[Direct site-specific cleavage of double-stranded DNA by conjugates of bleomycin A5 with triplex-forming oligonucleotide].

Monofunctional conjugates of 15-mer triplex-forming oligonucleotide (TFO) with covalently attached bleomycin A5 residue at the 5'-end (Blm-p15) were synthesized. Bifunctional conjugates of TFO containing, in addition to Blm, the residues of intercalator 6-chloro-2-methoxy-9-aminoacridine (Acr) or (N-(2-hydroxyethyl)phenazinium (Phn) were obtained for the first time. The Acr and Phn residues were attached to the 3'-phosphate group of TFO through L1 and L2 linkers, respectively, resulting in the compounds Blm-p15pL1-Acr and Blm-p15pL2-Phn. The values of dissociation constants of the corresponding triplexes were evaluated using the gel retardation method. The Acr residue in Blm-p15pL1-Acr was shown to enhance the stability of the formed triplex by one order of magnitude. It was demonstrated that all synthesized conjugates are capable of specifically and nonspecifically damaging a target DNA, forming direct breaks and alkaline-labile sites. The extent of the specific cleavage of the target DNA was 15% in the case of a fivefold excess of the conjugates over the DNA duplex. The site-specific triplex-mediated cleavage of a target DNA was shown for the first time to occur predominantly (> 90%) with the formation of the direct breaks of both DNA strands. The results show the availability of bleomycin-containing oligonucleotides as antigene compounds.

[Cleavage of RNA in a hybrid duplex by E. coli ribonuclease H. III. Substrate characteristics of hybrid duplexes of RNA and oligodeoxyribonucleotides containing a bleomycin A5 residue]. / Rasshcheplenie RNK v sostave gibridnykh dupleksov ribonukleazoi H E. coli. III. Substratnye svoistva gibridnykh dupleksov RNK i oligodezoksiribonukleotidov, soderzhashchikh ostatok bleomitsina A5.

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.

[Cleavage of RNA in hybrid duplexes by ribonuclease H from E. coli. I. Substrate properties of complexes formed by RNA and tandem of short oligodeoxyribonucleotides]. / Rasshcheplenie RNK v sostave gibridnykh dupleksov ribonukleazoi H E. coli. I. Substratnye svoistva kompleksov, obrazovannykh RNK i tandemom korotkikh oligodezoksiribonukleotidov.

We studied the E. coli RNase H cleavage of a 5'-labeled RNA fragment within two hybrid duplexes with identical sequences, one of which is formed by RNA and a 20-mer oligodeoxyribonucleotide (RNA/p20), whereas the second, by RNA and a tandem of short oligodeoxyribonucleotides (octanucleotide: (RNA/tandem). It was shown that RNA in the RNA/p20 complex is hydrolyzed from the 3'-end to yield consecutively the 17-, 14-, 11-, 8-, and 5-mer 5'-labeled fragments. On hydrolysis of RNA in complex RNA/tandem, the same products were registered, but their accumulation rates in this case differed. Thus, the initial rates of accumulation of the 17- and 8-mer were close. Moreover, the accumulation of the final 5-mer differed considerably: in the RNA/tandem complex it appeared within first minutes of the reaction, but only after a considerable lag period in complex RNA/p20. These data testify that the tandem is involved not only in the consecutive accumulation of the shortened products (which is characteristic of complexes including extended oligonucleotides) but also in the parallel accumulation. This results from hydrolysis of each duplex segment formed by RNA and the short oligonucleotide of the tandem. Although the order of recognition and cleavage of RNA target by ribonuclease H depends on the type of the hybrid duplex, the destruction of RNA target within complex RNA/tandem and in complex with the full-size oligonucleotide occurs with a close effectiveness.

[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.

[Increase in the effectiveness of site-specific cleavage of target DNA by tetranucleotide bleomycin derivatives using oligonucleotide-effectors]. / Povyshenie éffektivnosti sait-spetsificheskogo rasshepleniia DNK- misheni bleomitsinovym proizvodnym tetranukleotida s pomoshch'iu oligonukleotidov-éffektorov.

The efficiency of site-specific interaction of target DNA with bleomycin derivatives of short nucleotides can be significantly increased using flanking effector oligonucleotides. The cleavage of a 20-mer single-stranded target DNA by a tetranucleotide containing a bleomycin A5 residue at the 5'-end was studied in the presence of effector oligonucleotides bearing phenazine residues at the 5'- and 3'-ends. In the presence of two effectors, the extent of the target DNA modification at 37 degrees C increased from 20 to 70%. Site-specific cleavage occurs, by up to 90%, at a single site of the target DNA. The melting temperature of the complementary complex formed by the bleomycin A5-modified tetranucleotide and target DNA in the presence of two effectors was 45 degrees C, whereas in the absence of the effectors, below 7 degrees C.