[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.

[Sensitized photomodification of DNA with binary sysytems of oligonucleotide conjugates. VI. Effect of substituents in the anthracene residue of the sensitizer]. / Sensibilizirovannaia fotomodifikatsiia DNK binarnymi sistemami oligonukleotidnykh kon''iugatov. VI. Vliianie zamestitelei v antratsenovom ostatke sensibilizatora.

Photomodification of ssDNA by binary systems of oligonucleotide conjugates complementary to the adjacent sequences of the target DNA was studied. One of the conjugates comprised a substituted anthracene as a sensitizer; the other, p-azidotetrafluorobenzaldehyde 3-aminopropionylhydrazone as a photoreagent. The sensitized photomodification is initiated by the 365-580-nm light through an efficient energy transfer from the photoexcitated sensitizer onto the photoreagent in a complementary complex of the binary system with the DNA target where the sensitizer and the photoreagent are sterically converged. Influence of substituents in the anthracene residue on the efficiency of the DNA sensitized photomodification was considered. The oligonucleotide conjugate of anthracene-9-al 3-aminopropionylhydrazone allows highly specific initiation of the sensitized photomodification upon irradiation with visible light at > 460 nm in conditions generating no photoreaction in the sensitizer's absence.

Highly selective affinity labeling of DNA-polymerase from Thermus thermophilus B35 by a binary system of photoreactive agents.

The thermostable DNA-polymerase from Thermus thermophilus B35 (Tte-polymerase) was affinity labeled by a binary system of photoreagents comprising base-substituted TTP analogs. The 5;-[32P]-labeled primer was elongated by Tte-polymerase in the presence of a TTP analog containing the photoreactive 2,3,5, 6-tetrafluoro-4-azidobenzoyl group (FAB-4-dUTP). Then the reaction mixture was UV-irradiated (365-450 nm) in the presence or the absence of a photosensitizer (TTP analog containing a pyrene moiety, Pyr-dUTP). The initial rate of the Pyr-dUTP-sensitized photomodification was almost 10-fold higher than the rate of direct photomodification (in the absence of Pyr-dUTP); in the case of the sensitized modification, the product of covalent cross-linking of the photoreactive primer with Tte-polymerase was apparently homogenous according to the data of electrophoresis. The enzyme was protected from the photosensitized modification by dNTP. To confirm the selectivity of the photosensitized modification of Tte-polymerase, another DNA-binding protein (human replication factor A, RPA) was added to the reaction mixture. In the presence of the photosensitizer (Pyr-dUTP), RPA was not labeled and only Tte-polymerase was modified, whereas in the case of direct modification, Tte-polymerase and the p32 and p70 subunits of RPA were labeled. The suggested method enables highly selective affinity modification of DNA-polymerases.

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).

Refined high-field NMR solution structure of a binary-addressed pyrene/perfluoro-azide complementary DNA oligonucleotide system shows extensive distortion in the central nick region.

The structural analysis of the photoactivated binary system of complementary-addressing nucleic acid sequences (1:2:3) by high-resolution NMR spectroscopy and restrained molecular dynamics is reported. The binary system comprised a 12 base-pair target DNA sequence, pdGTATCAGTTTCT (1), and two hexanucleotides, (dAGAAACp-L-Az (2) and Pyr-pdTGATAC (3)), complementary to neighbouring sites in the target DNA. Oligonucleotide (2) is conjugated with a p-azidotetrafludrobenzyl group (Az) via a linker group (L), and the other oligonucleotide (3) is equipped with the photosensitizing pyrenyl-1-methylamino group (Pyr). We now extend the structural analysis of 1:2:3, which was previously based on qualitative 2D 1H-NMR data and thermodynamic analysis of complex formation from UV-visible thermal denaturation experiments. In the current work structural refinement was performed by separate molecular dynamics runs for six different starting structures based on 318 proton-proton distance-range constraints, evaluated from the 1H-NOESY spectrum (tau(mix) = 200 ms, 600 MHz) using complete relaxation matrix analysis (NMR/TRIAD/MARDIGRAS). Additional Watson-Crick hydrogen bond restraints were included in the calculations based on the detected signals from the exchangeable protons, using REFOPT(NY) methods. The final averaged structure obtained from the six refined co-ordinate sets showed a considerable degree of axis bend (62.5 degrees) with the bending point in the middle of the duplex in the region of the backbone nick between the two short oligonucleotides. The complex behaves dynamically as the equivalent of two short B-DNA-like duplexes displaying a hinge-like flexing at their junction. In all final structures the Pyr function location was very restricted, the aromatic group lying in the duplex minor groove near residues 4T, 5C and 2T. In contrast, the location of the perfluoroazido group was different in all the final structures, indicating the high flexibility of this group in the duplex. The only feature common to all six final azido group orientations was the outside location on the side of the major groove. The distance between the Pyr and Az groups varied from 11 A to 24 A for the six final structures (17 A, final average structure). The dynamics of duplex denaturation for 1:2:3 was probed by monitoring the temperature-induced NMR line broadening of the imino protons in a 1D variable temperature NMR experiment. The melting of 1:2:3 starts both from the ends and from the middle part of the duplex at the backbone break between the two short oligonucleotides reflecting the destabilisation of the pyrene-arylazido nick region in the duplex.

Structure of photoreactive binary system of oligonucleotide conjugates assembled on the target nucleotide sequence.

Recently we have developed an approach to superspecific photomodification of nucleic acids by binary systems of oligonucleotides conjugated to precursor groups capable of assembling into photoactivatable structure upon simultaneous binding of the conjugates to the target. We have investigated the solution structure of a model binary system 1:2:3, where 1 is the target 12-mer 5'-pdGTATCAGTTTCT, 2 is the photoreactive conjugate 5'-dAGAAACp-NH(CH2)2NH-Az and 3 is the sensitizing conjugate 5'-Pyr-pdTGATAC (Az is p-azidotetrafluorobenzoyl group and Pyr is the pyrenyl-1-methylamino group). The photoreaction within this complex results in crosslinking of reagent 2 with N7-position of the G7 residue of the target thus indicating that the photoreactive Az residue is located in the major groove near the G7 residue. The center-to-center distances between the Pyr and Az moieties in complex 1:2:3 independently determined by the Pyr-group fluorescence quenching and the Az-group sensitized photodecomposition were 11.2 and 12.6 A, respectively.

[Sensitized photomodification of DNA with binary systems of oligonucleotide conjugates. V. Effect of the structure of the target DNA. Quantitative photomodification]. / Sensibilizirovannaia fotomodifikatsiia DNK binarnymi sistemami oligonukleotidnykh kon''iugatov. V. Vliianie stroeniia DNK-misheni. Kolichestvennaia fotomodifikatsiia.

A sensitized photomodification of several single-stranded target DNAs by binary systems of oligonucleotide conjugates complementary to the adjacent regions of DNA was performed. One of the conjugates contained a sensitizer (pyrene, anthracene, or 1,2-benzanthracene), and another conjugate contained a photoreagent 4-azidotetrafluorobenzalhydrazone. The sensitized photomodification is initiated by irradiation at 365-580 nm due to effective energy transfer from the excited sensitizer to the photoreagent in a complementary complex of the binary system with the target DNA where the sensitizer and photoreagent are brought sterically together. Conditions for the quantitative photomodification of a single-stranded DNA by the binary system of oligonucleotide conjugates were found. The maximum degree of photomodification depends on the number of guanosine residues in the (pG)n sequence of the target DNA at the modification site: at n = 1 the yield of covalent adducts was 62-68%, at n = 2, 75-82%, and at n = 4, 98-99%.

[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 mammalian DNA polymerase beta. A new approach for highly selective affinity labeling of polymerases.

To enhance the specificity of polymerase photoaffinity labeling, a novel approach based on sensitized photomodification has been developed. A base-substituted analog of TTP containing a pyrene group (PyrdUTP) was synthesized and used as an active site-bound photosensitizer for photoaffinity modification of DNA polymerase beta (pol beta). 5'-[32P]-labeled primer was elongated in situ by pol beta with a photoreactive analog of TTP (FAB-4-dUTP). The pyrene sensitizer (PyrdUTP), excited by light (365-450 nm), can activate the photoreagent, cross-linking it to pol beta as a result of fluorescence resonance energy transfer. The initial rate of pol beta photomodification was shown to increase by a factor of ten. The selectivity of pol beta photosensitized modification was proved by adding human replication protein A.

Affinity labelling of the tick-borne encephalitis virus RNA replicase proteins by 4-N-exo-base-substituted photoreactive CTP analogs.

4-N-exo-base-substituted photoreactive analogs of CTP were designed and synthesized. Two flavivirus proteins NS5 and NS3 are shown to be labelled after RNA synthesis in the presence of the analogs, irradiation by UV-light (313 nm) and subsequent [alpha-32P]NTP incorporation.

[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%.

Structural studies by high-field NMR spectroscopy of a binary-addressed complementary oligonucleotide system juxtaposing pyrene and perfluoro-azide units.

Recently, a new approach has been proposed to improve the site-specificity and efficiency of the modification of nucleic acid target sequences, the binary system of complementary-addressing nucleic acid sequences. The binary system comprises two oligonucleotides, one modified with a photosensitizing group and the other with a photoreactive group. The sites of chemical modification are arranged to bring the two chemical functions close enough together in space to allow efficient energy transfer from the photo-excited photosensitizer to an arylazide moiety which expels N2 to form a nitrene which subsequently covalently labels the target nucleic acid. Structural analysis performed by high-resolution 2D NMR spectroscopy (400 MHz and 600 MHz) are reported for the model binary system 1:2:3, where 1 is the target 12-mer pdGTATCAGTTTCT, 2 is a photoactivatable fluoroazide derivative dAGAAACp-L-Az and 3 is the photosensitizer derivative Pyr-pdTGATAC (here: Az is the p-azidotetrafluorobenzyl group, Pyr the pyrenyl-1-methylamino group, L a linker group). The assignment of oligonucleotide and modifying group protons was performed using 1H COSY, TOCSY and NOESY experiments. Comprehensive analysis of 1H NOESY spectra of 1:2:3 showed that terminal fragments of the complex [5'p-1T-2G-3A-4T-], [-21A-22T-23A-24C], [-8T-9T-10T-11C-12T] and [13A-14G-15A-15A-17A-18C-] gave a continuous set of intra- and inter-nucleotide interactions, typical of regular double-stranded B-DNA. In contrast, the central region of the complex composed of 5C, 6A, 7G, 19T and 20G nucleotide residues, nearest the Pyr and Az groups, was found to be distorted. Thus some signals from aromatic and/or sugar-ring protons of the above nucleotide residues were extremely broadened or almost absent. Moreover, some intra- and/or inter-nucleotide interactions, typical of the regular DNA duplex, were not detected for the [-5C-6A-7G-] and [-19T-20G-] regions of the tandem system. Instead of that, some cross-peaks of low-intensity between the H2 proton of the Pyr group and 7G(H1'), 7G(H2'/H2"), 7G(H3'), 4T(H2"), 4T(H4') and 4T(H5'/H5") were observed. Additional 1H -1H NOE-interactions between methylene protons of the linker group L and some sugar ring protons of 18C nucleotide residue were detected. A preliminary structural model, constructed using proton-proton distances between Pyr and the DNA and Az-L and DNA obtained from a 1H NOESY experiment at 300 ms mixing time as constraints for the refinement of the structure, displayed significant distortion from B-DNA of the double-stranded helix in the middle of the complex, (-5C-6A-7G, -18C-19T-20G-). The Pyr group was located in what remains of the minor groove near 4T, 5C, 6A and 7G and the centroid of the azide ring less than 9A degrees from the centroid of the ring system of Pyr group.

Sensitized photomodification of single-stranded DNA by a binary system of oligonucleotide conjugates.

A photoactivatable binary system of oligonucleotide conjugates that form reactive species when assembling on a target nucleotide sequence has been developed. The binary system consists of two oligonucleotides. One contains a photosensitizing group, and the second contains a photoreactive group. Binding of the oligonucleotides to adjacent sequences in the target nucleic acid brings the groups in contact, which allows transfer of the absorbed energy from the sensitizer to the reagent and triggers crosslinking of the reagent to the target. One advantage of the binary system is the improved specificity, which is determined by independent binding of two oligonucleotides to the target sequence. Another advantage is the very high efficiency of the reaction achieved because each molecule of the target-bound sensitizing conjugate can activate many photoreactive oligonucleotide conjugate molecules bound to the target sequence.

[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.