RESUMEN
Novel amino linker and spacer phosphoramidites were synthesized from methoxyoxalamido (MOX) percursors possessing a secondary hydroxyl, which when phosphitylated endowed stability to the corresponding phosphoramidites. The synthetic strategy is robust, and the chemistry is reactive towards a variety of primary aliphatic diamines and amino alcohols to produce distinctly unique phosphoramidites. The selection of building blocks determines the length and physico-chemical properties of the phosphoramidite tethering arms, and the synthesis can be specifically tailored to suit individual requirement.
Asunto(s)
ADN Intergénico/síntesis química , Compuestos Organofosforados , Aminas , Estabilidad de Medicamentos , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Estructura MolecularRESUMEN
A number of novel biotin phosphoramidites, possessing exceptionally long and uncharged tethering arms, were synthesized from methoxyoxalamido (MOX) and succinimido (SUC) precursors. Included among these monomers is a uridine derivative with the biotin moiety attached through the 2'-position. Some of these phosphoramidites were used to make 5'-biotinylated primers, which were applied in direct sequencing of genomic DNA and capture of Sanger fragment pools.
Asunto(s)
Amidas/química , Biotina , Oligodesoxirribonucleótidos/síntesis química , Ácidos Fosfóricos/química , Amidas/síntesis química , Secuencia de Bases , Biotinilación , ADN/química , Indicadores y Reactivos , Estructura Molecular , Oligodesoxirribonucleótidos/química , Ácidos Fosfóricos/síntesis química , Relación Estructura-ActividadRESUMEN
Synthesis of new terminus modifiers, bearing, along with a phosphoramidite moiety, one, two or four methoxyoxalamido (MOX) precursor groups, is described. These modifiers are introduced onto the 5'-end of a synthetic oligodeoxyribonucleotide as the last step of an automated synthesis to form the MOX precursor oligonucleotide. The MOX groups are then post-synthetically derivatized with an appropriate primary amine to construct a 5'-modified oligonucleotide. The efficiency and simplicity of the novel modifying strategy were demonstrated in the synthesis of a number of 5'-functionalized oligonucleotides.
Asunto(s)
Oligodesoxirribonucleótidos/síntesis química , Ácido Oxámico , Indicadores y Reactivos , Modelos Moleculares , Conformación Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/química , Compuestos Organofosforados , Espectrometría de Masa de Ion Secundario , TiminaRESUMEN
Oligonucleotides bearing a terminal lipophilic group attached through a biodegradable ester bond should be useful as antisense pro-drugs with improved cellular uptake. The synthesis of 5'-ester oligonucleotides is, however, problematic due to lability of the ester bond during aqueous ammonia treatment that is commonly used for the deprotection of synthetic oligonucleotides. The synthesis of 5'-palmitoyl oligodeoxynucleotides was accomplished in good yield by the use of a combination of base-labile tert-butylphenoxyacetyl amino protecting groups (t-BPA), the oxalyl-CPG anchor group, and ethanolamine (EA) as a deprotecting reagent.
Asunto(s)
Ésteres/síntesis química , Oligodesoxirribonucleótidos/síntesis química , Oligonucleótidos Antisentido/síntesis química , Profármacos/síntesis química , Acilación , Secuencia de Bases , Etanolamina , Etanolaminas , Datos de Secuencia Molecular , Palmitatos/síntesis químicaRESUMEN
The efficiency of oligodeoxynucleotide deprotection is greatly enhanced using a combination of: (a) ethanolamine, and especially a mixture of hydrazine, ethanolamine and methanol, in place of the usual aqueous ammonia; (b) tert-butylphenoxyacetyl amino protecting groups, and (c) oxalyl link between the first nucleotide and the polymeric support. The extent of base modification, particularly of C, is shown to be extremely low, and the quality of deprotected oligonucleotides is as high as in the case of ammonia deprotection. This method is also shown to be applicable to the preparation of phosphorothioate and methylphosphonate oligodeoxynucleotides and oligoribonucleotides.
Asunto(s)
Oligonucleótidos/síntesis química , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Etanolamina , Etanolaminas/química , Hidrazinas/química , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/síntesis química , Reacción en Cadena de la Polimerasa , Tionucleótidos/síntesis química , Factores de TiempoRESUMEN
A rapid method for the removal of protecting groups from synthetic oligodeoxyribonucleotides, obtained by the phosphoroamidite and H-phosphonate methods, including the use of hydrazine solutions has been developed. The combination of this procedure with the application of isopropoxyacetyl N-protecting group and oxalyl ester linkage for the first nucleoside attachment to the polymer support allows to reduce to several minutes the time needed for the full oligonucleotide deprotection.
Asunto(s)
Hidrazinas/química , Oligodesoxirribonucleótidos/química , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia MolecularRESUMEN
The chemical-enzymatic synthesis of a gene coding for A2B2 repeats of the albumin-binding domain of streptococcal protein G has been accomplished. The codon usage of the natural gene has been modified to adapt an artificial sequence for the efficient translation in E. coli. The gene (238 b.p.) was cloned in the polylinker plasmid pUCL1 and then fused in frame to the 3'-terminus of the gene for the IgG-binding domain of staphylococcal protein A, which was earlier cloned in the expression plasmid pUCL2. A fused polypeptide composed of the E and B domains of protein A and A2B2 repeats of protein G was produced in E. coli cells under the lac promoter control. The resulted product was isolated by affinity chromatography on IgG-sepharose and (or) albumin-sepharose.
Asunto(s)
Albúminas/genética , Proteínas Bacterianas/genética , Escherichia coli/genética , Streptococcus/metabolismo , Albúminas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía de Afinidad , ADN , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Plásmidos , Secuencias Repetitivas de Ácidos NucleicosAsunto(s)
Bacillus thuringiensis/genética , Toxinas Bacterianas , Clonación Molecular/métodos , ADN Bacteriano/genética , ADN Viral/genética , ADN/genética , Endotoxinas , Escherichia coli/genética , Vectores Genéticos , Plásmidos , Fagos T/genética , Regiones Terminadoras Genéticas , Toxinas de Bacillus thuringiensis , Proteínas Bacterianas/genética , Proteínas Hemolisinas , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN/métodos , Transcripción GenéticaRESUMEN
Two new rapid procedures for the full deprotection of synthetic oligonucleotides has been developed. We have successfully used the mixture of ethanolamine and ethanol (1:1) or pure ethanolamine for deprotection of oligonucleotides, prepared by different methods. In the case of oligonucleotides prepared by commonly used beta-cyanoethyl phosphoramidite and H-phosphonates method deprotection takes half an hour at 70 degrees C. We have found also that mixture of hydrazine, ethanolamine and methanol (1:3:3, v/v/v) can serve as a very efficient reagent for deprotection of oligonucleotides, prepared by beta-cyanoethyl phosphoramidite method with isopropoxyacetyl protecting group for cytosine residues. In this case deprotection time is 12-17 min at room temperature.
Asunto(s)
Oligonucleótidos/síntesis química , Química/métodos , Indicadores y Reactivos , Organofosfonatos , FosfatosRESUMEN
Using oligonucleotide-directed mutagenesis and chemical-enzymatic DNA synthesis, genes for A and B insulin chains, C-peptide and Tyr-C-peptide have been constructed starting from synthetic gene for human proinsulin synthesized earlier. The genes for human preproinsulin, mini-proinsulin, single-chain insulin and their modifications were also synthesized. The constructions obtained were cloned in plasmid vectors.
Asunto(s)
Genes Sintéticos , Insulina/genética , Secuencia de Aminoácidos , Escherichia coli/genética , Ingeniería Genética , Humanos , Datos de Secuencia Molecular , Plásmidos , Proinsulina/genética , Precursores de Proteínas/genéticaRESUMEN
The chemical-enzymatic synthesis of a gene for IgG-binding fragment of the staphylococcal protein A has been carried out. The design of the gene, which consists of signal peptide and modified E and B domains, and strategy of the synthesis provided possibility of various degrees of polymerization of the gene fragment coding for B domain and of the whole gene. Several protein A-like polypeptides composed of the leader sequence, E domain and 1 to 4 copies of B domain were produced in E. coli cells under the lac promoter control.
Asunto(s)
Regulación de la Expresión Génica , Genes Sintéticos , Fragmentos de Inmunoglobulinas/genética , Proteína Estafilocócica A/genética , Staphylococcus aureus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Escherichia coli/genética , Datos de Secuencia Molecular , Staphylococcus aureus/inmunologíaRESUMEN
Plasmid-based promoter-probe vectors pPV4 and pPV5 have been constructed which are useful for comparing the relative efficiencies of bacterial promoters. The vectors utilize the beta-galactosidase (lacZ) gene of E. coli as an indicator gene. The latter was modified using synthetic DNA fragments. The promotor-probe system contains the ampicillin resistance gene and the origin of replication of plasmid pBR322. The plasmids pPV4 and pPV5 carry clustered unique restriction sites usable for promoter insertions, and SD sequence. A synthetic DNA fragment corresponding to transcription terminator was inserted downstream the lacZ gene. Presence of the terminator made it possible to clone strong promoters controlling transcription of the lacZ gene. To prevent any undesired promotor effect, the plasmid pPV5 has also second synthetic terminator upstream from the polylinker sequence. Using this promoter-probe system, relative efficiencies of a series of synthetic promoters, including PL promoter of phage lambda and its mutant, gene X promotor of phage fd and several model statistic promoters, have been compared.
Asunto(s)
Escherichia coli/genética , Galactosidasas/genética , Genes Bacterianos , Vectores Genéticos , Regiones Promotoras Genéticas , beta-Galactosidasa/genética , Resistencia a la Ampicilina/genética , Secuencia de Bases , Clonación Molecular , Regulación de la Expresión Génica , Datos de Secuencia Molecular , Plásmidos , Transcripción GenéticaRESUMEN
Rapid procedure for the synthesis of oligonucleotides by the phosphotriester method has been developed. It is based on the use of O-nucleophilic intramolecular catalysis. The application of this method to automated solid-phase oligonucleotide synthesis allows to perform one elongation cycle for 6-7 min.
Asunto(s)
Oligonucleótidos/síntesis química , Catálisis , Indicadores y Reactivos , MétodosRESUMEN
An effective procedure for the synthesis of oligonucleotides by the phosphotriester method has been developed. The procedure is based on the use of phosphate protecting groups enabling O-nucleophilic intramolecular catalysis in the reaction of internucleotide bond formation under the action of arylsulfonyl chlorides and their derivatives. Using this new procedure, the time needed to perform one elongation step on polymer support is 7-8 min. The effectiveness of the methodology has been demonstrated in the synthesis of many oligodeoxyribonucleotides of different length with high yields.