RESUMEN
1,2-Diol-oligoribonucleotides were prepared using fully protected 2'-O-[2-(2,3-dihydroxypropyl)amino-2-oxoethyl]uridine 3'-phosphoramidite. Incorporation of the 2'-modified uridine residue into oligonucleotide chains does not significantly affect the thermal stability of RNA and RNA-DNA duplexes. Periodate oxidation of the 1,2-diol results in reactive 2'-aldehyde oligoribonucleotides. Further application of these oligonucleotides for cross-linking with bacterial ribonuclease P was investigated.
Asunto(s)
Aldehídos/química , ADN/química , Ácidos Nucleicos Heterodúplex/química , Oligorribonucleótidos/química , Oligorribonucleótidos/síntesis química , ARN/química , Proteínas Bacterianas/química , Ribonucleasa P/químicaRESUMEN
The most promising approach for detection of random point mutations relies upon the DNA chemical cleavage near associated mismatching base pairs. In our study, the series of heteroduplexes with all types of mismatches and extrahelical nucleotide residues surrounded by both A x T and G x C pairs were performed via hybridization of 50-mer synthetic oligonucleotides differing in only one nucleotide at the central position. The chemical cleavage of DNA duplexes immobilized on magnetic beads by means of biotin-streptavidin interaction was carried out with chemicals, which able to attack only nucleobases flipped out of the base stack: potassium permanganate and hydroxylamine reacting to T and C respectively. The chemical reactivity of different mismatches was shown to correlate clearly with the target local structure in a particular sequence context. This work makes up for a deficiency in systematic study of DNA cleavage near mismatches in dependence on their type, orientation and flanking nucleotides. The model system elaborated may be applied to estimate the sensitivity of the methodology and to control the possibility of false-positive and false-negative result appearance, when different protocols for detection of DNA mutations are used. The modification of heteroduplex mixtures by potassium permanganate and hydroxylamine allows to reveal any non-canonical base pair and suggest its type and neighboring nucleotides from the nature of chemical as well as its localization from the length of cleavage products.
Asunto(s)
Disparidad de Par Base , División del ADN , ADN/química , Mutación Puntual , Nucleótidos de Citosina/química , ADN/genética , Análisis Heterodúplex , Hidroxilamina/química , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Permanganato de Potasio/química , Nucleótidos de Timina/químicaRESUMEN
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.
Asunto(s)
ADN/química , Disulfuros/química , Fosfatos de Azúcar/química , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de PoliacrilamidaAsunto(s)
Oligonucleótidos/genética , ARN Bacteriano/metabolismo , ARN Ribosómico 5S/metabolismo , Ribonucleasa H/metabolismo , Secuencia de Bases , Dicroismo Circular , Hidrólisis , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Hibridación de Ácido Nucleico , Oligonucleótidos/química , Oligonucleótidos/metabolismo , TemperaturaRESUMEN
An effective method for the oligonucleotide cyclization using BrCN-induced chemical ligation was developed. The novel idea to incorporate non-nucleotide inserts makes the process of cyclizations independent of the inner secondary structure of the linear precursor. An set of assays were developed to confirm the cyclic structure of the compounds obtained.
Asunto(s)
Oligodesoxirribonucleótidos/síntesis química , Secuencia de Bases , Bromuro de Cianógeno , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/químicaRESUMEN
Efficiency of the template-directed condensation of oligonucleotides depends on the nature of the nucleotide units to be joined. Fourteen out of sixteen dinucleotide combinations in double-stranded DNA were examined. Alterations of the reaction efficiency are identical for cyanogen bromide and 1-ethyl-3-(3'-dimethylaminopropyl)carbodiimide coupling reagents. Dependence of the chemical ligation efficiency on the DNA sequence-specific local conformation is discussed.
Asunto(s)
Ácidos Nucleicos/química , Oligonucleótidos/química , Secuencia de Bases , Bromuro de Cianógeno/química , Etildimetilaminopropil Carbodiimida/química , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Moldes GenéticosRESUMEN
The mutation system has been developed to study the mutagenic properties of modified oligonucleotide analogs. The mutagenic properties of oligonucleotides containing one ribonucleotide have been examined. The presence of a ribonucleotide is shown not to induce any mutations. But when the oligonucleotide induces two marker deletions detached by 6 nucleotides they may be repaired separately, in this case the deletion bordering with the ribonucleotide is predominantly repaired.
Asunto(s)
Reparación del ADN , Replicación del ADN , Mutagénesis Sitio-Dirigida , Oligonucleótidos/genética , Ribonucleótidos/genética , Secuencia de Bases , Datos de Secuencia MolecularRESUMEN
Chemical ligation of oligonucleotides in double stranded complexes has been considered in its structural-kinetic aspect. In a study of the reactivity of eleven synthetic water soluble carbodiimides the reaction rate has been found to be strongly affected by the substituents at N1 and N3 atoms. The chemical ligation appears to involve the linear form of carbodiimide. Model systems have been used to assess the rate constant for the phosphate activation. The ratio of kinetic constants for the productive and nonproductive reactions of the activated derivate apparently reflects the reactive site conformation; for a given duplex this parameter is virtually independent of the condensing agent structure.
Asunto(s)
ADN/química , Imidas/química , Ácidos Nucleicos Heterodúplex/química , Secuencia de Bases , Cinética , Datos de Secuencia Molecular , SolubilidadRESUMEN
To characterize structural factors affecting kinetic parameters of chemical ligation, DNA duplexes with various arrangements of reacting groups in the ligation site have been synthesized and studied. The modifications included replacement of a deoxythymidine with a uridine or a nucleoside having reversed configuration at C2' and/or C3' atoms, the modified residue being phosphate donor or acceptor; introduction of an "extra" nucleotide residue; replacement of a G.C pair second to the junction with a noncomplementary G.G pair. Thermal stability of the anomalous nucleotide duplexes has been characterized. Based on analysis of the kinetic parameters of chemical ligation, a suggestion is made about the conformation of the reactive site.
Asunto(s)
ADN/química , Ácidos Nucleicos Heterodúplex/química , Autorradiografía , Secuencia de Bases , Sitios de Unión , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Cinética , Datos de Secuencia Molecular , Conformación de Ácido NucleicoRESUMEN
Condensation of oligonucleotides containing ribonucleotide segments (from mononucleotide to full sequence) on DNA-template was studied, with cyanogen bromide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as condensing agents. Efficiency of the chemical ligation of RNA oligomers was much lower than that of DNA analogues. The yield of chemical ligation products was found to depend on the position of RNA segment in the hybrid duplexes and on the position of the phosphate group in the nick. The oligoribonucleotide's length does not affect the yield of condensation products. A nick in 5S RNA loop was repaired by means of the chemical ligation method.
Asunto(s)
Ácidos Nucleicos Heterodúplex , Autorradiografía , Secuencia de Bases , Fenómenos Químicos , Química , Dicroismo Circular , ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Oligonucleótidos/metabolismo , ARN/metabolismo , Moldes GenéticosRESUMEN
Structural defects, affecting T4 DNA ligase function, were revealed with the help of synthetic DNA duplexes, containing modifications at single nick. Changes of configuration at C2' and C3' atoms of furanose in the acceptor terminus lead to total blocking of the nick sealing activity of T4 DNA ligase. On the contrary, substitution of 3'-terminal deoxyribonucleotide for ribonucleotide doesn't affect the enzyme's action. The duplex looses all of it's substrate activity if the next from the nick G.C pair is substituted for the noncomplementary G.C pair. In DNA duplexes containing an unpaired base in the nick, elimination of the extrahelical nucleotide proceeds the ligation step. In these cases the duplex substrate activity decreases depending on the extent of extrahelical base stacking into the double stranded DNA.
Asunto(s)
ADN Ligasas/metabolismo , ADN Viral/metabolismo , Ácidos Nucleicos Heterodúplex/metabolismo , Polinucleótido Ligasas/metabolismo , Fagos T/enzimología , Catálisis , ADN Ligasa (ATP) , Electroforesis en Gel de Poliacrilamida , Especificidad por SustratoRESUMEN
Cyanogen bromide was found to induce a smooth and highly efficient condensation of oligonucleotide blocks within DNA duplexes. This template-directed reaction proceeds in the aqueous solution at 0 degree C for 1-2 min. Cyanogen bromide catalyzes the formation not only of phosphodiester, but also of unnatural phosphoramide and pyrophosphate interoligomer bonds.
Asunto(s)
Bromuro de Cianógeno , ADN , Oligodesoxirribonucleótidos , Fenómenos Químicos , Química , Indicadores y ReactivosRESUMEN
Three 35 bp-DNA duplexes have been assembled from synthetic oligonucleotides by means of DNA ligase or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in two parallel series of experiments. The "top" strands of these duplexes correspond either to the imperfect (natural) or perfect terminal inverted repeats of the IS1. Tm of DNA duplexes composed of 2 to 6 different oligonucleotides were investigated by UV spectroscopy. It was shown that DNA ligase effectively joined oligonucleotides even under conditions of DNA duplex instability. However, there is a minimum duplex size (within the range of 9-15 bp) below which the enzymatic ligation is ineffective. Chemical assembly of duplexes took place only if the double helix was stable. The yield was 50% after two successive ligation cycles. Efficiency of the chemical ligation depends on the nature of the nucleotide units to be joined.
Asunto(s)
Inversión Cromosómica , Elementos Transponibles de ADN , ADN/síntesis química , Genes Sintéticos , Secuencias Repetitivas de Ácidos Nucleicos , ADN/genética , Conformación de Ácido Nucleico , Desnaturalización de Ácido Nucleico , Oligonucleótidos/síntesis químicaRESUMEN
The optimal way of constructing a family of restriction endonuclease EcoRII substrates has been developed. The substrates are DNA-like duplexes containing regularly repeated native or modified sites of this enzyme as well as those of EcoRI and AluI. Synthesis of substrates was performed by water-soluble carbodiimide-induced polycondensation of two nonanucleotides, d(C-C-T-G-G-A-A-T-Tp) and d(C-C-A-G-G-A-G-C-Tp), as constituents of different complementary complexes. The products of reaction (degree of polymerization, 2-20) were isolated by G-200 gel-filtration. The yield of polymers was about 70%. The main products of reaction were dimers when dephosphorylated nonanucleotides (terminators of polycondensation) were used. The thermal stability of DNA-like duplexes is very high. The structure of the polymers obtained has been confirmed by UV-spectroscopy and by CD data as well as by the results of cleavage by EcoRI and AluI restriction endonucleases.
Asunto(s)
Enzimas de Restricción del ADN/genética , ADN Bacteriano/genética , Desoxirribonucleasas de Localización Especificada Tipo II , Escherichia coli/genética , Oligonucleótidos/síntesis química , Secuencias Repetitivas de Ácidos Nucleicos , Secuencia de Bases , Cromatografía en Gel , Dicroismo Circular , ADN Bacteriano/análisis , Escherichia coli/enzimología , Ácidos Nucleicos Heterodúplex/análisis , Oligonucleótidos/análisis , Especificidad por SustratoRESUMEN
Two types of DNA-duplexes containing the repeating fragments of natural promoters have been obtained starting from synthetic oligodeoxyribonucleotides TGCATTATAA, AACTAGTT, AGTTAACT. Deca- and octanucleotides have been synthesized by solid phase method with stepwise or blockwise chain elongation. UV- and CD-spectroscopy has been used to study the physico-chemical properties of the synthetic oligonucleotides. Polycondensation of oligonucleotides induced by water-soluble carbodiimide (chemical ligation) or T4-polynucleotide ligase allowed to synthesize the promoter models. The degree of polymerization varied from 2 to 8 in case of chemical ligation and from 2 to 30 in case of enzymatic ligation. A new chain length regulation technique has been developed by means of addition of a terminator of polycondensation (unphosphorylated oligonucleotide) in the reaction mixture.