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 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.     

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.     

Evaluating the specificity of antisense oligonucleotide conjugates. A DNA array analysis

Antisense oligonucleotides are potentially powerful tools for selective control of cellular and viral gene expression. Crucial to successful application of this approach is the specificity of the oligonucleotide for the chosen RNA target. Here we apply DNA array technology to examine the specificity of antisense oligonucleotide treatments. The molecules used in these studies consisted of phosphorothioate oligomers linked to the Antennapedia (Ant) delivery peptide. The antisense oligonucleotide component was complementary to a site flanking the AUG of the MDR1 message, which codes for P-glycoprotein, a membrane ATPase associated with multidrug resistance in tumor cells. Using a DNA array of 2059 genes, we analyzed cellular responses to molecules comprised of Ant peptide-oligonucleotide conjugates, as well as to the Ant peptide alone. Besides the expected reduction in MDR1 message level, 37 other genes (approximately 2% of those tested) showed changes of comparable magnitude. The validity of the array results was confirmed for selected genes using Northern blots to assess messenger RNA levels. These results suggest that studies using antisense oligonucleotide technology to modulate gene expression need to be interpreted with caution.     

Site-specific photomodification of nucleic acids with arylazide and perfluoroarylazide oligonucleotide derivatives. II. Specificity in relation to nucleosides]

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.     

Interactions of derivatives of short oligonucleotides with nucleic acids. VII. Effect of conformation changes in the duplex structure on on the specificity and efficacy of modification of target DNA by alkylating oligonucleotide derivatives]

The modification of a target DNA by alkylating oligonucleotide derivatives possessing various capacities for complex formation was studied. The binding properties of oligonucleotides were changed either by increasing their length (tetra-, octa-, and dodecamers) or by introducing a point substitution and/or an N-(2-hydroxyethylphenazinium) residue. It was found that conformational changes occurring in the structure of the target.reagent complex upon elevating the reaction temperature affect the efficiency and site-specificity of the alkylation. In the case of complete saturation of the target with the reagent, an increase in the hybridization ability of the reagent reduced the efficiency of the target modification. It was found that the modification by the tetranucleotide reagent (in the presence of an effector adjacent to the 3' end) occurs exclusively at an intracomplex target base. In the case of the dodecamer, which forms a stable, highly cooperative complex with the target, several bases of the target undergo alkylation, and an increase in temperature changes the site-specificity of alkylation. In this process, the redistribution of the target modification sites toward stronger nucleophilic centers enhances alkylation at temperatures near the melting temperature of the target.dodecanucleotide complex despite a decrease in the extent of target association.     

Direct site-specific cleavage of double-stranded DNA by conjugates of bleomycin A5 with triplex-forming oligonucleotide

Monofunctional conjugates of 15-mer triplex-forming oligonucleotide (TFO) with covalently attached bleomycin A5 residue at the 5′-end (Blm-p15) were synthesized. Bifunctional conjugates of TFO containing, in addition to Blm, the residues of intercalator 6-chloro-2-methoxy-9-aminoacridine (Acr) or N-(2-hydroxyethyl)phenazinium (Phn) were obtained for the first time. The Acr and Phn residues were attached to the 3′-phosphate group of TFO through L1 and L2 linkers, respectively, resulting in the compounds Blmp15pL1-Acr and Blm-p15pL2-Phn. The values of dissociation constants of the corresponding triplexes were evaluated using the gel retardation method. The Acr residue in Blm-p15pL1-Acr was shown to enhance the stability of the formed triplex by one order of magnitude. It was demonstrated that all synthesized conjugates are capable of specifically and nonspecifically damaging a target DNA, forming direct breaks and alkaline-labile sites. The extent of the specific cleavage of the target DNA was 15% in the case of a fivefold excess of the conjugates over the DNA duplex. The site-specific triplex-mediated cleavage of a target DNA was shown for the first time to occur predominantly (>90%) with the formation of the direct breaks of both DNA strands. The results show the availability of bleomycin-containing oligonucleotides as antigene compounds.     

Ethidium and azidoethidium oligonucleotide derivatives: Synthesis,complementary complex formation and sequence-specific photomodification of the single-stranded and double-stranded target oligo- and polynucleotides

Oligodeoxyribonucleotide derivatives containing ethidium or azidoethidium residues attached to 3′ and/or 5′ end were prepared. These derivatives formed tight specific complexes with complementary oligodeoxyribonucleotides where each attached ethidium residue led to an increase of complex T_m by 20–30°C. Tandem complexes of two oligodeoxyribonucleotides containing ethidium residues with an oligodeoxyribonucleotide having two adjacent complementary sequences for these Oligonucleotide were investigated. Photoinduced reactions of a number of ethidium and azidoethidium oligodeoxyribonucleotide derivatives with target complementary single-stranded and double-stranded oligo- and polydeoxyribonucleotides were investigated. The irradiation led to direct photocleavage of the target oligo- or polynucleotide, to formation of hidden (piperidine cleavable) modifications of the target of formation of covalent adducts between ethidium oligodeoxyribonucleotide derivative and the target. In a number of experiments, azidoethidium dyes were demonstrated to be considerably stronger photosensitizers than ethidium ones. Depending on the nature of the target (single- or double-stranded DNA) and on the irradiation conditions, the total damages to the target oligo- or polydeoxyribonucleotides ranged from 10–70% (for ethidium dyes) to 30–80% (for azidoethidium dyes).     

Ultrastructure of the conjugates of cytolytic T-lymphocytes and target cells]

Conjugates of target cells and of cytological T-lymphocytes obtained on the 11th day after alloimmunization were investigated. The conjugates formed small and medium lymphocytes; mature secretory granules, crystal-like structures and lipids were revealed in their cytoplasm. The lymphocyte is spherical, the area of contact with the target cell does not exceed 5 to 15%. Cytolysis of target cells is observed after 30 to 60 minutes of incubation. The lymphocyte becomes flattened, its nucleus acquires an oval form, and the area of contact with the target cell increases considerably. At the same time hypertrophy and change of orientation of Golgi's complex to the area of contact with the target cell, coalescence of the secretory granules with lipids and crystal-like structures, the appearance of immature secretory granules and vacuolar degeneration of mitochondria are demonstrated. The lymphocyte membrane becomes "desquamated"; structures connected with it, named "membranosomes" are described. It is suggested that the secretory processes in the cytoplasm of cytolytic T-lymphocytes are activated in their interaction with target cells.     

Effect of structural factors on the stability of duplexes formed by oligonucleotide conjugates with minor groove binders

The effect of structural factors on the stability of duplexes formed by DNA minor groove binders conjugated with oligonucleotide mono- or diphosphoramidates of the general formula Oligo-MGB_m (where Oligo is an oligonucleotide; m = 1 or 2; MGB is -L(Py)₂R, -L(Py)₄R, -L(Im)₄R, or -L(Py)₄NH(CH₂)₃CO(Py)₄R; Py is a 4-aminopyrrole-2-carboxylic acid residue; L is a -aminobutyric acid or an -aminocaproic acid residue, R = OEt, NH(CH₂)₆NEt₂, or NH(CH₂)₆N⁺Me₃) was studied by the method of thermal denaturation. The mode of binder interaction with the minor groove depends on the conjugate structure; it may be of the parallel head to head type for bisphosphoramidates and of the antiparallel head to tail type for monophosphoramidates of a hairpin structure. The effects of the duplexes with parallel orientation (bisphosphoramidates, MGB is L(Py)₄R, m = 2) and those of the hairpin structure with the antiparallel orientation (monophosphoramidates, MGB is L(Py)₄(CH₂)₃CO(Py)₄R, m = 1) on T _m values were close. The influence of the linker (L) and substituent (R) structures upon T _m was more pronounced for monophosphoramidate (MGB is L(Py)_nR, m = 1) than for bisphosphoramidate (MGB is L(Py)_nR, m = 2). No more than two oligopyrrolecarboxamide residues (either in parallel or antiparallel orientations) can be incorporated into the duplex minor groove. Moreover, it was shown by the example of monophosphoramidates (Oligo-L(Py)₄R and Oligo-L(Py)₄NH(CH₂)₃CO(Py)₄R) that the addition of a second ligand capable of incorporation into the minor groove increased T _m of the corresponding duplex in comparison with the duplex formed by the starting monophosphoramidate. At the same time, the introduction of a ligand incapable of incorporating decreased the T _m value. The mode of interaction of the conjugated binder with the oligonucleotide duplex is determined by its structure. For example, dipyrrolecarboxamide containing an ethoxy group at the binder C-end stabilizes the duplex due to stacking interaction with the terminal A · T pair, whereas tetrapyrrolecarboxamides stabilize the duplex by incorporation into the minor groove.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 159–166.Original Russian Text Copyright © 2005 by Ryabinin, Butorin, Elen, Denisov, Pyshnyi, Sinyakov.     

Effect of structural factors on the stability of duplexes formed by oligonucleotide conjugates with minor groove ligands

The effect of structural factors on the stability of duplexes formed by DNA minor groove binders conjugated with oligonucleotide mono- or diphosphoramidates of the general formula Oligo-MGBm (where Oligo is an oligonucleotide; m = 1 or 2; MGB is -L(Py)2R, L(Py)4R, -L(Im)4R, or -L(Py)4NH(CH2)3CO(Py)4R; Py is a 4-aminopyrrol-2-carboxylic acid residue, L is a gamma-aminobutyric acid or an epsilon-aminocaproic acid residue, R = OEt, NH(CH2)6NEt2, or NH(CH2)6N+Me3) was studied by the method of thermal denaturation. The mode of binder interaction with minor groove depends on the conjugate structure; it may be of the parallel head to head type for bisphosphoramidates and of the antiparallel head to tail type for monophosphoramidates of a hair-pin structure. The effects of the duplexes with parallel orientation (bisphosphoramidates, MGB is L(Py)4R, m = 2) and those of the hairpin structure with the antiparallel orientation (monophosphoramidates, MGB is L(Py)4(CH2)3CO(Py)4R, m = 1) on Tm values were close. The influence of the linker (L) and substituent (R) structures upon Tm was more pronounced for monophosphoramidate (MGB is L(Py)nR, m = 1) than for bisphosphoramidate (MGB is L(Py)nR, m = 2). No more than two oligopyrrolcarboxamide residues (either in parallel or antiparallel orientations) can be incorporated into the duplex minor groove. Moreover, it was shown by the example of monophosphoramidates (Oligo-L(Py)4R and Oligo-L(Py)4NH(CH2)3CO(Py)4R) that the addition of a second ligand capable of incorporation into the minor groove increased Tm of the corresponding duplex in comparison with the duplex formed by the starting monophosphoramidate. At the same time, the introduction of the ligand incapable of incorporating decreased the Tm value. The mode of interaction of the conjugated ligand with the oligonucleotide duplex is determined by its structure. For example, dipyrrolcarboxamide containing an ethoxy group at the ligand C-end stabilizes the duplex due to the stacking interaction with the terminal A*T pair, whereas tetrapyrrolcarboxamides stabilize the duplex by incorporation into the minor groove.     

Wavelength shifting oligonucleotide probe for the detection of adenosine of a target DNA with enhanced fluorescence signal

The modulated photophysical property of strong electronically coupled naphthyl uridine linked via a single C–C bond was explored in DNA detection via wavelength shifting and enhanced fluorescence emission by a simple ‘Just-Mix & Read’ strategy of homogeneous DNA detection.     

Quantitative determination of anthracycline antibiotic binding with DNA]

A new method of quantitative intravital assessment of anthracyclines accumulation and binding with DNA in alive cells has been developed. For this purpose DNA specific fluorescent dye Hoechst 33258 was used. Extent of fluorescence quenching of bound with DNA dye correlated with binding of daunomycin with cell DNA after mixing solutions of the drug and cells. It allowed us to determine quantity of drug molecules bound with DNA in the cells during the time.     

Effect of the structure of oligonucleotide substrates on interaction with methylase Eco dam

The interaction of Eco dam methylase with various synthetic oligonucleotide substrates was investigated. The "imperfect" duplexes contained a normal GATC recognition sequence in one chain of the enzyme recognition site and had some defects in the complementary chain, i.e., the absence of one or several nucleotide residues or the presence of S-methyl thiophosphate groups at the 3'-termini. The 3'-S-methyl thiophosphate residue has the same effect on the methylation of oligonucleotide complexes as does the absence of internucleotide phosphate in the analogous complexes. The presence of both GA dinucleotides in the recognition site is necessary for a productive enzyme-substrate interaction. The experimental data suggest that Eco dam methylase does form a symmetrical enzyme-substrate complex which is similar to that formed by type II restriction enzymes.     

Quantitative hybridization-arrest of mRNA in Xenopus oocytes using single-stranded complementary DNA or oligonucleotide probes.

The expression of heterologous mRNA in Xenopus oocytes was quantitatively inhibited by coinjection of single-stranded complementary DNA or synthetic complementary oligonucleotides. The lymphokines Interleukin-2 (IL-2) and Interleukin-3 (IL-3) were used as model systems to test the effectiveness of this procedure. Messenger RNA samples were hybridized to single stranded complementary DNA or oligonucleotides, injected into oocytes and the oocyte incubation medium assayed for the presence or absence of specific translation products 48 hours later. When IL-2 mRNA was hybridized to a large excess of long (490 bases) single stranded complementary DNA, the expression of IL-2 was effectively blocked (greater than 98%). Complementary oligonucleotides (18-23 bases) were almost as effective as the polynucleotide in inhibiting IL-2 activity (greater than 95%). Oligonucleotides derived from the 5' end, middle or 3' end of the coding sequence were all effective in arresting IL-2 mRNA translation. Oligonucleotide hybrid-arrest was effective even when no NaCl was present in the hybridization buffer, indicating that the annealing reaction could occur within the oocyte after injection. Definite proof that hybrid-arrest could occur in vivo was shown by the fact that oligonucleotides injected before or after mRNA injection, while not as effective as co-injection, still showed substantial inhibition of specific mRNA translation. The oligonucleotide hybrid-arrest method was equally effective in the case of IL-3, demonstrating its general applicability.     

Comparison of complex DNA mixtures with generic oligonucleotide microchips.

The reproducibility of melting curves for repeated hybridizations of target DNA with generic oligonucleotide microchips is shown experimentally to depend on the character of matching between fragments of target DNA and immobilized oligonucleotides. The reproducibility of melting curves is higher for the perfect match duplexes and decreases as the number of mismatched pairs within duplexes increases. This effect was applied to the comparative analysis of complex DNA mixtures. We developed a scheme in which we can identify and discriminate between the probe oligonucleotides responsible for the distinctions between target DNA mixtures. A scheme is illustrated by comparing DNA mixtures corresponding to V-D-J genes connected with populations of mRNAs CDR3 TCR Vb (T-cell receptor beta complementarity determining region 3) from the thymus and pancreas of NOD mice. Our results demonstrate that generic microchips can be applied efficiently to the analysis of DNA mixtures.     

The biophysics of DNA hybridization with immobilized oligonucleotide probes.

A mathematical model based on receptor-ligand interactions at a cell surface has been modified and further developed to represent heterogeneous DNA-DNA hybridization on a solid surface. The immobilized DNA molecules with known sequences are called probes, and the DNA molecules in solution with unknown sequences are called targets in this model. Capture of the perfectly complementary target is modeled as a combined reaction-diffusion limited irreversible reaction. In the model, there are two different mechanisms by which targets can hybridize with the complementary probes: direct hybridization from the solution and hybridization by molecules that adsorb nonspecifically and then surface diffuse to the probe. The results indicate that nonspecific adsorption of single-stranded DNA on the surface and subsequent two-dimensional diffusion can significantly enhance the overall reaction rate. Heterogeneous hybridization depends strongly on the rate constants for DNA adsorption/desorption in the non-probe-covered regions of the surface, the two-dimensional (2D) diffusion coefficient, and the size of probes and targets. The model shows that the overall kinetics of DNA hybridization to DNA on a solid support may be an extremely efficient process for physically realistic 2D diffusion coefficients, target concentrations, and surface probe densities. The implication for design and operation of a DNA hybridization surface is that there is an optimal surface probe density when 2D diffusion occurs; values above that optimum do not increase the capture rate. Our model predicts capture rates in agreement with those from recent experimental literature. The results of our analysis predict that several things can be done to improve heterogeneous hybridization: 1) the solution phase target molecules should be about 100 bases or less in size to speed solution-phase and surface diffusion; 2) conditions should be created such that reversible adsorption and two-dimensional diffusion occur in the surface regions between DNA probe molecules; 3) provided that 2) is satisfied, one can achieve results with a sparse probe coverage that are equal to or better than those obtained with a surface totally covered with DNA probes.