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.

[Complementary-addressed photomodification of DNA-targets by arylazide and perfluoroarylazide oligonucleotide derivatives. IV. Photomodification of ss- and ds-DNA-fragments]. / Komplementarno-adresovannaia fotomodifikatsiia DNK-mishenei arilazidnymi i perftorarilazidnymi proizvodnymi oligonukleotidov. IV. Fotomodifikatsiia ss- i ds-DNK-fragmentov.

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

[Complementary-addressed photomodification of nucleic acids by arylazide and perfluoroarylazide oligonucleotide derivatives. III. Oligonucleotide reagents with a photoactive group at the end or inside the chain; tandem reagents]. / Komplementarno-adresovannaia fotomodifikatsiia nukleinovykh kislot arilazidnymi i perftorarilazidnym proizvodnymi oligonukleotidov. III. Oligonukleotidnye reagenty s fotoaktivnoi gruppoi na kontse ili vnutri tsepi; tandemy reagentov.

Photomodification of target oligonucleotides with reagents bearing p-azidotetrafluorobenzamide group at various positions of the oligonucleotide address was investigated. The photoactive group was attached to the 5'- or 3'-terminal phosphate or at the C5-position of a deoxyuridine residue at the 5'-end or inside the oligonucleotide chain. The reagents with the internal photoactive group modified the target with 50-55% efficiency (fraction of covalent adducts reagent-target), whereas the derivatives with a terminal reactive group were more effective (70%). The main point of the modification was the guanosine residue of the target which located near to the photoactive group and was not involved into the duplex formation. Tandems of reagents which are complementary to neighbouring sites of the target modify predominantly the same guanosine residue, with up to 80% extent.

Human immunodeficiency virus type 1 reverse transcriptase. Affinity labeling of the primer binding site.

Affinity modification of the primer site of HIV1-RT was performed with an oligonucleotide derivative containing a photoreactive azido group at the 5' end of d(pT)10. The affinity of HIV1-RT for d(pT)10 and for its derivative was first estimated by measuring the Michaelis constants of these two oligonucleotides acting as primers in the retrotranscription of poly(rA). The enzyme was then inactivated under UV-irradiation at 303-365 nm in the presence of ArN3-d(U*T9); the dependence of the rate of inactivation on primer concentration was found to be consistent with the Km value. Last, selectivity of affinity modification was demonstrated through elongation of the covalently bound primer and selective protection of inactivation by d(pT)10 or tRNA(Lys).

[Site-specific photomodification of nucleic acids with arylazide and perfluoroarylazide oligonucleotide derivatives. II. Specificity in relation to nucleosides]. / Komplementarno adresovannaia fotomodifikatsiia nukleinovykh kislot arilazidnymi i perftorarilazidnymi proizvodnymi oligonukleotidov. II. Issledovanie spetsifichnosti po otnosheniiu k nukleozidam.

Oligonucleotide reagents bearing aromatic azido groups of different structures were shown to be suitable for nucleoside specific photomodification of nucleic acids. Modification of the pentadecanucleotide targets d(TAAGTGGAGTTTGGC), d(TAAGTGGAAAAAAAA), d(TAAGTGGACCCCCCC) and d(TAAGTGGATTTTTTT) was investigated with reagents d(UCH2OCH2CH2NHCORCCACTT) carrying a photoactive group R(R1-n-azidotetrafluorophenyl-reagent (I), R2-2-nitro-5-azidophenyl-reagent (II) and R3-n-azidophenyl-reagent (III)) at C-5-modified deoxyuridine. Photomodification did not exceed 5% for the targets in case of reagent (III); the modification extent was 25-50% depending on the target sequence for reagent (II); reagent (I) with perfluoro azido group was the most effective, that provided 60-70% of modification. Reagents (I) and (II) were found to be sensitive to the nucleoside sequence of the target: the most vulnerable sites for reagent (I) and (II) were guanine and cytosine residues, respectively. These bases were modified predominantly when being adjacent to the addressed site of the target.

[Effective complementarily-addressed photomodification of nucleic acids by oligonucleotide derivatives, containing aromatic azido groups]. / Effektivnaia komplemntarno adresovannaia fotomodifikatsiia nukleinovykhkislot proizvodnymi oligonukleotidov, soderzhashchikh aromaticheskuiu azidogruppu.

Highly effective site-specific photomodification of a DNA-target was carried out with oligonucleotide reagents carrying aromatic azido groups. Oligonucleotide derivatives with a photoactive function R on the 5'-terminal phosphate and at C-5 atom of deoxyuridine were synthesized: R1NH(CH2)3NHpd(TCCACTT) and d(ULNHRCCACTT), where R1 is p-azidotetrafluorobenzoyl, R2 is 2-nitro, 5-azidobenzoyl, R3 is p-azidobenzoyl; LNH = -CH2NH-, -CH2OCH2CH2NH- or -CH2NHCOCH2CH2NH-. The prepared compounds form stable complementary complexes and effect site-specific photomodification of the target DNA. The modification of pentadecanucleotide d(TAAGTGGAGTTTGGC) with the reagents was investigated. Maximum extent of modification strongly depended on the reagent's type, the photoreagent with R1 being the most effective. Whatever the binding site was, this agent provided a 65-70% modification in all cases except LNH = -CH2NH-, when the yield was twice lower. For the reagents bearing R1 the modification sites were identified. Selective modification at the G9 residue was detected in the case of LNH = -CH2OCH2CH2NH- and when a photoactive group was linked to the terminal phosphate.