Russian Journal of Bioorganic Chemistry, Vol. 23, No. 11, 1997, p. 796

Interaction of Short Oligonucleotide Derivatives with Nucleic Acids. IV. 1 Modification of DNA by an Akylating Tetranucleotide Reagent in the Presence of Effectors in Perfect and Imperfect Complexes

D. V. Pyshnyi, I. A. Pyshnaya, S .G. Lokhov, E. M. Ivanova, 2 and V. F. Zarytova

Novosibirsk Institute of Bioorganic Chemistry, Siberian Division, Russian Academy of Sciences , Novosibirsk , Russia

Abstract: It was demonstrated that any mismatches in a complex formed by an ssDNA target and a tetranucleotide at 25 or 37°C can be discriminated by alkylating the DNA with a tetranucleotide carrying a 4-[N-methylN-(2-chloroethyl)]aminobenzylmethylamine 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°C varied between 4 and 500 depending on the type of mismatch and its location in the complex and exceeded 400 at 37°C for all the investigated mismatches. The DNA target modification by the alkylating derivative of the 3'-phosphoestrone ester of tetranucleotide pCAGX (X = 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.

Russian Journal of Bioorganic Chemistry, Vol. 24, No. 1, 1998, p. 27

Interaction of Derivatives of Short Oligonucleotides with Nucleic Acids: VI. 1 Discrimination of Mismatch-Containing Complexes upon Ligation of a Tandem of Short Oligonucleotides on DNA Template

D. V. Pyshnyi*, A. A. Krivenko**, S. G. Lokhov*, E. M. Ivanova*, 2 G. M. Dymshits**, and V. F. Zarytova*

* Novosibirsk Institute of Bioorganic Chemistry, Siberian Division, Russian Academy of Sciences, pr. Akademika Lavrent’eva 8, Novosibirsk, 630090 Russia ** Institute of Cytology and Genetics, Siberian Division, Russian Academy of Sciences, pr. Akademika Lavrent’eva 10, Novosibirsk, 630090 Russia

Abstract: 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.

Russian Journal of Bioorganic Chemistry, Vol. 24, No. 1, 1998, đ . 20

Interaction of Derivatives of Short Oligonucleotides with Nucleic Acids: V. 1 Ligation of a Tandem of Short Oligonucleotides on the Complementary DNA Template

D. V. Pyshnyi*, A. A. Krivenko**, S. G. Lokhov*, E. M. Ivanova*, 2 G. M. Dymshits**, and V. F. Zarytova*

*Novosibirsk Institute of Bioorganic Chemistry, Siberian Division, Russian Academy of Sciences, pr. Akademika Lavrent’eva 8, Novosibirsk, 630090 Russia **Institute of Cytology and Genetics, Siberian Division, Russian Academy of Sciences, pr. Akademika Lavrent’eva 10, Novosibirsk, 630090 Russia

Abstract: 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.

Russian Journal of Bioorganic Chemistry, Vol. 24, No. 2, 1998, đ . 117

Interaction of Derivatives of Short Oligonucleotides with Nucleic Acids. VII. 1 Effect of Conformational Changes in the Duplex Structure on Site Specificity and Efficiency of Modification of Target DNA by Alkylating Oligonucleotide Derivatives

D. V. Pyshnyi, S. G. Lokhov, E. M. Ivanova, 2 and V. F. Zarytova

Novosibirsk Institute of Bioorganic Chemistry, Siberian Division, Russian Academy of Sciences, pr. Akademika Lavrent’eva 8, Novosibirsk, 630090 Russia

Abstract: 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.

Russian Journal of Bioorganic Chemistry, Vol. 24, No. 3, 1998, đ . 179

Interaction of Derivatives of Short Oligonucleotides with Nucleic Acids: VIII. 1 Some Features of Modification of Target DNA by Alkylating Oligonucleotide Derivatives in Tandem Complexes

D. V. Pyshnyi, S. G. Lokhov, E. M. Ivanova, 2 and V. F. Zarytova

Novosibirsk Institute of Bioorganic Chemistry, Siberian Division, Russian Academy of Sciences, pr. Akademika Lavrent’eva 8, Novosibirsk, 630090 Russia

Abstract: 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 was 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 alkylation 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.