[Monomers for oligonucleotide synthesis with linkers carrying reactive residues: II. Synthesis of phosphoamidites based of uridine and cytosine and containing a linker with methoxyoxalamide groups in position 2']. / Monomery dlia oligonukleotidnogo sinteza s linkerami, nesushchimi reaktsionnosposobnye ostatki 2. Sintez fosfamiditov na osnove uridina i tsidina, soderzhashchikh v 2'-polozhenii linker s metoksioksalilamidnymi gruppami.

A convenient preparative synthesis of 2'-amino-2'-deoxyuridine was developed. Starting from 2'-amino-2'-deoxyuridine and 2'-amino-2'-deoxycytidine, monomers for the phosphoamidite oligonucleotide synthesis were obtained that carry a linker with methoxyoxalamide groups in position 2'. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.

[Monomers for oligonucleotide synthesis with linkers carrying reactive residues: I. Synthesis of deoxynucleoside derivatives with methoxyoxalamide groups in heterocyclic bases]. / Monomery dlia oligonukleotidnogo sinteza s linkerami, nesushchimi reaktsionnosposobnye ostatki 1. Sintez proizvodnnykh dezoksinukleotidov s metoksioksalilamidnymi gruppami, prisoedinennymi k geterotsiklicheskim osnovaniiam.

A number of monomers for the standard phosphoamidite oligodeoxynucleotide synthesis that carry reactive methoxyoxalamide groups attached to the thymidine, 2'-deoxycytidine, and 2'-deoxyadenosine heterocyclic bases were prepared. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.

[Chemical ribonucleases. 4. Analysis of the fragment structure of chemical ribonucleases based on 1,4-diazabicyclo[2.2.2]octane]. / Khimicheskie ribonukleazy. 4. Analiz fragmentnoi struktury khimicheskikh ribonukleaz na osnove 1,4-diazabitsiklo[2.2.2]oktana.

Artificial ribonucleases of the ABLkCm series were synthesized. They consist of a lipophilic alkyl radical (Et, n-C14H29, or C15H31) A, an "RNA-binding domain" B (bisquaternary salt of 1,4-diazabicyclo[2.2.2]octane), a "catalytic domain" Cm [histamine (C1) or histidine (C3) residue], and a "linker" Lk that joins the "domains" B and Cm [here, k is the number of methylene units (one or three) in the linker]. The effect of the "domain structure" on the catalytic properties of the chemical ribonucleases was analyzed using seven compounds of this series (ABL1C1, ABL3C1, ABL3C3, AC1, AB, BL2, and BL3C3). The catalytic activity of the compounds was assessed in the reaction of hydrolysis of the in vitro transcripts of human tRNA(Lys) and yeast tRNA(Asp) under physiological conditions. It was shown that only chemical ribonucleases that involve all the fragments of the ABLkCm construct can hydrolyze the substrate tRNA at a high rate (90% of tRNA is hydrolyzed for 10 h at 37 degrees C). The activity of the compounds is largely determined by the presence of a long lipophilic radical linked to 1,4-diazabicyclo[2.2.2]octane and a long linker, which joins the RNA-hydrolyzing and RNA-binding fragments. The results indicate an important role of hydrophobic interactions in the acceleration of the RNA hydrolysis reaction. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.

[Reagents for modification of protein-nucleic complexes. III. Site-specific photomodification of elongation complex of DNA polymerase beta with arylazide derivatives of primers sensitized with fluorescent ATP gamma-amide]. / Reagenty dlia modofikatsii belkovo-nukleinovykh kompleksov. III. Sait-spetsificheskaia fotomodifikatsiia élongiruiushchego kompleksa DNK-polimerazy beta arilazidnymi proizvodnymi praimerov, sensibilizirovannaia fluorestsentnymi gamma-amidami ATP.

ATP gamma-amides containing in gamma-N-position 1-methylpyrene, 9-methylanthracene, 10-chloro-9-methylanthracene, and 3-methylperylene residues were synthesized and characterized. These compounds were used as sensitizers of site-specific photomodification of the reconstituted elongating complex of the mammalian DNA polymerase beta. The photomodification was carried out with the use of photoaffine reagents, which were synthesized in situ by the 5'-(32)P-labeled primers extension with photoactive analogues of dCTP containing in the exo-N-position of cytosine various perfluoroarylazide groups. The effect of structures of the sensitizers and photoactive reagents on the efficiency and selectivity of photolinking of primers to the enzyme and template, as well as formation of a number of other photomodification products was studied. It was shown that the sensitizers containing 10-chloro-9-methylanthracene and 3-methylperylene residues allow preparation of photolinks in such irradiation conditions when photomodification in their absence is not essentially observed.

[Reagents for modification of protein-nucleic acids complexes. II. Site-specific photomodification of DNA-polymerase beta complexes with primers elongated by the dCTP exo-N-substituted arylazido derivatives]. / Reagenty dlia modifikatsii belkovo-nukleinovykh kompleksov. II. Sait-spetsificheskaia fotomodofikatsiia kompleksov DNK-polimerazy beta praîmerami, élongirovannymi ékzo-N-zamashchennymi arilazidnymi proizvodnymi dCTP.

Substrate properties of the earlier synthesized and characterized dCTP derivatives bearing in the exo-N-position of cytosine 2-(4-azido-2,3,5,6-tetrafluorobenzoylamino)ethyl (I), 2-(2-nitro-5-azidobenzoylamino)ethyl (II), 2-(4-azido-2,3,5,6-tetrafluorobenzylideneaminooxymethylcarbonylamino)ethyl (III), 4-(4-azido-2,3,5,6-tetrafluorobenzylideneaminooxy)butyloxy (IV), or 4-(4-azido-2,3,5,6-tetrafluorobenzylidenehydrazinocarbonyl)butyl- carbonylamino (V) groups were studied in the primer extension reaction catalyzed by rat DNA polymerase beta. Unlike the earlier results obtained with HIV reverse transcriptase, dCTP derivatives (I)-(III) were not recognized by rat DNA polymerase beta as dTTP analogues, and all the five nucleotides were utilized as dCTP analogues. When compared with dCTP, Km values for the synthesized dCTP derivatives were higher by a factor of 4-20; Vmax were 1-2.3 times higher for (I)-(III) and (V) but 20-fold lower for derivative (IV). Site-specific photomodifications of the primer-template-DNA polymerase beta complexes were carried out using photoreactive reagents PRI-PRV, obtained in situ by extension of 5'-32P-labeled primers with dCTP analogues (I)-(V), respectively, when exposed to UV irradiation at 303-313 nm. Reagents PRI and PRIV provided the maximum photocrosslinking of the 5'-32P-labeled primer to the DNA template (56%) and to the enzyme (20%), respectively. The lowest efficiency of photocrosslinking was observed for PRII (about 1%).

[Chemical ribonucleases. 3. Synthesis of organic catalysts of hydrolysis of phosphodiester bonds based on quaternary salts of 1,4-diazabicyclo(2.2.2)octane]. / Khimicheskie ribonukleazy. 3. Sintez organicheskikh katalizatorov gidroliza fosfodiéfirnykh sviazei na osnove chetvertichnykh solei 1,4-diazabitsiklo(2.2.2)oktana.

On the basis of imidazole and bisquaternary salts of 1,4-diazabicyclo[2.2.2]octane, a number of highly effective catalysts of the nDm series (here, n is the number of positive charges at neutral pH values and m is the digital code of the catalytically active fragment: 1, histamine, and 2, histidine methyl ester) were synthesized for the cleavage of the phosphodiester bonds in ribonucleic acids. A general method for the synthesis of chemical ribonucleases was suggested, which helps vary both the number of positive charges in their RNA-binding domain and the catalytic center. By the example of hydrolysis under physiological conditions of the in vitro transcript of tRNA(Lys) from human mitochondria, it was shown that the RNA cleavage rate with the nDm conjugates increases approximately 30-fold along with the increase in the number of positive charges from two to four.

Proteins neighboring 18S rRNA conserved sequences 609-618 and 1047-1061 within the 40S human ribosomal subunit.

The structure of human 40S ribosomal subunits has been probed by a cross-linking strategy based on the use of oligonucleotide derivatives that modify proteins in the vicinity of specific 18S rRNA sequences. The oligonucleotide derivatives carried a p-azidoperfluorobenzamide group at the 5' ends and were complementary to 18S rRNA sequences 609-618 and 1047-1061, homologous to the highly conserved regions designated as the "530 stem-loop" and "790 stem-loop", respectively, in Escherichia coli 16S rRNA. Ribosomal proteins surrounding these sequences were the main targets of the cross-linking. Proteins S3 and S5 were cross-linked to the derivative complementary to the sequence 609-618, and proteins S2 and S3 were modified by the derivative complementary to the sequence 1047-1061. Cross-linking was not affected by binding of 40S subunits to either poly(U) or poly(U) and Phe-tRNA(Phe).

[Sensitized photomodification of DNA with binary systems of oligonucleotide conjugates. IV. Photoinduced electron transfer]. / Sensibilizirovannaia fotomodifikatsiia DNK binarnymi sistemami oligonucleotidnykh kon''iugatov. IV. Foroinditsirovannyi perenos élektronov.

The photomodification of single-stranded DNA sensitized to visible light (450-580 nm) by a binary system of oligonucleotide conjugates complementary to adjacent DNA sequences was studied. One oligonucleotide carries a residue of the photoreagent p-azidotetrafluorobenzaldehyde hydrazone at its 3'-terminal phosphate, and the other has a residue of the sensitizer, perylene or 1,2-benzanthracene, at the 5'-terminal phosphate. The rate of photomodification sensitized by the perylene derivative is 300,000-fold higher than the rate of photomodification in the absence of the sensitizer. Since the excitation energy of perylene is lower than the energy necessary for the initiation of azide photodecomposition, it is likely that the sensitization in the complementary complex occurs by electron transfer from the azido group of the photoreagent to the excited sensitizer. The sensitization by the 1,2-benzanthracene oligonucleotide derivative occurs by means of singlet-singlet energy transfer, which enables this sensitizer to act as a unconsumable catalyst each molecule of which is able to initiate the photomodification of more than 20 DNA molecules. By both mechanisms, the photomodification occurs with high specificity on the G11 residue of the target DNA. The degree of sensitized photomodification reaches 72%.

[Sensitized photomodification of DNA with binary systems of oligonucleotide conjugates. III. Double-quantum sensitization]. / Sensibilizirovannaia fotomodifikatsiia DNK binarnymi sistemami oligonucleotidnykh kon''iugatov. III. Dvykhkvantovaia sensibilizatsiia.

Site-specific modification of single-stranded DNA by oligonucleotide derivatives of p-azido-O-(4-aminobutyl)tetrafluorobenzaldoxime sensitized by an oligonucleotide derivative of pyrenylethylamine was studied. Upon irradiation with the long-wave UV light (365-390 nm) of a DNA target-oligonucleotide reagent complementary complex, a considerable increase in the rate of sensitized photomodification at the G11 residue of the target relative to the direct photomodification was observed owing to the singlet-single energy transfer from the sensitizer onto the photoreagent. Upon simultaneous irradiation of the complex with UV and visible light in the region of the triplet-triplet absorption of pyrene (360-580 nm), an additional increase in the modification rate and a change in its site-direction (from the G11 to T13 residue) occurred through the two-photon triplet-triplet sensitization. The total extent of the structure photomodification amounted to 80%.

[Sensitized photomodification of DNA with binary systems. II. Spectral photosensitivity. One- and two quantum sensitization]. / Sensibilizirovannaya fotomodifikatsiya DNK binarnymi sistemami. II. Spektral'naia svetochuvstvitel'nost'. Odno- i dvukhkvantovaia sensibilizatsiia.

The efficiency of the photomodification of target single-stranded DNA with a decanucleotide derivative of p-azidotetrafluorobenzamide (direct photomodification) and with its complexes with decanucleotide derivatives of pyrene complementary to the adjacent segment of the target (sensitized photomodification) was studied as a function of the wavelength of long-wave UV light. The sensitized photomodification occurs mainly by singlet-singlet energy transfer from pyrene to azide in their complementary complex, which allows a significant increase in the rate and level of photomodification. When irradiation occurred simultaneously in the UV and visible regions (365-580 nm), two-photon triplet-triplet sensitization was revealed for the first time, which leads to a still greater acceleration of the target modification and a change of its site-direction from the G11 to T13 residue. The change of the mode of sensitization depending on the irradiation conditions allows the regulation of the reactivity of the binary system of oligonucleotide derivatives without altering their composition.

[New photoreactive N(4)-substituted dCTP analogues:synthesis, photochemical characteristics, and substrate properties in HIV-1 reverse transcriptase catalyzed DNA synthesis]. / Novye fotoaktivnye N(4)-zameshchennye analogi dCTP: poluchenie, fiziko-khimicheskie i substratnye svoistva pri sinteze DNK, kataliziruemoi obratnoi transkriptazoi VICh-1.

Photochemical characteristics and substrate properties of four newly synthesized dCTP analogues: N4-[2-(2-nitro-5-azidobenzoylamino)ethyl]-, N4-[2-(4-azidotetrafluorobenzylideneaminooxymethylcarbamoyl)ethyl] -, N4-[4-(4-azidotetrafluorobenzylideneaminooxy)butyloxy]-, and N4-[4-(4-azidotetrafluorobenzylidene hydrazinocarbonyl)butylcarbamoyl]-, and N4-[4-(4-azidotetrafluorobenzylideneaminooxy)butyloxy]-2'-de oxycytidine 5'-triphosphates as well as those of the earlier described N4-[2-(4-azidotetrafluorobenzoylamino)ethyl]- and 5-[E-3-(4-azidotetrafluorobenzoylamino)-1-propenyl)]-2'-deoxycytid ine 5'-triphosphates were compared. When being irradiated with UV light at a wavelength of 303-313 nm, the new analogues demonstrated greater than 10-fold higher photoactivity as compared with the old compounds. The first three new compounds were utilized by HIV-1 reverse transcriptase as dCTP and dTTP, while the last derivative was recognized only as dTTP. Once incorporated into the primer 3'-terminus, none of the analogues synthesized terminated further primer elongation with natural triphosphates.

[Sensitized photomodification by binary systems. I. Synthesis of oligonucleotide reagents, and the effect of their structure on the efficacy of target modification]. / Sensibilizirovannaia fotomodifikatsiia DNK binarnymi sistemami. I. Sintez oligonukleotidnykh reagentov, vliianie ikh stroeniia na éffektivnost' modifikatsii mishenei.

A highly effective sensitized photomodification of the target DNA by a binary system of oligonucleotide reagents complementary to adjacent regions of the target was accomplished. One of the oligonucleotides carries a photoregent p-azidotetrafluorobenzamide, and the other carries a pyrene sensitizer. Synthesis of the oligonucleotide derivatives was described. The rate and efficacy of the direct and sensitized target photomodifications depending on the location of the photoreagent and sensitizer at the 3'- and 5'-terminal phosphates and on the length of the linker between the sensitizer and addressed nucleotide were studied. The oligonucleotide derivatives with the photoreagent at the 3' terminus proved to be more effective (yield of the covalent adducts 70%). The rate of photomodification sensitized by UV light (365-390 nm) is 100-1500-fold higher than that of the direct site-specific modification and decreases with an increasing length of the linker. In all cases, modification occurs at the guanosine residue located near the photoreagent.

dNTP binding to HIV-1 reverse transcriptase and mammalian DNA polymerase beta as revealed by affinity labeling with a photoreactive dNTP analog.

The dNTP binding pocket of human immunodeficiency virus type 1 reverse transcriptase (RT) and DNA polymerase beta (beta-pol) were labeled using a photoreactive analog of dCTP, exo-N-[beta-(p-azidotetrafluorobenzamido)-ethyl]-deoxycytidine-5'- triphosphate (FABdCTP). Two approaches of photolabeling were utilized. In one approach, photoreactive FABdCTP and radiolabeled primer-template were UV-irradiated in the presence of each enzyme and resulted in polymerase radiolabeling. In an alternate approach, FABdCTP was first UV-cross-linked to enzyme; subsequently, radiolabeled primer-template was added, and the enzyme-linked dCTP analog was incorporated onto the 3'-end of the radiolabeled primer. The results showed strong labeling of the p66 subunit of RT, with only minor labeling of p51. No difference in the intensity of cross-linking was observed with either approach. FABdCTP cross-linking was increased in the presence of a dideoxyterminated primer-template with RT, but not with beta-pol, suggesting a significant influence of prior primer-template binding on dNTP binding for RT. Mutagenesis of beta-pol residues observed to interact with the incoming dNTP in the crystal structure of the ternary complex resulted in labeling consistent with kinetic characterization of these mutants and indicated specific labeling of the dNTP binding pocket.

Site-specific photomodification of single-stranded DNA targets by arylazide and perfluoroarylazide derivatives of oligonucleotides.

Highly efficient site-specific photomodification of single-stranded DNA targets was achieved with oligonucleotide reagents bearing aromatic azido groups (R (R1 = p-azidotetrafluorobenzoyl, R2 = 2-nitro-5-azidobenzoyl, R3 = p-azidobenzoyl) at either the terminal phosphate or at the C5 position of deoxyuridine at the end or inside of the oligonucleotide chain. The extent of modification strongly depends on the reagent type. It does not exceed 5% in the case of the reagent with R3. It was 25%-50% and 60%-70% for the reagents with R2 and R1 depending on the target structure. The reagent with perfluoroarylazido group R1 appeared to be most efficient. The extent of covalent adduct formation amounts to 70% for all reagents bearing a perfluoroarylazine group at the end of the oligonucleotide chain, independently of whether it was attached to the 3'- or 5'-phosphate or to the C5 of deoxyuridine. The reagents with the reactive group within the chain provided fewer cross-links (50%-55%). The reagents with R1 and R2 were found to be sensitive to the nucleotide structure of the target. Guanine and cytosine residues were modified preferentially when adjacent to the R1 or R2 group of the reagent, respectively.

Sequence-specific photomodification of single-stranded and double-stranded DNA fragments by oligonucleotide perfluoroarylazide derivative.

A highly efficient, sequence-specific photomodification of single-stranded (ss) and double-stranded (ds) DNA fragments was carried out with a hexadecathymidilate derivative, R approximately p(T)16 (R-perfluoroarylazido group), using 27-base pair DNA fragments as a target [table: see text] The main points of modification were G7 and G24 of the A-rich strand of the ss target and G7 and G22 of the A-rich and T-rich strands, respectively, for the ds target. The extent of photomodification was 60%-77% for ss DNA and 10%-53% for ds DNA depending on the reaction conditions. Photomodification increased in buffer with a high ionic strength (1.0 M) and at low temperature (4 degrees C) when presumably the triplexes were more stable.