RESUMEN
Patients with post-stress pathologies display the signs of inflammation in the peripheral blood as well as in the brain. The mechanisms of such post-stress neuroimmune changes, their contribution to the behavior, the relationship of the intensity of inflammation with genetically determined features have not been clarified. The goal of this work was to evaluate the dynamics of post-stress inflammation in the blood and hippocampus of rats which differ in level of excitability of the nervous system. Rats of two strains (high/low excitability threshold) were subjected to stress according to the K. Hecht protocol and their behavior, neutrophil:lymphocyte ratio and the number of Iba+ cells in the hippocampus were analysed 24 hours, 7 and 24 days after stress exposure. Highly excitable animals show an increase in anxiety-like behavior, in the number of neutrophils compared to lymphocytes as well as in the number of Iba1+ cells in CA1, CA3 and DG areas of the hippocampus in response to stress. Thus, hereditary high excitability of the nervous system is a possible risk factor for the development of post-stress pathologies.
Asunto(s)
Hipocampo , Neuronas , Estrés Psicológico , Animales , Masculino , RatasRESUMEN
We propose an original method for controlling BP by administration of Si~ODN nanocomposites containing antisense oligonucleotides fixed on silicon-organic nanoparticles. ODN in nanocomposites are targeted to mRNA of the genes encoding angiotensin-converting enzyme (ACE1) and type 1 angiotensin-II receptor (AT1A). The experiments were performed on hypertensive ISIAH rats, a genetic model of hypertension. Single inhalation or intraperitoneal administration of the nanocomposites targeted to ACE1 mRNA or ATA1 mRNA, respectively, led to a pronounced decrease (by ~30 mm Hg) in systolic BP in ISIAH rats over a week. The use of scrambled ODN in the nanocomposites had no effect. A decrease in the expression of ACE1 and AT1A genes under the effect of the corresponding antisense ODN was demonstrated, which attested to directed effect of the test preparations.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Hipertensión/tratamiento farmacológico , Nanopartículas/química , Nanopartículas/uso terapéutico , Oligonucleótidos Antisentido/química , Oligonucleótidos Antisentido/uso terapéutico , Sistema de Transporte de Aminoácidos A/genética , Sistema de Transporte de Aminoácidos A/metabolismo , Animales , Presión Sanguínea/genética , Hipertensión/genética , Hipertensión/metabolismo , Masculino , Peptidil-Dipeptidasa A/genética , ARN Mensajero/metabolismo , Ratas , SilicioRESUMEN
We have previously described nanocomposites containing conjugates or complexes of native oligodeoxyribonucleotides with poly-L-lysine and TiO2 nanoparticles. We have shown that these nanocomposites efficiently suppressed influenza A virus reproduction in MDCK cells. Here, we have synthesized previously undescribed nanocomposites that consist of TiO2 nanoparticles and polylysine conjugates with oligonucleotides that contain phosphoryl guanidine or phosphorothioate internucleotide groups. These nanocomposites have been shown to exhibit antiviral activity in MDCK cells infected with H5N1 influenza A virus. The nanocomposites containing phosphorothioate oligonucleotides inhibited virus replication ~130-fold. More potent inhibition, i.e., ~5000-fold or ~4600-fold, has been demonstrated by nanocomposites that contain phosphoryl guanidine or phosphodiester oligonucleotides, respectively. Free oligonucleotides have been nearly inactive. The antiviral activity of oligonucleotides of all three types, when delivered by Lipofectamine, has been significantly lower compared to the oligonucleotides delivered in the nanocomposites. In the former case, the phosphoryl guanidine oligonucleotide has appeared to be the most efficient; it has inhibited the virus replication by a factor of 400. The results make it possible to consider phosphoryl guanidine oligonucleotides, along with other oligonucleotide derivatives, as potential antiviral agents against H5N1 avian flu virus.
Asunto(s)
Antivirales/farmacología , Guanidinas/química , Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Nanocompuestos/química , Organofosfatos/farmacología , Oligonucleótidos Fosforotioatos/farmacología , Animales , Antivirales/síntesis química , Perros , Relación Dosis-Respuesta a Droga , Portadores de Fármacos , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/crecimiento & desarrollo , Lípidos/química , Células de Riñón Canino Madin Darby , Oligonucleótidos/química , Oligonucleótidos/farmacología , Organofosfatos/química , Oligonucleótidos Fosforotioatos/química , Polilisina/química , Titanio/química , Replicación Viral/efectos de los fármacosRESUMEN
TiO2-based nanocomposites were prepared to deliver oligonucleotides into cells. The nanocomposites were designed by the immobilization of polylysine-containing oligonucleotides on TiO2-nanoparticles (TiO2·PL-DNA). We showed for the first time the possibility of using the proposed nanocomposites for treatment of hypertensive disease by introducing them into hypertensive ISIAH rats developed as a model of stress-sensitive arterial hypertension. The mRNA of the gene encoding angiotensin I-converting enzyme (ACE1) involved in the synthesis of angiotensin II was chosen as a target. Administration (intraperitoneal injection and inhalation) of the nanocomposite showed a significant (by 20-30 mm Hg) decrease in systolic blood pressure when the nanocomposite contained the ACE1 gene-targeted oligonucleotide. When using the oligonucleotide with a random sequence, no effect was observed. Further development and improvement of the inhalation nanocomposite drug delivery to systemic hypertensive disease treatment promises new possibilities for clinical practice.
Asunto(s)
Terapia Genética , Hipertensión/terapia , Oligonucleótidos/administración & dosificación , Peptidil-Dipeptidasa A/genética , Animales , Masculino , Ratas , Ratas Endogámicas , Titanio/administración & dosificaciónRESUMEN
The objective of the present study that involved 176 children at the age varying from 2 to 12 years presenting with chronic ENT diseases was etiological diagnostics and etiotropic therapy of these pathologies taking into consideration the duration of the disease of less than one year (n=72), from 1 to 2 years (n=54), and over 2 years (n=50). The bacteriological method was employed to identify microflora from the upper respiratory tract and the molecular-biological methods for the detection of Epstein-Barr virus DNA, cytomegalovirus, and 6 types of human herpes virus in the blood and saliva. All the children were treated with the recombinant interferon preparations given for 1-1.5 months. For 41% of the children this treatment was combined with antibacterial therapy followed by immunocorrective therapy with interferon inducers (in 79.4% of the patients) or bacterial lysates (20.6%). The study revealed the predominant role of types 4, 5, and 6 type herpes viruses in the development of chronic ENT pathologies in the children with the gradual lowering of activity of these infections over 2 years. Staphylococcus aureus and Streptococcus pyogenes as well as fungi of the genus Candida were the commonest bacterial and fungal pathogenic agents isolated from the naso- and oropharynx of the children suffering from chronic ENT pathology.The effectiveness of etiotropic therapy was shown to decrease with time, from 78% during 1 year after the onset of the disease to 30% within the next 2 years.
Asunto(s)
Enfermedades Otorrinolaringológicas/tratamiento farmacológico , Niño , Preescolar , Enfermedad Crónica/terapia , Femenino , Humanos , Masculino , Enfermedades Otorrinolaringológicas/microbiología , Enfermedades Otorrinolaringológicas/virologíaRESUMEN
Antiviral activity of TiO2 * PL * DNA nanobiocomposites was studied on the MDCK cell culture infected with influenza A virus (subtype H3N2). DNA fragments in the nanocomposites are electrostatically bound to titanium dioxide nanoparticles pre-covered with polylysine. It was shown that TiO2 * PL * DNA(v3') nanocomposite bearing the DNA(v3') fragment targeted to the 3'-end of the noncoding region of segment 5 of viral RNA specifically inhibited the virus reproduction with the efficiency of 99.8 and 99.9% (or by factors of~400 and 1000) at a low concentration of DNA(v3') in nanocomposite (0.1 and 0.2 µM, respectively). The TiO2 * PL * DNA(r) nanocomposite containing oligonucleotide noncomplementary to viral RNA or the oligonucleotide unbound to the nanoparticles show very low antiviral activity (inhibition by factors of~3.5 and 1.3, respectively).
Asunto(s)
Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Nanopartículas/administración & dosificación , Oligonucleótidos/administración & dosificación , Titanio/administración & dosificación , Antivirales , Línea Celular , Humanos , Subtipo H3N2 del Virus de la Influenza A/patogenicidad , Nanopartículas/química , Oligonucleótidos/química , Polilisina/química , ARN Viral/efectos de los fármacos , Electricidad Estática , Titanio/químicaRESUMEN
Methods of noncovalent immobilization of DNA fragments onto titanium dioxide nanoparticles (TiO2) were developed, which led to TiO2-DNA nanocomposites capable of penetrating through cell membranes. TiO2 nanoparticles of different forms (amorphous, anatase, brookit) with enhanced agglomeration stability were synthesized. The particles were characterized by X-ray diffraction, small angle X-ray scattering, infrared spectroscopy and atomic force microscopy. Three approaches to the preparation of nanocomposites are described: (1) sorption of polylysine-containing oligonucleotides onto TiO2-nanoparticles, (2) the electrostatic binding of oligonucleotides to TiO2 nanoparticles bearing immobilized polylysine, and (3) sorption of oligonucleotides on TiO2 nanoparticles in the presence of cetavlon. All three methods provide an efficient and stable immobilization of DNA fragments onto nanoparticles, which leads to nanocomposites with a density for an oligonucleotide up to 40 nmol/mg. It is shown that DNA fragments in nanocomposites retain their ability to form complementary complexes and can be delivered into cells without transfection agents and other methods of exposure.
Asunto(s)
ADN/química , Nanopartículas del Metal/química , Titanio/química , Permeabilidad de la Membrana Celular , Células HeLa , Humanos , Microscopía de Fuerza Atómica , Nanocompuestos/química , Oligonucleótidos/química , Difracción de Rayos XRESUMEN
Influenza A viruses take a significant place in human and animal pathology causing epidemics and epizootics. Therefore, the development of new antiflu drugs has become more and more urgent. Deoxyribozymes can be considered as promising antiviral agents due to their ability to efficiently and highly specifically cleave RNA molecules. In this study, a number ofgenomic sequences of the most relevant influenza A virus subtypes, H5N1, H3N2, and H1N1, were analyzed. Conservative regions were revealed in five the least variable segments of the fragmented viral RNA genome, and potential sites of their cleavage with "10-23" deoxyribozymes were determined. 46 virus-specific 33-mer deoxyribozymes with the general structure of 5'N8AGGCTAGCTACAACGAN9 were designed and synthesized. Screening of the antiviral activity of these agents in conjugation with lipofectin on the Madin-Darby Canine Kidney cells infected with highly pathogenic avian influenza virus A/chicken/Kurgan/05/2005 (H5N1) revealed 17 deoxyribozymes, which suppressed the titer of virus cytopathicity by more than 2.5 IgTCID50/mL (i.e. the virus neutralization index was more than 300), with five of them suppressing the virus titer by a factor of 1000 and more. The most active deoxyribozymes appeared to be specific to segment 5 of the influenza A virus genome, which encoded nucleoprotein (NP).
Asunto(s)
Antivirales/farmacología , ADN Catalítico/farmacología , ADN de Cadena Simple , Gripe Humana , Animales , Antivirales/síntesis química , ADN Catalítico/síntesis química , ADN de Cadena Simple/síntesis química , ADN de Cadena Simple/farmacología , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H3N2 del Virus de la Influenza A/efectos de los fármacos , Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/patogenicidad , Gripe Humana/tratamiento farmacológico , Gripe Humana/genética , Células de Riñón Canino Madin Darby/efectos de los fármacos , ARN Viral/química , ARN Viral/genética , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
Process of methylation of histone H3 for lysine 4 (H3K4) was studied in hippocampal pyramidal neurons of rats--intact and submitted to emotional-painful stress with active and inactivated channels of NMDA-receptors with taking into account the interhemisphere lateralization and in connection with the genetically determined level of excitability of the animals' nervous system. There were revealed interstrain differences in the basal level of the H3K4 methylation whose direction depends on structural-functional peculiarities of hippocampal fields and lateralization. Under action of stress the direction of the observed changes in the degree of the H3K4 methylation depended on the functional state of channels of NMDA-receptors. On the background of active receptors the proportion of immunopositive cells predominantly increased. In the CA1 field those changes were not connected to excitability and lateralization, whereas in the CA3 field it had a complex character and depended on those two factors. At inactivation of channels of NMDA-receptors the portion of immunopositive nuclei as a result of the stress action, on the contrary, predominantly decreased; interstrain specificity of these changes was connected to lateralization, while its direction in different hippocampal fields was different. Action of the short-time emotional-painful stress did not lead to a change of shape of interhemisphere asymmetry at active state of receptors, whereas at inactivation of receptors it changes depending on the structural-functional organization of hippocampus and on excitability of the nervous system.
Asunto(s)
Potenciales de Acción , Región CA1 Hipocampal/metabolismo , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Células Piramidales/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Estrés Psicológico/metabolismo , Animales , Región CA1 Hipocampal/citología , Región CA1 Hipocampal/fisiología , Lateralidad Funcional , Metilación , Células Piramidales/fisiología , Ratas , Ratas Wistar , Umbral SensorialRESUMEN
Glutamate receptors in the central nervous system play a significant role in the mechanisms of differential adaptation to the environmental conditions. However, structural and functional parameters of kainate receptors (KR) under normal conditions and during exposure to stress are not well characterized. Therefore, the aim of this research was to 1) study the distribution and the quantity of KR GluR 5/6/7 subunits; 2) examine their changes in the pyramidal cell layer of the hippocampus in rat strains with have genetically determined distinctions in the levels of nervous system excitability following the exposure to short-term emotional-painful stress; 3) estimate the sensitivity of hippocampal pyramidal neurons to the action of KR agonist -kainic acid. It was demonstrated that GluR 5/6/7 KR are localized mainly in the region of hippocampal CA2 area; in the animals with low excitability their quantity was greater than in those with high excitability. Short-term emotional-painful stress resulted in the increase of KR in hippocampal CA2 area only in highly excitable rats. Selective sensitivity of pyramidal neurons in different hippocampal fields to the action of kainic acid was demonstrated and it was found to depend on animal strain characteristics of of the nervous system excitability.
Asunto(s)
Región CA3 Hipocampal/metabolismo , Dolor/metabolismo , Receptores de Ácido Kaínico/metabolismo , Estrés Psicológico/metabolismo , Animales , Región CA3 Hipocampal/patología , Inmunohistoquímica , Ácido Kaínico/farmacología , Fenómenos Fisiológicos del Sistema Nervioso , Neuronas/metabolismo , Neuronas/patología , Dolor/patología , Ratas , Ratas Endogámicas , Especificidad de la Especie , Estrés Psicológico/patologíaRESUMEN
In this study we examine the possibility that TiO2 nanoparticles and their conjugates can penetrate into cultivated cells without any special transfection procedures. Oligonucleotides and their derivates were conjugated with the TiO2 nanoparticles, which were obtained as colloidal solutions at a concentration of TiO2 0.3M by TiCl4 hydrolysis. The electronic microscopy of various cell cultures (KCT, Vero, and MDCK) treated with nanoparticle solutions (20 µg/µl) showed that nanoparticles could enter the cells and accumulate in the vacuoles and phagosomes and form inclusions in cytoplasm. Thus, we demonstrated the penetration of TiO2 nanoparticles and their oligonucleotide conjugates into intracellular space without any auxiliary operations. Most other researches used electroporation techniques for similar purposes [1, 2, 5].
RESUMEN
A simple and efficient method of synthesis of polyamine-oligonucleotide conjugates in high yields (up to 95%) was suggested. The terminal phosphate group of deprotected oligonucleotides was selectively activated with the redox pair triphenylphosphine-dipyridyl disulfide in the presence of a nucleophilic catalyst, and the activated oligonucleotide derivative was subjected to the reaction with a polyamine.
Asunto(s)
Oligonucleótidos/síntesis química , Poliaminas/síntesis química , Oligonucleótidos/química , Poliaminas/químicaRESUMEN
A new type of coating for manufacturing DNA chips was constructed of the basis of an organic-inorganic nanocomposite based on the polyvinylbutyral-tetraethoxysilane copolymer. The organosilicon composite was functionalized by introduction of ethanolamine vinyl ether copolymers, which contain amino groups and anchor vinyloxide units capable of reacting with silanol groups of the nanocomposite. The resulting coatings form a film on glass slides with a high surface density of amino groups (up to 700 groups/nm2) suitable for three-dimensional immobilization of oligonucleotides. The use of bifunctional reagents (e.g., phenylene diisothiocyanate) for the attachment of oligonucleotides bearing amino linkers to the amino-containing surface provides an immobilization density of 0.5-1.6 pmol/mm2. Immobilization with a higher density (10-12 pmol/mm2) was achieved for attachment to amino-containing glass slides upon the use of oligonucleotides containing selectively activated terminal phosphate groups. The activation of oligonucleotides was carried out with the triphenylphosphine-dithiodipyridine pair in the presence of dimethylaminopyridine N-oxide. The resulting DNA chips were shown to be useful in principle for DNA detection.
Asunto(s)
Vidrio/química , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Polivinilos/química , Silanos/química , ADN/análisis , Nanoestructuras/química , Oligonucleótidos/química , Compuestos Organofosforados/química , Compuestos de Organosilicio/química , Piridinas/químicaRESUMEN
Substrate properties of various morpholinonucleoside triphosphates in the reaction of DNA elongation catalyzed by DNA polymerase beta, reverse transcriptase of human immunodeficiency virus (HIV-1 RT), and reverse transcriptase of Moloney murine leukemia virus (M-MuLV RT) were compared. Morpholinonucleoside triphosphates were utilized by DNA polymerase beta and HIV-1 reverse transcriptase as substrates, which terminated further synthesis of DNA, but were virtually not utilized by M-MuLV reverse transcriptase. The kinetic parameters of morpholinoderivatives of cytosine (MorC) and uridine (MorU) were determined in the reaction of primer elongation catalyzed by DNA polymerase beta and HIV-1 reverse transcriptase. MorC was a more effective substrate of HIV-1 reverse transcriptase and significantly less effective substrate of DNA polymerase beta than MorU. The possible use of morpholinonucleoside triphosphates as selective inhibitors of HIV-1 reverse transcriptase is discussed.
Asunto(s)
ADN Polimerasa beta/antagonistas & inhibidores , Desoxirribonucleótidos/farmacología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Inhibidores de la Transcriptasa Inversa/farmacología , Catálisis , ADN Polimerasa beta/metabolismo , Reparación del ADN/efectos de los fármacos , ADN Viral/biosíntesis , Desoxirribonucleótidos/metabolismo , Transcriptasa Inversa del VIH/metabolismo , VIH-1/enzimología , Cinética , Virus de la Leucemia Murina de Moloney/enzimología , Morfolinas/metabolismo , Morfolinas/farmacología , ADN Polimerasa Dirigida por ARN/metabolismo , Proteínas Recombinantes , Inhibidores de la Transcriptasa Inversa/metabolismo , Relación Estructura-ActividadRESUMEN
Heptadeoxynucleotides were obtained that contained an aliphatic amino group in position 8 of the deoxyadenosine residue: ALNH2 CTTTCT, CTCALNH2 CTT, and ACACTCALNH2 where L = NH(CH2)n, n = 3, 5, or 7. A 4-azidotetrafluorobenzoyl residue was attached to the amino group in the oligonucleotides, and photomodification of a DNA target by the resulting reagents was carried out. It was shown that the length of the spacer influences the photomodification extent of the target; a spacer with n = 5 is optimum. The maximum modification extent (65%) was reached when a reagent containing a photoreactive group at the 5'-terminal deoxyadenosine residue was used.
Asunto(s)
ADN/efectos de los fármacos , Desoxiadenosinas/química , Oligodesoxirribonucleótidos/síntesis química , ADN/efectos de la radiación , Oligodesoxirribonucleótidos/química , FotoquímicaRESUMEN
High efficiency was demonstrated for the photomodification of a DNA target by a 5'-p-azidotet-rafluorobenzoyl reagent based on a tetranucleotide and its 3'-phosphoestrone ester in the presence of a pair of flanking effectors. These effectors are oligonucleotide derivatives with N-(2-hydroxyethyl)phenazinium groups or those connected to cholesterol residues at the terminal phosphates.
Asunto(s)
Aminoácidos/química , Colesterol/química , Oligonucleótidos/química , Secuencias Repetitivas de Ácidos Nucleicos , Electroforesis en Gel de Poliacrilamida , Sondas de Oligonucleótidos , Oligonucleótidos/síntesis química , Fosfatos/química , Fotoquímica , TemperaturaRESUMEN
A comparison of Km values and maximal rates of extension (Vmax) for primers containing different modified bases or mismatches, and fully complementary primers of the same length catalyzed by the Klenow fragment of E coli DNA polymerase I was carried out. Base modifications include T-T dimers and apurinic sites. In the case of mismatch, the number of complementary bases from the 3'-terminus to the non-complementary nucleotide determines the efficiency of substrate incorporation, which is a measure of degree of interaction of the enzyme with its primer template. Differently, removal of one base in any position from the 3'-terminus of the primer is equivalent to shortening of the primer by one nucleotide unit, and decreases the affinity to the enzyme by 1.8-fold. Since apurinic sites fail to interfere with the efficiency of DNA synthesis, we suppose that the Klenow fragment of E coli DNA polymerase I does not participate in the correction of DNAs containing apurinic nucleotides units. Finally, the efficiency of elongation of the d(p primer was shown to decrease with an increase in T-T dimers in the primer. When the d(pT)10m primer contains about 2.6 T-T dimers per molecule, the efficiency of its elongation decreases by a factor of 8-18.
Asunto(s)
ADN Polimerasa I/metabolismo , Cartilla de ADN/metabolismo , Escherichia coli/enzimología , Ácido Apurínico , ADN/biosíntesis , Cartilla de ADN/química , Cinética , Ácidos Nucleicos Heterodúplex , Dímeros de Pirimidina , Moldes GenéticosRESUMEN
Oligodeoxynucleotide conjugates of netropsin (Nt) and distamycin A (Dst) were synthesized, and the thermal stability of several model DNA duplexes containing conjugates was studied. Two Dst residues conjugated at both ends of the oligonucleotide were needed for substantial increase in the melting temperature of the corresponding duplex (delta Tm > 30 degrees C). Two attached Dst residues had a greater effect on the Tm value than did two free molecules of Dst per duplex. In contrast to Dst, one Nt molecule linked to the oligonucleotide was enough to influence the thermal stability of the duplexes. Like Dst, the attached Nt appeared to stabilize duplexes much more than free Nt molecules. Attachment of Nt to either the 5'- or 3'-end of the different nonadeoxynucleotides containing 5' ...TTAAA... or 5' ...TATA... sites increased Tm of their duplexes by 21 degrees C-25 degrees C, whereas delta Tm for free Nt was 8 degrees C-15 degrees C (delta delta Tm = 10 degrees C-14 degrees C). The same phenomenon was shown for oligonucleotide phosphorothioates (delta Tm were 18 degrees C-22 degrees C and 9 degrees C-13 degrees C for attached and free Nt, respectively; delta delta Tm = 9 degrees C). This effect was even more pronounced for a hairpin oligonucleotide (delta delta Tm = 18 degrees C).
Asunto(s)
ADN/metabolismo , Distamicinas/química , Netropsina/química , Oligonucleótidos/síntesis química , Animales , ADN/química , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Distamicinas/síntesis química , Distamicinas/metabolismo , Humanos , Netropsina/síntesis química , Netropsina/metabolismo , Oligonucleótidos/química , Oligonucleótidos/metabolismoRESUMEN
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.
Asunto(s)
ADN de Cadena Simple/química , Oligonucleótidos/química , Azidas/química , Sitios de Unión , Reactivos de Enlaces Cruzados/química , ADN de Cadena Simple/genética , Oligonucleótidos/genética , Fotoquímica , Relación Estructura-ActividadRESUMEN
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.