Sensitized photomodification of DNA with binary systems of oligonucleotide conjugates. IV. Photoinduced electron transfer

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 sysytems of oligonucleotide conjugates. VI. Effect of substituents in the anthracene residue of the sensitizer

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.     

Sensitized photomodification of DNA with binary systems of oligonucleotide conjugates. V. Effect of the structure of the target DNA. Quantitative photomodification]

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 site-specific photomodification of ssDNA by binary systems of oligonucleotide conjugates: VI. Effect of substituents in the sensitizer anthracene residue

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. For Part V, see [1]; prefix “d” in designations of oligonucleotides is omitted.     

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.     

Photomodification of nucleic acids by N-(2-hydroxyethyl)phenazine derivatives of oligonucleotides

Photomodification of a 302-membered single-stranded DNA fragment by 5'-mono- and 3',5'-di-N-(2-oxyethyl)phenazine (Phn) derivatives of oligonucleotides has been investigated. Under strong laser irradiation (lambda 532 nm; power density 2,5 GV/cm2, irradiation dose 30 J) the DNA fragment in the presence of Phn-reagents was significantly destructed (up to 70-95%). The level of complementary addressed modification (24-51%) is a direct function of the length of oligonucleotide address of the photoreagent and the amount of Phn residues, stabilizing the complementary complex. The character of the nonaddressed modification is close to the statistic one, although for a number of photoreagents a rather efficient nonspecific modification of 5'-terminal sequence of target DNA has been detected. Of interest also is an unusually broad positional direction of the DNA fragment photomodification in the area of perfect complementary coupling of 5'-Phn-reagents.     

Synthesis of nucleotide derivatives containing 1-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamido)-2,3-propanediol for photoaffinity modification of proteins and nucleic acid

1-(4-(3-(Trifluoromethyl)-3H-diazirin-3-yl)benzamido)-3-O-(4,4'- dimethoxytrityl)-2,3-propanediol phosphoramidite was synthesized and used as a modified unit in the automatic synthesis of oligodeoxyribonucleotides. Pentadecathymidylates with various numbers of 2,3-propanediol moieties substituted with aryl(trifluoromethyl)diazirinyl (ATFMD) were obtained, and the thermal stability of their duplexes with (dA)15 were studied. One ATFMD-propanediol residue was shown to reduce the thermal stability of the duplex by 8-9 degrees C. The irradiation of the ATFMD-containing duplexes by UV light with the wavelength of 350 nm was found to cause the cross-linking reaction of the ATFMD-containing strand with the complementary strand and the formation of the cross-linked duplexes. The photomodification efficiency was independent of the oligonucleotide sequence, with each ATFMD group providing for 5% cross-linking. The irradiation of an ATFMD-containing duplex, a substrate of the HIV-1 integrase, in the presence of this enzyme resulted in the covalent DNA-protein complex. The oligonucleotides with the 1-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamido)-2,3-propanediol moiety in their chains can be used for the photoaffinity modification of both nucleic acids and proteins that recognize them. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.     

Synthesis of Oligonucleotide Derivatives with Aryl(trifluoromethyl)diazirine Moiety for the Photoaffinity Modification of Proteins and Nucleic Acids

1-(4-(3-(Trifluoromethyl)-3H-diazirin-3-yl)benzamido)-3-O-(4,4"-dimethoxytrityl)-2,3-propanediol phosphoramidite was synthesized and used as a modified unit in the automatic synthesis of oligodeoxyribonucleotides. Pentadecathymidylates with various numbers of 2,3-propanediol moieties substituted with aryl(trifluoromethyl)diazirinyl (ATFMD) were obtained, and the thermal stability of their duplexes with (dA)₁₅ were studied. One ATFMD-propanediol residue was shown to reduce the thermal stability of the duplex by 8–9°C. The irradiation of the ATFMD-containing duplexes by UV light with the wavelength of 350 nm was found to cause the cross-linking reaction of the ATFMD-containing strand with the complementary strand and the formation of the cross-linked duplexes. The photomodification efficiency was independent of the oligonucleotide sequence, with each ATFMD group providing for 5% cross-linking. The irradiation of an ATFMD-containing duplex, a substrate of the HIV-1 integrase, in the presence of this enzyme resulted in the covalent DNA–protein complex. The oligonucleotides with the 1-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzamido)-2,3-propanediol moiety in their chains can be used for the photoaffinity modification of both nucleic acids and proteins that recognize them.     

UV-induced vanadate-dependent modification and cleavage of skeletal myosin subfragment 1 heavy chain. 1. Evidence for active site modification

Ultraviolet irradiation above 300 nm of the stable MgADP-orthovanadate (Vi)-myosin subfragment 1 (S1) complex resulted in covalent modification of the S1 and in the rapid release of trapped MgADP and Vi. This photomodified S1 had Ca2+ATPase activity 4-5-fold higher than that of the non-irradiated control S1, while the K+EDTA-ATPase activity was below 10% of controls. There was a linear correlation between the activation of the Ca2+ATPase and the release of both ADP and Vi with irradiation time. Analysis of the total number of thiols and the ability of photomodified S1 to retrap MgADP by cross-linking SH1 and SH2 with various bifunctional thiol reagents indicated that the photomodification did not involve these reactive thiols. Irradiation of the S1-MgADP-Vi complex caused a large increase in absorbance of the enzyme at 270 nm which was correlated with the release of Vi from the active site, suggesting an aromatic amino acid(s) was (were) involved. However, analysis by three different methods showed no loss of tryptophan. All the irradiation-dependent phenomena could be prevented by replacing Mg2+ with either Co2+, Mn2+, or Ni2+. Unlike previous irradiation studies of Vi-dynein complexes [Lee-Eiford, A., Ow, R. A., & Gibbons, I. R. (1986) J. Biol. Chem. 261, 2337-2342], no peptide bonds were cleaved in photomodified S1. Photomodified S1 was able to retrap MgADP-Vi at levels similar to unmodified S1. Upon irradiation of the photomodified S1-MgADP-Vi complex, MgADP and Vi were again released from the active site, resulting in heavy chain cleavage to form NH2-terminal 21-kDa and COOH-terminal 74-kDa peptides. All evidence indicates that this new photomodification and subsequent chain cleavage occur specifically at the active site.     

Activation by laser irradiation of a "switched on" oligonucleotide reagent for an addressed modification of nucleic acids

Properties of oligonucleotide reagents containing an alkylating group of regulated reactivity (nitrogen mustard residue activatable upon mild borohydride reduction of the aromatic formyl group) have been studied. It was shown that these reagents can also be activated by irradiation with nitrogen laser light (lambda 337 nm). Activation of the reagent in complex with a target polydeoxyribonucleotide resulted in the addressed chemical modification of the target. The positional direction of the modification depended on the way of the activation (borohydride reduction or laser irradiation).     

Effective complementarily-addressed photomodification of nucleic acids by oligonucleotide derivatives, containing aromatic azido groups]

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.     

Wavelength dependence for the photoreactions of DNA-psoralen monoadducts. 2. Photo-cross-linking of monoadducts

The photoreactions of HMT [4'-(hydroxymethyl)-4,5',8-trimethylpsoralen] monoadducts in double-stranded DNA have been studied with complementary oligonucleotides. The HMT was first attached to the thymidine residue in the oligonucleotide 5'-GAAGCTACGAGC-3' as either a furan-side monoadduct or a pyrone-side monoadduct. The HMT-monoadducted oligonucleotide was then hybridized to the complementary oligonucleotide 5'-GCTCGTAGCTTC-3' and irradiated with monochromatic light. In the case of the pyrone-side monoadducted oligonucleotide, photoreversal was the predominant reaction, and very little cross-link was formed at all wavelengths. The course of the photoreaction of the double-stranded furan-side monoadducted oligonucleotide was dependent on the irradiation wavelength. At wavelengths below 313 nm, both photoreversal and photo-cross-linking occurred. At wavelengths above 313 nm, photoreversal of the monoadduct could not be detected, and photo-cross-linking occurred efficiently with a quantum yield of 2.4 X 10(-2).     

Site-specific and photo-induced alkylation of DNA by a dimethylanthraquinone-oligodeoxynucleotide conjugate.

A dialkyl-substituted anthraquinone derivative was synthesized and ligated to a sequence-directing oligodeoxynucleotide to examine its efficiency and specificity for cross-linking to complementary sequences of DNA. The anthraquinone appendage stabilized spontaneous hybridization of the target and probe sequences through non-covalent interactions, as indicated by thermal denaturation studies. Covalent modification of the target was induced by exposure to near UV light (lambda > 335 nm) to generate cross-linked duplexes in yields as great as 45%. Reaction was dependent on the first unpaired nucleotide extended beyond the duplex formed by association of the target and probe. A specificity of C > T > A = G was determined for modification at this position. The overall site and nucleotide selectivity seems to originate from the chemical requirements of cross-linking and does not likely reflect the dominant solution structure of the complex prior to irradiation.     

Photomodification of human immunocompetent blood cells

Near-UV irradiation (280-365 nm) at non-lethal doses increased lymphocyte E and EAC rosette-forming capacity, reduced cell proliferation in response to mitogen (PHA), induced an increase in the content of lipid peroxidation products in the cell culture medium. An antioxidant (alpha-tocopherol, 10(-7) M) administered before or immediately after near UV irradiation of lymphocytes reduced the above effects. The addition of an antioxidant to the culture medium 90 min after cell irradiation failed to reduce lymphocyte rosette-forming capacity. Near-UV irradiation of the blood reduced cell proliferative response to PHA. alpha-tocopherol (10(-7) M) administered before and immediately after the blood photomodification blocked the suppression of cell proliferation in response to mitogen.     

Biochemical and photometric studies of modification of collagen structure induced by UV irradiation

The influence of UV irradiation (270–380 nm) on the biochemical, fluorescence and colorimetric properties of collagen was studied. The long-term UV irradiation (120 h) was accompanied by the increase of the structural stability of collagen to specific and nonspecific proteolytic enzymes, by formation of new additional fluorophore containing compounds, by the increased amount of carbonyl groups in the collagen, and by significant changes in the distribution pattern of products of alkaline hydrolysis during gel chromatography. The coordinates of color of the collagen films have been also changed. These changes of collagen suggest that UV irradiation induces photomodification and photooxidation processes in collagen.     

Cooperative Interactions in Photosensitized Modification of DNA with Oligonucleotide-derived Binary Reagents

Quantitative characteristics of thermodynamic and kinetic cooperativity arising in the process of photomodification of a single-stranded DNA fragment with binary systems of oligonucleotide conjugates forming an active site on the target were studied. Oligonucleotides of the binary system were complementary to adjacent segments of the DNA target, and contained arylazide (X) and perylene (S) residues covalently attached to their terminal phosphates. Upon irradiation at the perylene absorption wavelength, the target was modified by the arylazide residue, which was activated owing to the contiguity with the sensitizing perylene group in the tandem complex. Basing on the kinetic data, the constants of association of both derivatives of oligonucleotides with the target were determined: K _x = 1.13 · 10⁶ M^–1, K _s = 1.49 · 10⁴ M^–1. It was determined that association of both oligonucleotides with the target proceeded with a positive cooperativity characterized by parameter = 45. The kinetic cooperativity parameter was found to be approximately equal to 200; this characterized the acceleration of target modification in complex with the binary reagent versus that in the absence of sensitizer.     

Light-induced enzyme system leading to pigmentation.

Alpha-Chymotrypsin was light sensitized by acylating with cis-cinnamoyl ester, a substrate interconvertible to the trans form by ultraviolet (UV) light. The degree of acylation by this method was complete leaving no residual activity of the enzyme. Upon UV irradiation the inhibited enzyme regained about 70% of its original activity, thereby adding light-sensitiveness to the proteolytic enzyme. In seeking a photographic application of the light-sensitized enzyme, a pigmenting enzyme was incorporated with it. The coupled enzyme system was shown to exhibit a light signal in the form of dark pigment slurry.     

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.     

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.     

The effect of UV irradiation and of UV-irradiated autologous blood on the functional state of human peripheral blood lymphocytes

The effect of UV irradiation (UVI, 254 nm) and of UV-irradiated autologous blood on the spontaneous and mitogen-induced DNA-synthetic activity of intact lymphocytes has been studied. Lymphocytes were isolated from nonirradiated and irradiated blood, and from the mixture of UV-irradiated blood with the intact one in the volume ratio close to that in the blood stream during UV-irradiated blood autotransfusion (1:10, 1:40, 1:160). It has been shown that UVI of the whole blood caused in some donors the increase in spontaneous DNA synthesis, while in others the decrease or no statistically significant changes were observed. The analysis of the results obtained shows an inverse relation of the UVI effect to the initial level of spontaneous DNA synthesis (r = -0.68). In contrast to direct UVI effect, an addition of UV-irradiated blood to the autologous intact one resulted in an increase in spontaneous DNA synthesis in lymphocytes of all the samples examined. A 7-day cocultivation of lymphocytes, isolated from irradiated and nonirradiated blood samples, revealed a 1.8 times increase compared to the calculated value. The mitogen-induced DNA synthesis has a low sensitivity to UV rays, since the mitogens and the irradiation of optical range have presumably the common targets. It is assumed that photomodification of HLA-D/DR antigens can be a trigger mechanism for activation of immunocompetent cells by UVI.