a tandem of three short oligonucleotides (pN8 + pN4 + pN8' Bio) in solution, with subsequent UV-immobilization of the hybridization product on a nylon membrane and its colorimetric detection with the streptavidin-alkaline phosphatase technique. Owing to the high selectivity of ligation, the 20-mer ligation product was detected on a membrane only when it was completely complementary to a template fragment. The results showed that any single-nucleotide substitution in the tetramer-binding site can be localized and identified with the use of all 12 possible tetramers.
The effect of the bleomycin A5 residue linked to four-, eight-, and twelve-mer oligodeoxyribonucleotides on the substrate properties of their tandem and continuous (with or without unmodified octanucleotide effectors) hybrid duplexes was studied using E. coli RNase H. The bleomycin derivatives of oligodeoxyribonucleotides were shown to form hybrid duplexes with practically the same thermostability as those formed by unmodified oligodeoxyribonucleotides. The RNA in the bleomycin-containing hybrid duplexes is cleaved by the E. coli RNase H; however, the initial hydrolysis rate (v0) is 2.6-3.4-fold reduced for the continuous duplexes. In the case of tandem hybrid complexes, the effect of bleomycin on v0 was less pronounced. We hypothesized that steric factors play a key role in the bleomycin inhibition and effectors probably determine the substrate properties of such hybrid complexes. Of all the tandem systems studied, the RNA duplex with the bleomycin-containing tetranucleotide flanked with two effectors displayed the best substrate properties. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.