RESUMO
Nucleic acids have made a long and arduous journey "from the bench to the bedside." At present, it can be assumed that drugs based on modified oligonucleotides will find a worthy application in personalized medicine of the future.
RESUMO
Eukaryotic dimeric nuclear factor-κB (NF-κB) is one of the main transcription factors that activate expression of genes, products of which play the key role in development of cardiovascular pathologies, carcinogenesis, and inflammatory and viral diseases. In this review, the main attention is given to modulation of the transcription factor NF-κB activity by antisense oligonucleotides and oligonucleotide decoys. Also, current concepts about interactions between NF-κB dimers and DNA and general problems that arise in experimental use of synthetic oligonucleotides in vivo are discussed.
Assuntos
NF-kappa B/metabolismo , Oligonucleotídeos/farmacologia , Animais , Regulação da Expressão Gênica , Humanos , Camundongos , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Oligonucleotídeos/química , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/farmacologia , Multimerização Proteica , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/genéticaRESUMO
DNA duplexes bearing an aldehyde group at the 2'-position of the sugar moiety were used for affinity modification of (cytosine-5)-DNA methyltransferase SsoII. It is shown that lysine residues of M.SsoII N-terminal region are located in proximity to DNA sugar-phosphate backbone of a regulatory sequence of promoter region of SsoII restriction-modification enzyme coding genes. The ability of the two M.SsoII subunits to interact with DNA regulatory sequence has been demonstrated by affinity modification using DNA duplexes with two 2'-aldehyde groups. Changes in nucleotide sequence of one half of the regulatory region prevented cross-linking of the second M.SsoII subunit. The results on sequential affinity modification of M.SsoII by two types of modified DNA ligands (i.e. by 2'-aldehyde-containing and phosphoryldisulfide-containing) have demonstrated the possibility of covalent attachment of the protein to two different DNA recognition sites: regulatory sequence and methylation site.
Assuntos
Domínio Catalítico , Enzimas de Restrição-Modificação do DNA/química , DNA-Citosina Metilases/química , DNA/química , Regiões Promotoras Genéticas , DNA/metabolismo , Enzimas de Restrição-Modificação do DNA/metabolismo , DNA-Citosina Metilases/metabolismo , Ligação ProteicaRESUMO
We have applied bioinformatic analysis of X-ray 3D structures of complexes of transcription factor NF-kappaB with DNAs. We determined the number of possible Van der Waals contacts and hydrogen bonds between amino acid residues and nucleotides. Conservative contacts in the NF-kappaB dimer-DNA complex composed of p50 and/or p65 NF-kappaB subunit and DNA sequences like 5 -GGGAMWTTCC-3 were revealed. Based on these results, we propose a novel scheme for interactions between NF-kappaB p50 homodimer and the kappaB region of the immunoglobulin light chain gene enhancer (Ig-kappaB). We applied a chemical cross-linking technique to study the proximity of some Lys and Cys residues of NF-kappaB p50 subunit with certain reactive nucleotides into its recognition site. In all cases, the experimentally determined protein-DNA contacts were in good agreement with the predicted ones.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , NF-kappa B/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Humanos , Ligação de Hidrogênio , Dados de Sequência Molecular , NF-kappa B/química , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
DNA duplexes containing a single phosphoryldisulfide link in place of the natural internucleotide phosphodiester bond were employed in affinity modification of Cys142 in cytosine-C5 DNA methyltransferase SsoII (M.SsoII). The possibility of duplex-M.SsoII conjugation as a result of disulfide exchange was demonstrated. The crosslinking efficiency proved to depend on the DNA primary structure, modification position, and the presence of S-adenosyl-L-homocysteine, a nonreactive analog of the methylation cofactor. The SH group of M.SsoII Cys142 was assumed to be close to the DNA sugar-phosphate backbone in the DNA-enzyme complex.
Assuntos
Cisteína/metabolismo , DNA-Citosina Metilases/metabolismo , DNA/metabolismo , Compostos Organofosforados/metabolismo , Sequência de Aminoácidos , Sequência de Bases , DNA/química , Metilação de DNA , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
DNA duplexes containing the iodoacetamido group at position 2' of the ribose moiety were proposed for affinity modification of Cys in DNA-binding proteins. Reactive DNA derivatives were obtained with iodoacetic anhydride and synthetic oligodeoxyribonucleotides containing 2'-amino-2'-deoxyuridine in place of thymine at various positions. The derivatives were tested for reaction with amino acids and peptides and shown to specifically interact with Cys-containing proteins. The possibility of using the modified DNA duplexes to probe the protein SH group close to the DNA sugar-phosphate backbone in DNA-protein complexes was demonstrated with the example of subunit p50 of human transcription factor NF-kappa B.
Assuntos
DNA/química , Indicadores e Reagentes/química , Iodoacetatos/química , Ácidos Nucleicos Heteroduplexes/química , Proteínas/química , Nucleotídeos de Uracila/química , Cisteína/química , Dimerização , Glutationa Transferase/química , Glutationa Transferase/genética , Humanos , NF-kappa B/química , NF-kappa B/genética , Subunidade p50 de NF-kappa B , Oligonucleotídeos/química , Ribose/químicaRESUMO
The synthesis of oligodeoxyribonucleotides bearing mono- and diphosphoryldisulfide internucleotide links was optimized. Oligonucleotide 3'-thiophosphorothioates were modified using the thiophosphoryl-disulfide exchange with preactivated 5'-deoxy-5'-mercaptooligonucleotides or 5'-phosphorothioate derivatives both with and without a complementary template. The lack of template was shown to differently affect the product ratio (homo- and heterodimers) in the reactions of mono- and diphosphoryldisulfide-containing oligonucleotides. A replacement of one natural phosphodiester bond in 15-16-mer duplexes by a mono- or diphosphoryldisulfide group causes a slight thermal destabilization of the corresponding duplex. The disulfide recombination of the resulting compounds was studied.
Assuntos
Dissulfetos/química , Oligodesoxirribonucleotídeos/síntese química , Sequência de Bases , Oligodesoxirribonucleotídeos/químicaRESUMO
Novel modified DNA duplexes with single bridging 5'-SS-monophosphoryldithio links [-OP(=O)-O(-)-SS-CH(2)-] were synthesized by autoligation of an oligonucleotide 3'-phosphorothioate and a 5'-mercapto-oligonucleotide previously converted to a 2-pyridyldisulfide adduct. Monophosphoryldisulfide link formation is not a stringent template-dependent process under the conditions used and does not require strong binding of the reactive oligomers to the complementary strand. The modified internucleotide linkage, resembling the natural phosphodiester bond in size and charge density, is stable in water, easily undergoes thiol-disulfide exchange and can be specifically cleaved by the action of reducing reagents. DNA molecules containing an internal -OP(=O)-O(-)-SS-CH(2)- bridge are stable to spontaneous exchange of disulfide-linked fragments (recombination) even in the single-stranded state and are promising reagents for autocrosslinking with cysteine-containing proteins. The chemical and supramolecular properties of oligonucleotides with 5'-sulfhydryl groups were further characterized. We have shown that under the conditions of chemical ligation the 5'-SH group of the oligonucleotide has a higher reactivity towards N-hydroxybenzotriazole-activated phosphate in an adjacent oligonucleotide than does the OH group. This autoligation, unlike disulfide bond formation, proceeds only in the presence of template oligonucleotide, necessary to provide the activated phosphate in close proximity to the SH-, OH- or phosphate function.
Assuntos
Reagentes de Ligações Cruzadas/química , DNA/química , Oligodesoxirribonucleotídeos/química , Tionucleotídeos/química , Cromatografia Líquida de Alta Pressão , Reagentes de Ligações Cruzadas/síntese química , Reagentes de Ligações Cruzadas/metabolismo , DNA/síntese química , Dissulfetos/química , Ditiotreitol/química , Eletroforese em Gel de Poliacrilamida , Etildimetilaminopropil Carbodi-Imida/química , Modelos Químicos , Desnaturação de Ácido Nucleico , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/metabolismo , Moldes GenéticosRESUMO
Effective methods of the directed introduction of diphosphoryl disulfide bridges into hairpin DNA duplexes in place of natural phosphodiester groups were developed using the H2O2-effected ligation of 3'- and 5'-thiophosphorylated oligonucleotides or the autoligation of a preactivated oligonucleotide derivative with a phosphorothioate-bearing oligomer. The postsynthetic recombination of the disulfide-linked oligonucleotide fragments was characterized. It was shown that, along with template-directed reactions, out-of-duplex formation and exchange of diphosphoryl disulfide bonds in the DNA sugar-phosphate backbone may occur. In modified hairpin DNA, a spontaneous exchange of disulfide-linked fragments virtually does not take place because of the intramolecular duplex formation.
Assuntos
DNA/química , Dissulfetos/química , Fosfatos Açúcares/química , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de PoliacrilamidaRESUMO
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).
Assuntos
DNA/metabolismo , Distamicinas/química , Netropsina/química , Oligonucleotídeos/síntese química , Animais , DNA/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Distamicinas/síntese química , Distamicinas/metabolismo , Humanos , Netropsina/síntese química , Netropsina/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/metabolismoRESUMO
Protected 2'-deoxy-2'-fluorouridine and 2'-deoxy-2'-fluorocytidine suitable for incorporation into oligonucleotides via the phosphoramidite approach have been prepared. Five modified and two unmodified oligonucleotides have been synthesized to investigate the regiospecific cleavage of a 5S RNA from Escherichia coli by RNase H. In order to show whether the modified oligonucleotides are able to hybridize with the RNA the physico-chemical properties (melting curves, CD spectra) of analogous DNA/oligodeoxyribonucleotide duplexes have been examined. The modified oligonucleotides are shown to form stable duplexes with a DNA-matrix which exist in an A-like form. Two of the modified probes containing four 2'-deoxy-2'-fluorocytidines or two 2'-deoxy-2'-fluorouridines direct the splitting by RNase H of only one phosphodiester bond of the RNA.
Assuntos
Desoxicitidina/análogos & derivados , Floxuridina/metabolismo , Sondas de Oligonucleotídeos/metabolismo , RNA Bacteriano/metabolismo , Ribonuclease H/metabolismo , Sequência de Bases , Desoxicitidina/metabolismo , Escherichia coli/enzimologia , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Ribossômico 5S/metabolismo , RNA de Transferência de Fenilalanina/metabolismoRESUMO
The efficiency of the cleavage of RNA involved in perfect as well as imperfect hybrid duplexes composed of three components: (1) homogeneous RNA's or polyribonucleotides; (2) corresponding complementary synthetic oligodeoxyribonucleotides; (3) E. coli RNase H was investigated. The predominant RNA hydrolysis was shown to take place within the perfect hybrid duplexes formed by the target RNA and the complementary oligodeoxyribonucleotide probes. RNase H was found to cleave effectively a number of imperfect hybrid duplexes containing a central base pair mismatch.
Assuntos
RNA/metabolismo , Ribonuclease H/metabolismo , Autorradiografia , Sequência de Bases , Catálise , Eletroforese em Gel de Poliacrilamida , Escherichia coli/enzimologia , Hidrólise , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Sondas de OligonucleotídeosRESUMO
A synthesis of synthons which allow one to introduce 2'-deoxy-2'-fluoropyrimidine derivatives into the oligodeoxynucleotide chain by means of the standard solid phase phosphoramidite method has been developed. Oligonucleotides with 1-beta-D-arabinofuranosylcytosine were synthesized using either aC derivative with the unprotected 2'-OH group or O2,2'-anhydro-4-thiouridine. The synthesis of seven modified oligonucleotides (7 to 11 nucleotide residues) is described and their ability to form duplexes with complementary DNA have investigated as well as RNase H hydrolysis of hybrids formed by the E. coli 5S RNA and the obtained oligonucleotide probes.
Assuntos
Citarabina/metabolismo , Desoxirribonucleosídeos/metabolismo , Sondas de Oligonucleotídeos , RNA/metabolismo , Ribonuclease H/metabolismo , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Hidrólise , Dados de Sequência Molecular , Ácidos Nucleicos Heteroduplexes , Hibridização de Ácido Nucleico , RNA Ribossômico 5S/metabolismoRESUMO
A one-step procedure for estimating the activity of ribonuclease H from E. coli has been developed. This method is based on continuous registration of the increment in the UV adsorption of the substrate in the course of the enzymatic reaction. The heteroduplex Am.dT20 (m = 18-24) was found to be the optimal substrate for the enzyme. A comparative analysis of the rates of the enzymatic reaction as determined by UV spectroscopy and ion-pair HPLC was carried out. The kinetic parameters of the Am hydrolysis in Am.dT20 catalyzed by E. coli RNase have been determined for the first time (Km = 44 +/- 11 nM, Vmax = 0.0363 +/- 0.0053 E). The method sensitivity is 0.01-0.05 E which makes it possible to determine the RNAse H within the concentration range of 0.5-2.5 u./ml.
Assuntos
Escherichia coli/enzimologia , RNA Bacteriano/metabolismo , Ribonuclease H/metabolismo , Cromatografia Líquida de Alta Pressão , Hidrólise , Cinética , Hibridização de Ácido Nucleico , Espectrofotometria Ultravioleta , Especificidade por SubstratoRESUMO
Several oligo(ribodeoxyribo)nucleotides to be used as probes in the RNase H-induced hydrolyses of 5S ribosomal RNA E. coli were synthesized on the Victoria-4M automatic gene synthesizer by the phosphoramidite approach, which allows for introducing ribonucleotides into any position of the oligomer. 2'-Hydroxy function was protected by tert-butyldimethylsilyl group whose hydrophobicity simplified isolation of the oligonucleotides by reverse-phased HPLC.
Assuntos
Sondas de Oligonucleotídeos/síntese química , Autorradiografia , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Hibridização de Ácido NucleicoRESUMO
New oligonucleotide probes for regiospecific cleavage of RNA molecules by hybridase (RNase H) are suggested. RNase H from E. coli is shown to site-specifically split eight phosphodiester bonds in RNA in the heteroduplex, formed by 5S rRNA and d(ACCACCGCGCT). The partial substitution of deoxycytidines in position 5, 6, 8, 10 of the probe by 2'-O-methylcytidines leads to unique (regiospecific) RNA cleavage between U25 and C26.
Assuntos
Endorribonucleases/metabolismo , Sondas de Oligonucleotídeos , RNA/metabolismo , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Hidrólise , Dados de Sequência Molecular , Ácidos Nucleicos Heteroduplexes/metabolismo , Ribonuclease HRESUMO
Modified oligodeoxyribonucleotides containing 3'-terminal 2'-deoxy-2'-fluorouridine (UF) or 2'-fluorothymidine (TF) were successfully applied for specific RNA hydrolysis by RNase H from E. coli. The nonanucleotides d(CACCGCGCTF) and d(CACCGCGCUF) were synthesized using the phosphoramidite solid support method. The modified nucleosides were immobilized on the CPG support and provided the starting nucleoside residues. Model experiments were carried out using the 5S RNA from E. coli ribosomes and its 1-41 fragment. It was found that the use of this type of modified probes did not decrease neither the efficiency nor the specificity of the RNase H reaction.
Assuntos
Endorribonucleases/metabolismo , Ácidos Nucleicos Heteroduplexes/metabolismo , Sondas de Oligonucleotídeos/síntese química , RNA de Cadeia Dupla/metabolismo , RNA Ribossômico 5S/metabolismo , RNA Ribossômico/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Escherichia coli/análise , Floxuridina , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/metabolismo , RNA Bacteriano/metabolismo , Ribonuclease HRESUMO
The TMV RNA molecule can be cleaved at a single site by RNase H directed by chimeric oligo(deoxyribo-ribo)nucleotide with an internucleotide pyrophosphate bond.
Assuntos
Endorribonucleases/metabolismo , Oligodesoxirribonucleotídeos , RNA Viral/metabolismo , Vírus do Mosaico do Tabaco , Sítios de Ligação , Códon , Eletroforese em Gel de Poliacrilamida , Ácidos Nucleicos Heteroduplexes , Ribonuclease HRESUMO
Chimeric oligo(ribo-deoxyribo)nucleotides with an internucleotide pyrophosphate bond are novel probes for regiospecific hydrolysis of RNA by RNase H. It has been shown that the use of d(TGTGTAT)ppGCCAU leads to unique hydrolysis of the TMV RNA fragment pAAUGGCAUACAC between C10 and A11.