[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.

[Structure of an oligonucleotide of bleomycin A5 from 13C-NMR data]. / Struktura oligonukleotidnogo proizvodnogo bleomitsina A5 po dannym spektroskopii 13C-IaMR.

The localization of the covalent bond in the conjugates of bleomycin A5 and oligonucleotides was established by 13C NMR using the bleomycin derivative of uridine-5'-phosphate synthesized as a model compound. The phosphate group of the nucleotide was shown to form a phosphamide bond with the primary amino group of the spermidine moiety of bleomycin A5. The formation of the P-N bond causes the downfield shift of the signals of the neighboring carbon atoms of the spermidine fragment by 1.8 and 4.2 ppm and the splitting of the signal of the C-2 atom of the spermidine fragment with J 6.8 Hz due to vicinal spin-spin coupling with the phosphorous atom.

[Catalytic cleavage of target DNA in a complementary complex by a bleomycin oligonucleotide derivative]. / Kataliticheskoe rasshcheplenie DNK-misheni bleomitsinovym proizvodmym oligonukleotida v sostave komplementarnogo kompleksa.

For the first time, reagents that are capable of catalytic site-specific cleavage of DNA were synthesized based on oligonucleotides. One molecule of reagent (Blm-R)pd(CCAAACA) containing bleomycin A5 residue (Blm-RH) could degrade three molecules of target DNA pd(TGTTTGGCGAAGGA). The reagent displayed maximum catalytic activity in a temperature range that was close to the melting temperature of the reagent-target DNA complex. The number of the target DNA molecules that could be degraded by the reagent was limited by the degradation of the antibiotic residue itself during the oxidative degradation of the target DNA. The reagent catalyzed the subsequent degradation of residues G7, T5, T4, and T3 of the target DNA at an equimolar reagent-target DNA ratio. When the tenfold excess of the target DNA was used, the reagent degraded primarily the single G7 residue of the target DNA by 30%.

[Cleavage of a double-stranded DNA target by bleomycin derivatives of oligonucleotides, forming a ternary complex]. / Rasshcheplenie dvukhtsepochechnoi DNK-misheni bleomitsinovymi proizvodnymi oligonukleotidov, obrazuiushchimi troinoi kompleks.

Hexadecathymidylate derivatives, containing covalently-bound antitumor antibiotic bleomycin A5, were shown to form a triple-helix complex with double-strand 30-bp DNA-target and to carry out within this complex complementary-addressed DNA modification. Fivefold excess of reagent in relation to target leads to non-specific cleavage mainly of pyrimidine-rich DNA strand. Total degrees of the target-strand cleavage by 5'- and 3'-bleomycin derivatives of hexadecathymidylate were 25 and 35% for purine-rich strand and 47 and 36% for pyrimidine-rich strand. Degrees of non-specific cleavage by 5'-bleomycin derivative of hexadecanucleotide that does not form triple-helix were 6 and 16% for purine- and pyrimidine-rich strands, respectively. Comparison of these data has shown that site-specific cleavage prevailed nonspecific one. Triplex of 5'-bleomycin derivative with DNA melted by 5 degrees C lower (m.p. 40 degrees C) than the similar triplex of hexadecathymidylate. Temperature lowering from 50 to 20 degrees C increases the DNA-cleavage degree according to the increase in the part of target molecules involved in triple-helix formation.

Comparison of interactions of 5'-derivatives of deoxyoctathymidylate with human DNA polymerize alpha and HIV reverse transcriptase.

Km and Vmax values for d(pT8) and its derivatives containing various 5'-end groups were estimated in the reaction of DNA polymerization alpha catalyzed by DNA polymerase alpha and HIV-RT. The effect of 5'-end modification of primer is more pronounced in the case of HIV-RT. Strong influence is observed for an intercalating (ethidium) group. The affinity of EtpT8 is 200-fold higher than that of d(pT8). Attachment of Phn-, Dnm- and Hem-groups results in the increase of affinity of modified primer from 10 up to 20 times. For DNA polymerase alpha the influence of modifiers on primer affinity is much weaker. The effect of 5'-end residues on the Vmax values is also more pronounced for HIV RT. The way to improve selective interaction of oligonucleotide derivatives with the primer site of HIV RT is suggested.

Synthesis and high stability of complementary complexes of N-(2-hydroxyethyl)phenazinium derivatives of oligonucleotides.

Two simple methods for the synthesis of oligonucleotides bearing a N-(2-hydroxyethyl)phenazinium (Phn) residue at the 5'- and/or 3'-terminal phosphate groups are proposed. By forming complexes between a dodecanucleotide d(pApApCpCpTpGpTpTpTpGpGpC), a heptanucleotide d(pCpCpApApApCpA), and Phn derivatives of the latter, it is shown that the introduction of a dye at the end of an oligonucleotide chain strongly stabilizes its complementary complexes. The Tmax and the thermodynamic parameters (delta H, delta S, delta G) of complex formation were determined. According to these data, coupling of a dye with the 5'-terminal phosphate group is the most advantageous: delta G(37 degrees C) is increased by 3.59 +/- 0.04 kcal/mol compared to 2.06 +/- 0.04 kcal/mol for 3'-Phn derivatives. The elongation of the linker, which connects the dye to the oligonucleotide, from a dimethylene up to a heptamethylene usually leads to destabilization of the oligonucleotide complex. The complementary complex formed by the 3',5'-di-Phn derivative of the heptanucleotide was found to be the most stable among all duplexes investigated. Relative to the unmodified complex the increase in free energy was 4.96 +/- 0.04 kcal/mol. The association constant of this modified complex at 37 degrees C is 9.5 x 10(6) M-1, whereas the analogous value for the unmodified complex is only 3 x 10(3) M-1.

Sequence-specific cleavage of single-stranded DNA by oligonucleotides conjugated to bleomycin.

Cleavage of a single-stranded DNA fragment by complementary oligonucleotides conjugated to bleomycin A5 has been investigated. The conjugates efficiently cleave the DNA at the GT sequences near the oligonucleotide binding site. The temperature dependence of the reaction and the composition of the degradation products indicate that the oligonucleotide-linked bleomycin attacks the available double-stranded DNA regions within the oligonucleotide-DNA duplex and in the hairpin DNA region in the vicinity of the carrier oligonucleotide binding site.

[Direct breaks in a single-stranded DNA fragment by a bleomycin-derived oligonucleotide]. / Priamye razryvy odnotsepochechnogo fragmenta DNK bleomitsinovym proizvodnym oligonukleotida.

A method for coupling bleomycin to oligonucleotides is suggested. The reaction was carried out between the amino group of the spermidine residue of the bleomycin A5 Cu(II)-complex (Cu(II)Blm-RH) and the 5'-phosphate group of the oligonucleotide pd(CCAAACA) (I) activated with a mixture of triphenylphosphine and 2,2'-dipyridyldisulphide in the presence of 4-N,N-dimethylaminopyridine-1-oxide. The resultant compound (Ia) (yield 70%) forms more stable complementary complexes than the parent oligonucleotide (delta Tm = 11 degrees C). When Cu(II) ion was removed from (Ia), compound (Ib) formed which effectively (80%) cleaved pd(TGTTTGGCGAAGGA). Neither pd(TCCTTCG) nor the oligonucleotide tail of the reagent (Ib) were destroyed under the cleavage conditions. Free Blm-RH and bleomycin bound in the reagent (Ib) damage different regions of the target.

5'-derivatives of oligonucleotides as primers of DNA polymerization catalyzed by AMV reverse transcriptase and Klenow fragment of DNA polymerase 1.

The Km and Vmax values for d(pT)8 and its derivatives containing various 5'-end groups were estimated in the reaction of polymerization catalyzed with AMV-RT and FK. The change in affinity of modified primers was more pronounced in the case of AMV-RT than in the case of FK. Introducing in d(pT)8 of intercalators such as phenazinium, ethidium and daunomycin residues results in 2.7-, 8.7- and 11-fold increases in the primer affinity to AMV-RT, respectively. However, in the case of hemin and cholesterol derivatives the Km values were 3 and 5 times higher than those for d(pT)8. Compared to d(pT)8, the affinity of FK to all the above analogs was 2.3-3.6 times higher with the exception of cholesterol derivative to which it was 2.4-fold lower. The effect of the 5'-end residues on the Vmax values of d(pT)8 was small and ranged from 44% to 120% of that for d(pT)8. Therefore such reactive derivatives of oligonucleotides can be used as effective primers of AMV-RT and FK. Possible reasons for various effects of the 5'-end residues of the primer on its interaction with FK or AMV-RT in the presence of poly(A) are discussed.

[Synthesis, structure and properties of rubomycin derivatives of mono- and oligonucleotides]. / Sintez, struktura i svoistva rubomitsinovykh proizvodnykh mono- i oligonukleotidov.

Daunomycin derivatives of pT(DT) and oligodeoxynucleotides were synthesized using reactive zwitter-ionic 4-N,N-dimethylaminopyridine derivatives of the terminal phosphate group. Daunomycin oligodeoxynucleotide analogues form more stable complementary complexes than the corresponding non-modified oligonucleotides. Both one- and two-dimensional (2D NOESY and 2D COSY) NMR spectra of DT were recorded and the proton signals assigned. From the detected cross-relaxation between H6 of thymidine and H1', H2', H2" of the carbohydrate residue of daunomycin it was concluded that, in DMSO, the DT molecule has a rather stable conformation, apparently due to the stacking interaction between the mononucleotide and daunomycin residues.