RESUMO
The concept of solid-phase synthesis of oligoribonucleotides using T4 RNA ligase and T4 polynucleotide kinase has been proposed and tested with model homo-oligoribonucleotides. The method consists of the immobilization of the first oligomer block at the 3'-terminus on a solid support followed by a chain elongation in the 5'-direction with trinucleoside diphosphates using T4 RNA-ligase and phosphorylation using polynucleotide kinase. Hydrazides of Biogel P-300, Sepharose 4B and cellulose were tested as solid supports for immobilization of initial oligomers. The properties of supports were rated on reactivities of immobilized 5'-phosphorylated oligomers as phosphate donors in the solid phase reactions, hydrodynamical properties and capacity to eliminate donor molecules spontaneously during reactions. Hydrazide of Sepharose 4B appeared to be a more suitable support because of better hydrodynamic properties and highest reactivities of immobilized donors. Saturated concentrations of RNA ligase and polynucleotide kinase and optimal time of joining reaction were determined. In a model experiment ApApA was twice attached to the immobilized hydrazide of Sepharose 4B donor (pA)6pAox. The yield of (Ap)12 was 25%.
Assuntos
Bacteriófago T4/enzimologia , Oligorribonucleotídeos/síntese química , Polinucleotídeo 5'-Hidroxiquinase/química , RNA Ligase (ATP)/química , Animais , Celulose/química , Humanos , Hidrazinas/química , Oligorribonucleotídeos/metabolismo , Fosforilação , Polinucleotídeo 5'-Hidroxiquinase/metabolismo , RNA Ligase (ATP)/metabolismo , Sefarose/química , Fatores de TempoRESUMO
A series of novel N1 alkylated purine nucleic acids were polymerized either enzymatically or by automated synthesis to further establish the SAR requirements for HIV, RT, and HCMV activity. Out of the series, two constructs, 2'-O-methyl-1-allylinosinic acid phosphorothioate 33-mer (16) and an oligomer incorporating 1-propyl-6-thioinosinic acid residues (20), were found to be highly active under all three assay conditions. SAR studies indicate that sulfur incorporation, high molecular weight, and low steric bulk at N1 all can be important for activity.
Assuntos
Antivirais/síntese química , Polinucleotídeos/síntese química , Compostos Alílicos/síntese química , Compostos Alílicos/química , Compostos Alílicos/farmacologia , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Antivirais/química , Antivirais/farmacologia , Sobrevivência Celular , Citomegalovirus/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Humanos , Inosina Monofosfato/química , Oligorribonucleotídeos/síntese química , Oligorribonucleotídeos/química , Oligorribonucleotídeos/farmacologia , Polímeros , Polinucleotídeos/química , Polinucleotídeos/farmacologia , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacologia , Relação Estrutura-Atividade , Tionucleotídeos/síntese química , Tionucleotídeos/química , Tionucleotídeos/farmacologia , Células Tumorais CultivadasRESUMO
RNA oligomers greater than 35-40 mers in length form in one day in the montmorillonite clay-catalyzed reaction of unblocked RNA monomers at 25 degrees C in aqueous solution.
Assuntos
Adenina/análogos & derivados , Nucleotídeos/química , Oligorribonucleotídeos/síntese química , RNA/síntese química , Bentonita/química , Biopolímeros/química , Desoxirribonuclease I , Estrutura Molecular , Uridina/químicaRESUMO
The precursor of an RNA molecule from T4-infected E. coli cells (p2Sp1 RNA) has the capacity to cleave itself at specific positions [(UpA (139-140) and CpA (170-171)], within a putative loop and stem structure. This sequence-specific cleavage requires at least a monovalent cation and non-ionic detergents. We studied the self-cleavage reaction of an RNA fragment (GUUUCGUACAAAC) (R1) with the sequence corresponding to the p2Sp1 RNA in the presence of Mg2+ and non-ionic detergents. It requires Mg2+ and is aided by a non-ionic detergent, Brij 58. The cleavage reaction is time, temperature, and pH-dependent. The cleavage occurs at the phosphodiester bond between UpA and CpA on the RNA fragment (GUUUCGUACAAAC) (R1). Furthermore, the maximum of cleavage of R1 occurs at a very low Mg2+ concentration (< or = 5 mM).
Assuntos
Cetomacrogol/química , Magnésio/química , Oligorribonucleotídeos/síntese química , Precursores de RNA/metabolismo , Tensoativos/química , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Oligorribonucleotídeos/metabolismo , Precursores de RNA/química , Temperatura , Fatores de TempoRESUMO
Bis-phosphoimidazolides of an analogue of adenosine (in which ribose is replaced by an acyclic chain) and of two related analogues of guanosine undergo oligomerization in the presence of complementary polynucleotide templates. Data on the template- and nontemplate-directed reactions are presented, and the possible relevance to origins of life is discussed.
Assuntos
Oligorribonucleotídeos/síntese química , Moldes Genéticos , Imidazóis , Estrutura Molecular , Compostos Organofosforados , PolímerosRESUMO
The 2-(methylthio)phenylthiomethyl (MPTM) group was developed as a new type of 2'-hydroxyl protecting group in oligoribonucleotide synthesis. The building monomer units of uridine and cytidine for the phosphotriester approach were synthesized from 2'-O-(1,3-benzodithiol-2-yl)-3',5'-O- (1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)uridine and successfully utilized for the synthesis of CpUpG.
Assuntos
Oligorribonucleotídeos/síntese química , Indicadores e Reagentes , Siloxanas , Compostos de Trialquitina , Uridina/análogos & derivadosRESUMO
Recent results concerning the synthesis of oligoribonucleotides via the phosphotriester method, such as functionalization of ribonucleosides, new phosphorylating agents, 5'-O-sulfonylation and chromatography on Sephadex LH-20 for monitoring the removal of internucleotide phosphotriester groups, are presented. To show that efficiency of a new approach to the synthesis of oligoribonucleotides the pentamer /Up/4U was obtained.
Assuntos
Oligonucleotídeos/síntese química , Oligorribonucleotídeos/síntese química , Ésteres , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Métodos , SiloxanasRESUMO
For further improvement in the investigation to utilize a cellulose acetate derivative as a novel type of polymer-support for the synthesis of oligonucleotides, the investigations on utilizing another spacer; on protecting groups for O6-position of guanosine unit, ribothymidine, and pseudouridine; and on a novel protecting group for the introduction of phosphate function at 5'-terminal position, targeting the syntheses of 13-mer, ApApGpGpApApApApUpUpApUpG, 11-mer, pCpUpCpGpUpCpCpApCpCpA, and 12-mer, UpCpCpGpGprTp- psipCpGpApUpU, found in the partial structures of a yeast tRNA(Ala), will be described in detail.
Assuntos
Oligorribonucleotídeos/síntese química , Sequência de Bases , Celulose/análogos & derivados , Indicadores e Reagentes , Dados de Sequência Molecular , Pseudouridina , RNA de Transferência de Alanina/síntese química , Saccharomyces cerevisiaeRESUMO
Cholesterol was linked to 2'-O-methyl-oligoribonucleotides (2'-OMe-RNA) via a disulfide bond by reacting the 3'-(pyridyldithio)-modified 2'-OMe-RNA with thiocholesterol in dichloromethane-methanol solution. This ligation reaction was made possible by a novel strategy in which the highly charged oligonucleotide was rendered soluble in nonaqueous solvent through conversion to a lipophilic amidinium salt. The biodegradable lipophilic modification of 2'-OMe-RNA resulted in a large increase in incorporation of such oligonucleotides into liposomes prepared by reversephase evaporation. Furthermore, association of these modified oligonucleotides with cultured TIB 73 cells was 100-fold higher than that seen with unmodified 2'-OMe-RNA in serum-free medium and about 10 to 30-fold higher in the presence of 10% calf serum. During incubation with cells, release of the internalized oligonucleotide from the thiocholesteryl moiety can be demonstrated.
Assuntos
Colesterol/análogos & derivados , Lipossomos/metabolismo , Oligorribonucleotídeos/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Colesterol/metabolismo , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Dissulfetos , Metilação , Dados de Sequência Molecular , Oligorribonucleotídeos/síntese químicaRESUMO
Advances in oligoribonucleotide synthesis have lagged behind those in oligodeoxyribonucleotide synthesis because of the difficulty in identifying orthogonal protecting groups for the 2'- and 5'-hydroxyls. Adaptation of the phosphoramidite method for DNA synthesis to RNA synthesis has greatly improved our understanding of RNA. It allows site-specific introduction of modified nucleosides to any position in an RNA molecule, as well as introduction of variations at multiple sites in the molecule. This overview discusses issues that are relevant to RNA synthesis by the phosphoramidite approach, including supports used, activation of the ribonucleoside phosphoramidites, and protection of the nucleobase, phosphate, and 2'- and 5'-hydroxyls.
Assuntos
Bioquímica/métodos , Oligorribonucleotídeos/síntese química , Compostos Organofosforados/química , Radical Hidroxila/química , Oligorribonucleotídeos/química , Fosfatos/química , Polímeros/química , Ribonucleosídeos/química , Compostos de Tritil/químicaRESUMO
Some new 2',5'-oligonucleotide trimers containing 5'-terminal 5'-amino-5'-deoxy- and 5'-amino-3',5'-dideoxyadenosine derivatives have been synthesized. Some of the trimers showed biological inhibitions of HIV-1 reverse transcriptase (RT), HIV-1 induced syncytia formation and PCR amplification.
Assuntos
Nucleotídeos de Adenina/síntese química , Didesoxiadenosina/química , Oligorribonucleotídeos/síntese química , Inibidores da Transcriptase Reversa/síntese química , Nucleotídeos de Adenina/farmacologia , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Biopolímeros , Transcriptase Reversa do HIV/antagonistas & inibidores , Oligorribonucleotídeos/farmacologia , Reação em Cadeia da Polimerase , Inibidores da Transcriptase Reversa/farmacologiaRESUMO
The synthesis of oligoguanylates [oligo(G)s] is catalyzed by a template of oligocytidylates [oligo(C)s] containing 2',5'- and 3',5'-linked phosphodiester bonds with and without incorporated C5'ppC groupings. An oligo(C) template containing exclusively 2',5'-phosphodiester bonds also serves as a template for the synthesis of complementary oligo(G)s. The oligo(C) template was prepared by the condensation of the 5'-phosphorimidazolide of cytidine on montmorillonite clay. These studies establish that RNA oligomers prepared by mineral catalysis, or other routes on the primitive earth, did not have to be exclusively 3',5'-linked to catalyze template-directed synthesis, since oligo(C)s containing a variety of linkage isomers serve as templates for the formation of complementary oligo(G)s. These findings support the postulate that origin of the RNA world was initiated by the RNA oligomers produced by polymerization of activated monomers formed by prebiotic processes.