RESUMO
Antisense oligonucleotides (ONs) are designed to hybridize target mRNA in a sequence-specific manner and inhibit gene expression by preventing translation, either by activation of RNase H or steric blockage of the ribosome complex. Second-generation ONs, which possess greater binding affinity for target RNA relative to the isosequential phosphodiester (PO) ONs, have been developed and include, among others, peptide nucleic acids (PNA) and N3' P5' phosphoramidate oligonucleotides (npONs). In the present study, PNA and npON derivatives were targeted to the coding portion of the complementary mRNA of the N protein of the vesicular stomatitis virus (VSV) in order to evaluate their ability to arrest translation in an in vitro rabbit reticulocyte lysate system. High-affinity hybridization of ONs lacking RNase H activity was not sufficient to block translation in this test system. Only antisense ONs acting via an RNase H mechanism or by steric hindrance through covalent attachment (via transplatin modification) to the target mRNA were found to definitively arrest translation in this study.
Assuntos
Hibridização de Ácido Nucleico , Proteínas do Nucleocapsídeo , Nucleocapsídeo/genética , Oligonucleotídeos Antissenso/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , RNA Mensageiro/antagonistas & inibidores , Ribonuclease H/farmacologia , Animais , Sistema Livre de Células , Células L , Substâncias Macromoleculares , Camundongos , Estrutura Molecular , Desnaturação de Ácido Nucleico , RNA Mensageiro/genética , Coelhos , Reticulócitos/efeitos dos fármacos , Reticulócitos/metabolismo , Ribossomos/fisiologia , Relação Estrutura-Atividade , TemperaturaRESUMO
In the context of developing an approach to irreversibly and specifically link oligonucleotides to RNA, the purpose of this work was to determine the factors interfering with the rate of the rearrangement of the transplatin 1,3-intrastrand crosslinks into interstrand crosslinks, rearrangement triggered by the formation of a double helix between platinated oligo-2'-O-methyl-ribonucleotides and their complementary strands. The rate of the rearrangement has been studied as a function of the length of the hybrids, the location of the intrastrand crosslinks, the nature of the oligonucleotide backbone, and the nature of the doublet replacing the triplet complementary to the intrastrand crosslinks. The thermal stability of the platinated hybrids has been determined in various salt conditions. The results are discussed in relation to the mechanism of the rearrangement. It is shown that the cellular proteins present weaker nonspecific interactions with single-stranded platinated oligo-2'-O-methyl-nucleotides than with the isosequential oligodeoxyribonucleotides.
Assuntos
Cisplatino , Reagentes de Ligações Cruzadas , Oligorribonucleotídeos/química , RNA/química , Sequência de Bases , Estabilidade de Medicamentos , Isomerismo , Metilação , Hibridização de Ácido Nucleico , Compostos Organofosforados , TermodinâmicaRESUMO
We report a new case of fatal familial insomnia, characterized by mutation of codon 178 of prion protein gene and by methionine homozygosity at codon 129. This homozygotic form is revealed by severe insomnia and dysautonomia. Microscopic lesions, neuronal loss and gliosis, are limited to a part of the thalamus (dorso-median and anterior nuclei). From genetic analysis of a blood stain, retrospective diagnosis of fatal familial insomnia was made on the patient's mother who died three years earlier with clinical features suggestive of Creutzfeldt Jakob disease. In her case the mutation of the codon 178 was associated with the methionine/valine heterozygosity at codon 129. This genotypic polymorphism may account for conformation variation of prion protein isoform that could explain clinical and pathological differences.
Assuntos
Códon/genética , Doenças Priônicas/genética , Príons/genética , Adulto , Encéfalo/patologia , Síndrome de Creutzfeldt-Jakob/genética , Heterozigoto , Humanos , Masculino , Mutação , Fenótipo , Polimorfismo Genético , Doenças Priônicas/patologia , Fatores de TempoRESUMO
Our aim was to determine whether a single transplatin monofunctional adduct, either trans-[Pt(NH3)2(dC)Cl]+ or trans-[Pt(NH3)2(dG)Cl]+ within a homopyrimidine oligonucleotide, could further react and form an interstrand cross-link once the platinated oligonucleotide was bound to the complementary duplex. The single monofunctional adduct was located at either the 5' end or in the middle of the platinated oligonucleotide. In all the triplexes, specific interstrand cross-links were formed between the platinated Hoogsteen strand and the complementary purine-rich strand. No interstrand cross-links were detected between the platinated oligonucleotides and non-complementary DNA. The yield and the rate of the cross-linking reaction depend upon the nature and location of the monofunctional adducts. Half-lives of the monofunctional adducts within the triplexes were in the range 2-6 h. The potential use of the platinated oligonucleotides to modulate gene expression is discussed.