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.     

Modulation of HIV-1 integrase activity by single-stranded oligonucleotides and their conjugates with eosin

Integration of the DNA copy of the genomic RNA into an infected cell genome is one of the key steps of the replication cycle of all retroviruses. It is catalyzed by the viral enzyme, integrase. We have shown that conjugates of short single-stranded oligonucleotides with eosin efficiently inhibit the catalytic activity of the HIV-1 integrase. In this article, we have found that the dependence of the integrase catalytic activity on the concentration of oligonucleotides has a bell-shaped pattern. The modulation of HIV-1 integrase activity correlated with the oligonucleotide length and was not associated with specific sequences. Moreover, a similar mode of the oligonucleotide action was found for integrase from the prototype foamy virus. This dual effect of the oligonucleotide and their conjugates with eosin might be explained by their binding with retroviral integrase in two different sites; the oligodeoxynucleotide binding in the first site results in integrase activation, whereas interactions with another one lead to inhibition of the enzyme activity. Eosin coupling to oligonucleotides did not change the mode of their action but enhanced their affinity to both binding sites. The affinity increase was found to be much more important for the site responsible for the integrase inhibition, thus explaining the high inhibitory potency of oligonucleotide-eosin conjugates.     

Association of Chernobyl-derived 239+240Pu, 241Am, 90Sr and 137Cs with different molecular size fractions of organic matter in the soil solution of two grassland soils

Radiocesium is normally bound only rather weakly and unspecifically by humic substances, in contrast to the actinides Pu and Am. Recently, however, it was observed that fallout ¹³⁷Cs in the soil solution from an Of-horizon of a podzol forest soil (slightly decomposed plant material) was associated essentially only with one single size fraction of the humic substances. In deeper soil layers with well humified material (AOh-horizon), radiocesium was associated with all size fractions of the dissolved organic matter (DOM). To examine whether this unexpected behaviour is also observable for DOM isolated from other soils, we determined the association of fallout ¹³⁷Cs,⁹⁰Sr,²³⁸Pu, ^239+240Pu and ²⁴¹Am with various size fractions of DOM from in situ soil solutions isolated from two layers (0–2 cm and 2–5 cm) of two grassland soils (a soddy podzolic soil and a peat soil) within the 10 km zone of the nuclear reactor at Chernobyl (Ukraine). The four size fractions of DOM as obtained by gel filtration of the soil solution were (mean nominal molecular weight in daltons): fraction I: ≥2000, fraction II: 1300; fraction III: 560, fraction IV: inorganic compounds. The results for the well humified DOM (humus accumulation horizon of podzol, deeper layer of peat soil) showed that Pu and Am are essentially associated with the high molecular weight fractions, while Sr is present only in the `inorganic' fraction. Radiocesium is found in all the size fractions separated. A quite similar pattern was also found for Pu, Am, and Sr in the soil solution from only slightly decomposed plant material (0–2 cm of peat soil), but not for radiocesium. This radionuclide was again essentially only observable in one single low molecular weight fraction of DOM. The above results thus support our recent observations in the different horizons of a forest podzol mentioned above, even though no reason for the different binding of radiocesium by well humified soil organic matter and by only slightly decomposed plant material can be given at present. The data demonstrate, however, that information on only the total amount of a radionuclide in the soil solution will not be sufficient to interpret or predict its fate adequately in the soil. Received: 13 February 1998 / Accepted in revised form: 14 July 1998     

SFPQ and NONO Proteins and Long Non-Coding NEAT1 RNA: Cellular Functions and Role in the HIV-1 Life Cycle

Molecular Biology - About 20 years ago, large RNA–protein complexes called paraspeckles were discovered in cell nuclei. The main components of these complexes are SFPQ and NONO proteins and...     

Gene IL6 G(-174)C and gene IL10 G(-1082)A polymorphisms are associated with unfavourable outcomes in patients with acute coronary syndrome

We investigated the association of gene IL6 G(-174)C polymorphism and gene IL10 G(-1082)A polymorphism with coronary artery disease (CAD) in the Russian population. A total of 1145 patients with CAD diagnose on the basis of clinical studies in cardiological hospitals of Moscow, St -Petersburg, Kazan, Chelyabinsk, Perm, Stavropol and Rostov-on-Don. Supervision term was 9.10 +/- 5.03 months (the maximum term 18 months). In case of gene IL10 G(-1082)A polymorphism we determined that patients with CAD diagnose and A alleles gene IL10 had unfavorable outcome more often than patients with homozygous G alleles. Survival time from end point from carrier genotype GA and AA is 11.68 +/- 0.67 months against 12.69 +/- 0.65 months from carrier phenotype GG gene IL10 (chi2 = 4.13, p = 0.042). The group studied do not differ significantly with respect to the distributions of gene IL6 G(-174)C alleles and genotypes. However in case combined group studies of gene IL10 G(-1082)A polymorphism and IL6 G(-174)C polymorphism we determined that patients with CAD diagnose and carrier genotype GG gene IL6 and genotype GA and AA gene IL10 had unfavorable outcome more often (survival time 11.01 +/- 1.24 months) than patients with genotype CC and CG gene IL6 and genotype GG gene IL10 (survival time 13.28 +/- 0.83 months) chi2 = 10.23, p = 0.017. The obtained data allows assuming the important role of the IL6 and IL10 genes which are responsible for functioning of inflammation system, in the accelerated formation of failures at the patients who had a coronary syndrome.     

The polymorphisms G(−174)C in IL6 gene and G(−1082)A in IL10 gene are associated with poor outcomes in patients with acute coronary syndrome

Association between the rates of poor outcomes in the patient cohort with acute coronary syndrome and polymorphisms G(?174)C in the IL6 gene and G(?1082)A in the IL10 gene were determined. In total, 1145 patients hospitalized for coronary artery disease to cardiological hospitals of Moscow, St. Petersburg, Kazan, Chelyabinsk, Perm, Stavropol, and Rostov-on-Don were examined. The mean observation period was 9.10 ± 5.03 months (maximal, 18 months). Analysis of the survival of the patients with acute coronary syndrome that carried allele A has demonstrated that the presence of IL10 gene polymorphism G(?1082)A is associated with more frequent poor outcomes as compared with GG genotype. The survival time to endpoint for the carriers of GA and AA genotypes was 11.68 ± 0.67 months versus 12.69 ± 0.65 months for the carriers of GG genotype in IL10 gene (χ² = 4.13, p = 0.042). As for the IL6 gene polymorphism G(?174)C, survival rate analysis did not detect any significant association with the risk for poor outcome. However, joint analysis of these polymorphisms in both genes has demonstrated that characteristic of the patients with acute coronary syndrome that carry GG genotype of IL6 gene and GA and AA genotypes of IL10 is a higher rate of poor outcomes (time to endpoint, 11.01 ± 1.24 months) as compared with the carriers of IL6 gene CC and CG genotypes and IL10 gene GG genotype (time to endpoint, 13.28 ± 0.83 months (ξ² = 10.23, p = 0.017). These data suggest that the genes IL6 and IL10, whose products are involved in the control of inflammatory response, play an important role by increasing the probability of poor outcomes in the patients with acute coronary syndrome.     

Polymorphic markers Ala455Val of the THBD gene and Arg353Gln of the F7 gene and association with unfavorable outcomes of coronary atherosclerosis in patients with a history of acute ischemic heart disease

The polymorphic markers Ala455Val of the THBD gene and Arg353Gln of the F7 gene were tested for association with the frequency of unfavorable outcomes in patients with a history of acute ischemic heart disease. The study involved 1145 patients hospitalized in cardiology clinics of Moscow, St. Petersburg, Kazan, Chelyabinsk, Perm, Stavropol, and Rostov-on-Don because of acute ischemic heart disease. The patients were followed up for up to 62.5 months. None of the markers displayed a significant association with the time to an endpoint. The patients were then grouped by sex. In females, the frequency of unfavorable outcomes (fatal or nonfatal myocardial infarction and fatal or nonfatal stroke) was higher in carriers of allele Val of the Ala344Val polymorphic marker of the THBD gene and carriers of genotype Arg/Arg of the Arg353Gln polymorphic marker of the F7 gene, but the difference was not statistically significant. Such an increase in frequency was not observed in males. To study the combined effect of the polymorphic markers of the THBD and F7 genes, the course of ischemic heart disease was compared for two female subgroups. One included carriers of allele Val of the Ala344Val polymorphic marker of the THBD gene and genotype Arg/Arg of the Arg353Gln polymorphic marker of the F7 gene; the other subgroup included carriers ofgenotype Ala/Ala of the Ala455Val polymorphic marker of the THBD gene and allele Gln of the Arg353Gln polymorphic marker of the F7 gene. The frequency of unfavorable outcomes in the first subgroup was higher than in the second one. The time to an endpoin was 40.5 months (95% confidence interval (CI) 33.5-47.6) in the first subgroup and 51.6 months (95% CI 45.0-58.1) in the second subgroup (chi2 = 4.15, P = 0.042). The results made it possible to assume that the F7 and THBD genes play an important role in genetic predisposition to unfavorable outcomes in patients with a history of acute ischemic heart disease.     

Interactions of HIV-1 DNA heterocyclic bases with viral DNA

Human immunodeficiency virus type 1 integrase is one of three viral enzymes, and it realizes a key process of the viral replication cycle, i.e. viral DNA integration into infected cell genome. Integrase recognizes nucleotide sequences located at the ends of the viral DNA U3 and U5 LTRs and catalyzes 3'-processing and strand transfer reactions. To study the interactions between integrase and viral DNA at present work, we used modified integrase substrates mimicking the terminal U5 LTR sequence and containing non-nucleoside insertions in one or/and both strands. It is shown that the substrate modifications have no influence on the integrase binding rate, while the heterocyclic bases removal in the 5th and 6th substrate positions and in the 3rd position of the substrate processed strand distinctly inhibits the integrase catalytic activity. This fact demonstrates these bases significance for the active enzyme/substrate complex formation. On the contrary, modification of the 3rd position within substrate non-processed strand stimulates 3'-processing. Since heterocyclic base elimination results in disruption of the DNA complementary and staking interactions, this result shows that DNA double helix destabilization close to the cleaved bond promotes the 3'-processing.     

Association of the polymorphic markers G(−455)A in the FGB gene and C(−1654)T in the PROC gene with hereditary predisposition to unfavourable outcome in patients with history of acute coronary syndrome

Associations of polymorphisms of genes FGB G(?455)A and PROC C(?1654)T with the frequency of poor outcomes in patients with the history of acute coronary syndrome (ACS) were studied in the Russian population. A total of 1145 patients admitted to cardiological hospitals of Moscow, St. Petersburg, Kazan, Chelyabinsk, Perm, Stavropol, and Rostov-on-Don with ischemic heart disease exacerbation were examined. The mean follow-up time was 1.14 ± 0.33 years, and the maximum follow-up time was 3.2 years. The risk of poor outcome did not depend on the carriership of genotypes of the polymorphic G(?455)A marker in the FGB gene. However, the PROC C(?1654)T polymorphism patients with ACS history and allele T of the PROC gene had a poor outcome more often than patients homozygous for allele C. The survival time to the endpoint for carriers of the TT and CT genotypes of the PROC gene was 2.19 ± 0.18 years vs. 2.46 ± 0.16 years for carriers of the CC genotype. On the base of these results it is suggested that hemostasis-related genes play an important role in early failures in patients with ACS history.