RESUMO
The aim of this work was to construct an anti-messenger targeted to the pim-1 oncogene transcript, based on circular or clamp oligodeoxyribonucleotides. The formation of bimolecular triplexes by clamp or circular oligonucleotides was investigated using single-stranded targets of both DNA (5'-CCCTCCTTTGAAGAA-3') and RNA type (5'-CCCUCCUUUGAAGAA-3'). The third, 'Hoogsteen' strand of the triplex was represented by G,T-rich sequences. The secondary structures of the complexes were determined by thermal denaturation, circular dichroism and gel mobility shift experiments and shown to depend on the nature of the target strand. With DNA as target, the sequence of a clamp (or circular) oligonucleotide that formed the triple helix was 3'-GGGAGGAAACTTCTTTT-TTGTTGTTT-TT-GGTGGG-5', where the first TT dinucleotide (in italics) is a linker and the second TT (bold) represents the bridge through which the 'Hoogsteen' strand switches from one strand of the Watson-Crick duplex to the other, once the duplex is formed by the corresponding portion of the anti-messenger (underlined). The portion of the 'Hoogsteen' sequence of the triplex between the two TT dinucleotides binds to the 3' extremity of the target strand and runs parallel to it. The portion situated at the 5' end of the oligonucleotide switches to the purine tract of the complementary strand of the duplex and is antiparallel to it. In contrast, with RNA as target, for a branched clamp oligonucleotide that formed a triple helix over its entire length (5'-TTCTTCAAAGGAGGG-3' 3'-GGGTGGTTT-T-GTTGTT-5') the portion of the 'Hoogsteen' sequence that bound to the 3' extremity of the target strand had to be antiparallel to it.
Assuntos
DNA de Cadeia Simples/química , Técnicas Genéticas , Purinas/química , Pirimidinas/química , RNA/química , Dicroísmo Circular , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Espectrofotometria UltravioletaRESUMO
The influence of the secondary structure of oligonucleotides having a natural phosphodiester backbone on their ability to interact with DNA and RNA targets and on their resistance to the nucleolytic digestion is investigated. Oligonucleotides having hairpin, looped and snail-like structure are found to be much more stable to nuclease degradation in different biological media and inside cells than the linear ones. The structured oligonucleotides can also hybridise with their DNA and RNA targets.
Assuntos
Conformação de Ácido Nucleico , Oligonucleotídeos Antissenso/química , Células 3T3 , Animais , Eletroforese em Gel de Poliacrilamida , Endonucleases/metabolismo , Exonucleases/metabolismo , Vírus da Leucemia Murina de Friend/genética , Genes env/genética , Células HeLa , Humanos , Camundongos , Desnaturação de Ácido Nucleico , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligorribonucleotídeos/química , Ribonuclease H/metabolismo , Espectrofotometria Ultravioleta , Termodinâmica , TransfecçãoRESUMO
This investigation is devoted to design of short "switch" oligonucleotides mono- or bi-functionnalized with intercalating agents capable to form a stable triplex with HIV integrase-cognate sequences and inhibit selectively HIV integration. Methods of intercalator incorporation at 5'- and/or 3'-terminal positions or one of the pyrimidine heterocyclic bases are developed.
Assuntos
Inibidores de Integrase de HIV/farmacologia , HIV-1/genética , Substâncias Intercalantes/química , Oligonucleotídeos/química , Integração Viral/efeitos dos fármacos , Sequência de Bases , Inibidores de Integrase de HIV/química , Repetição Terminal Longa de HIVRESUMO
Oligonucleotide-peptide conjugates have several applications, including their potential use as improved antisense agents for interfering with the RNA function within cells. In order to provide robust and generally applicable conjugation chemistry, we developed a novel approach of fragment coupling of pre-synthesized peptides to the 2'-position of a selected nucleotide within an otherwise protected oligonucleotide chain attached to a solid support.
Assuntos
Bioquímica/métodos , Nucleoproteínas/química , Oligonucleotídeos Antissenso/química , Oligonucleotídeos/química , Peptídeos/química , Adenosina/análogos & derivados , Adenosina/química , Arabinonucleosídeos/química , Cromatografia Líquida de Alta Pressão , Nucleoproteínas/síntese química , Oligonucleotídeos/síntese química , Oligonucleotídeos Antissenso/síntese química , Peptídeos/síntese química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The chemical ligation of 17 50-54-membered nicked DNA dumbbells with different closing fragments, nick positions, and nucleotides facing the nick were investigated. T4, T5, GTA4C, GCGA2GC, and GCGA3GC sequences were chosen as the closing fragments. The nicks were placed in the center of the duplex stem or were adjacent to the closing fragments. N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and cyanogen bromide were used as the condensing agents. We showed that the ligation efficiency is 10%-90% depending on the sequence of the closing fragments, nick position, and nucleotides facing the nick. Coupling yields of 80%-90% were observed when the nick was situated in the middle of the molecule between two T residues or was adjacent to GCGA2GC or GCGA3GC minihairpins. In the last case, the reacting 3'-phosphate and 5'-hydroxy groups were brought close together by only two base pair minihairpins. The coupling yields did not depend on the nature of the condensing agent. On the basis of the results obtained, we believe a rational design of nicked DNA dumbbells has been developed for efficient chemical synthesis of closed dumbbells.
Assuntos
Proteínas de Ligação a DNA , DNA/síntese química , Proteínas Nucleares , Conformação de Ácido Nucleico , Polidesoxirribonucleotídeos/química , Pareamento de Bases , Sequência de Bases , Brometo de Cianogênio/química , DNA/química , DNA/genética , Desenho de Fármacos , Etildimetilaminopropil Carbodi-Imida/química , Fator 1 Nuclear de Hepatócito , Fator 1-beta Nuclear de Hepatócito , Cinética , Polidesoxirribonucleotídeos/genética , Elementos de Resposta/genética , Moldes Genéticos , Termodinâmica , Fatores de Transcrição/metabolismoRESUMO
To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented.
Assuntos
DNA/química , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/química , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Oligodesoxirribonucleotídeos/química , Sequência de Bases , Sítios de Ligação , Substâncias Macromoleculares , Magnésio/química , Modelos Teóricos , Oligodesoxirribonucleotídeos/metabolismoRESUMO
The method of protein affinity modification by DNA analogues was used to study the characteristic features of restriction endonuclease MvaI interaction with DNA. Oligonucleotide duplexes containing a monosubstituted pyrophosphate internucleotide bond were used for cross-linking to the enzyme. The conditions of the reaction of MvaI endonuclease with these reagents were investigated. On the basis of data obtained, the model of successive inclusion of two Mg2+ ions into MvaI endonuclease-substrate complex was proposed and confirmed by the kinetic scheme of the process.
Assuntos
DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Magnésio/química , Algoritmos , Reagentes de Ligações Cruzadas , Cinética , Oligonucleotídeos/química , Conformação ProteicaRESUMO
A 30-membered DNA duplex containing the recognition site of restriction endonuclease SsoII and a 30-membered RNA--DNA hybrid duplex, a substrate of E. coli RNase H, were synthesized. The cleavage of the 30-membered fragments of nucleic acids catalyzed by endonucleases in the presence of Co-phthalocyanine complex [CoPc(COONa)8] containing eight carboxyl groups at the periphery of the ligand was studied. It was shown that the efficiency of enzyme catalysis decreases in the presence of the metal complex for both endonucleases. By addition of a 100-fold excess of Co-phthalocyanine complex with respect to DNA duplex the initial rate of substrate hydrolysis by restriction endonuclease SsoII is observed to decrease twice. An equimolar ratio of the metal complex and hybrid duplex leads to essentially complete inhibition of RNA cleavage by RNase H from E. coli. The inhibition of catalytic activity of enzymes recognizing the double-stranded nucleic acids in the presence of Co-phthalocyanine complex is assumed to be caused by the ability of the latter to interact with DNA, RNA, and DNA--RNA duplexes.
Assuntos
Metais/farmacologia , Ácidos Nucleicos/metabolismo , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Sequência de Bases , Sítios de Ligação , DNA/química , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/antagonistas & inibidores , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Indóis/farmacologia , Cinética , Ácidos Nucleicos/química , Compostos Organometálicos/farmacologia , RNA/química , RNA/metabolismo , Ribonuclease H/antagonistas & inibidores , Ribonuclease H/metabolismo , Especificidade por SubstratoRESUMO
DNA duplex and dumbbells containing chemically active acylphosphate internucleotide groups were synthesized. To obtain these compounds the chemical ligation method was used. The acylphosphate group was inserted into a DNA duplex and dumbbells as a result of template-directed condensation of 5'-phosphate and especially introduced 3'-carboxy groups of oligonucleotides. 1-Ethyl-3-(3'-dimethylaminopropyl)carbodiimide (EDC) was used as a condensing agent. Oligonucleotides containing a carboxy group were obtained by the interaction of their 3'-phosphate with glycine methyl ester under the action of EDC, followed by ester hydrolysis. The yields of acylphosphate-containing double-stranded oligonucleotides achieved 15-25% depending on the structure of their precursors. It was shown that these compounds are acylating agents and are efficiently cleaved in near-physiological conditions under the action of ethylenediamine or N-methylimidazole. These results indicate that double-stranded oligonucleotides carrying acylphosphate internucleotide groups could constitute new crosslinking reagents for affinity modification of DNA recognizing proteins.
Assuntos
DNA/síntese química , Fosfatos/química , Sequência de Bases , DNA/químicaRESUMO
Cyclic and polycyclic oligonucleotides were synthesized using chemical ligation. Two types of catenanes with one and several intertwinings were produced. The yield of these molecules depended on the ligation conditions and nucleotide sequence of the ligated oligonucleotide and its template. Structure of ligation products was investigated and confirmed using restriction endonuclease MvaI. Interaction of the synthesized molecules with restriction endonucleases SsoII, EcoRII and HindIII was also studied.
Assuntos
DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , DNA/metabolismo , DNA Circular/química , DNA Circular/metabolismo , Ácidos Nucleicos Heteroduplexes/metabolismo , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/metabolismoRESUMO
The main purpose of the present work is to search for the optimal design of a DNA duplex containing an active group for crosslinking and irreversible inhibition of the transcription factor NF-kappa B. Modified DNA duplexes with an identical nucleotide sequence but different internucleotide phosphates replaced by the trisubstituted pyrophosphate internucleotide group were synthesized. Crosslinking of the human NF-kappa B p50 subunit with the modified DNA duplexes was carried out. It was shown that only four modified duplexes crosslinked with the NF-kappa B p50 subunit. The specificity of these reactions was confirmed. A position of the phosphate in the NF-kappa B recognition site was found where replacement on the active trisubstituted pyrophosphate group resulted in a 50% yield of crosslinking. The fact that DNA duplexes containing the trisubstituted pyrophosphate group specifically react with the NF-kappa B p50 subunit in the Escherichia coli total lysate supports the idea that such modified DNA can be used as high specific inhibitors for DNA-recognizing proteins.
Assuntos
DNA/metabolismo , NF-kappa B/antagonistas & inibidores , Oligonucleotídeos Antissenso/farmacologia , Animais , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Reagentes de Ligações Cruzadas , Cristalografia por Raios X , Difosfatos , Desenho de Fármacos , Escherichia coli , Humanos , Substâncias Macromoleculares , Camundongos , NF-kappa B/química , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/química , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/químicaRESUMO
A chemical ligation procedure has been developed for the synthesis of oligoribonucleotides carrying a trisubstituted pyrophosphate (tsp) linkage in place of a single phosphodiester. Good yields of tsp were obtained when a single 2'-deoxynucleoside 5' to the tsp was used in the ligation reaction. A tsp linkage was found to be reasonably stable to hydrolysis but cleaved by treatment with ethylenediamine or lysine to give phosphoamidate adducts. A model human immunodeficiency virus type 1 (HIV-1) TAR RNA duplex containing an activated tsp was able to bind to HIV-1 Tat protein with only 3-fold reduced affinity and to a Tat peptide (residues 37-72) with identical affinity compared to that of an unmodified duplex. Tsps incorporated at sites previously identified as being in close proximity to Tat protein were able to cross-link to Tat peptide (37-72) to form a covalent phosphoamidate conjugate. Endopeptidase cleavage followed by MALDI-TOF (matrix-assisted laser desorption ionization time of flight) mass spectrometric analysis provided strong evidence that a TAR duplex containing a tsp replacing the phosphate at 38-39 had reacted specifically with Lys51 in the basic region of Tat peptide (37-72). The new chemical cross-linking method may be generally useful for identifying lysines in close proximity to phosphates in basic RNA-binding domains of proteins.
Assuntos
Reagentes de Ligações Cruzadas/farmacologia , Difosfatos/química , Produtos do Gene tat/metabolismo , Repetição Terminal Longa de HIV , HIV-1 , Modelos Químicos , RNA Viral/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Difosfatos/metabolismo , Eletroforese em Gel de Poliacrilamida , Produtos do Gene tat/síntese química , Dados de Sequência Molecular , Oligonucleotídeos/síntese química , Oligonucleotídeos/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Moldes Genéticos , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
Cyclization of a 38-mer oligodeoxyribonucleotide on a cyclic template was studied by the chemical and enzymic ligation methods. Both structures and yields of the reaction products depended on the ligation method and the nucleotide and template sequences. The chemical ligations resulted in the formation of catenanes, whose structures were confirmed by hydrolysis with the MvaI restriction endonuclease. Presence of G/C-rich clusters near the formed internucleotide bond favored the catenane formation.
Assuntos
Nucleotídeos Cíclicos/síntese química , Oligodesoxirribonucleotídeos/síntese química , Moldes GenéticosRESUMO
Cyanogen bromide has been found to induce the template-guided condensation of oligonucleotides only in the presence of N-substituted morpholines. Based on 31P, 1H and 13C NMR spectroscopy data, the mechanism of the phosphomonoester group activation by cyanogen bromide in N-substituted morpholine buffers is suggested. It has also been shown that BrCN can be used for the synthesis of oligonucleotide derivatives in aqueous solution.
Assuntos
Brometo de Cianogênio/química , Evolução Molecular , Morfolinas/química , Oligodesoxirribonucleotídeos/química , Oligonucleotídeos/síntese química , Soluções Tampão , DNA/química , Moldes GenéticosRESUMO
A method for preparing a modified derivative of 2'-amino-2'-deoxy-arabino-adenosine ready for directed insertion into an oligonucleotide chain during solid-phase synthesis was elaborated. A series of the title oligonucleotides (6-25-mers) containing a 2'-amino-2'-deoxy-arabino-adenosine fragment were prepared. A high reactivity of the 2'-amino group during the acylation with carboxylic acid anhydrides was demonstrated. It was shown that the insertion of the modified fragments into oligonucleotides did not inhibit the formation of the DNA duplex.