RESUMO
The 5' cap structure of mRNA is a N7 methylated guanosine residue that is linked by a 5'-5' triphosphate linkage to the 5'-terminus of cellular and viral RNAs synthesized by RNA polymerase II. This unique structure facilitates several processes of mRNA metabolism, including splicing, nucleocytoplasmic transport,initiation of translation, and degradation. Previous research has demonstrated that the lanthanide macrocycle complex, Eu(THED)3+, effectively cleaves the 5' cap structure of mRNA in solution by nucleophilic attack of the triphosphate linkage via the metal-activated hydroxyethyl group of the THED ligand. This report shows that attachment of a Eu(THED)3+analog to the 3'-terminus of an antisense oligonucleotide, which targets the 5'-terminus of the intercellular adhesion molecule 1 mRNA, potentiates the inhibitory activity of the antisense oligonucleotide in cytokine-treatedendothelial cells.
Assuntos
Európio/farmacologia , Molécula 1 de Adesão Intercelular/genética , Oligonucleotídeos Antissenso/farmacologia , Compostos Organometálicos/farmacologia , Capuzes de RNA/metabolismo , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismoRESUMO
Little is known about the mechanisms that account for inhibition of gene expression by antisense oligonucleotides at the level of molecular cell biology. For this purpose, we have selected potent 2'-O-(2-methoxy)ethyl antisense oligonucleotides (IC50 = 2 and 6 nM) that target the 5' cap region of the human intercellular adhesion molecule 1 (ICAM-1) transcript to determine their effects upon individual processes of mRNA metabolism in HUVECs. Given the functions of the 5' cap structure throughout mRNA metabolism, antisense oligonucleotides that target the 5' cap region of a target transcript have the potential to modulate one or more metabolic stages of the message inside the cell. In this study we found that inhibition of protein expression by these RNase H independent antisense oligonucleotides was not due to effects on splicing or transport of the ICAM-1 transcript, but due instead to selective interference with the formation of the 80 S translation initiation complex. Interestingly, these antisense oligonucleotides also caused an increase in ICAM-1 mRNA abundance in the cytoplasm. These results imply that ICAM-1 mRNA turnover is coupled in part to translation.
Assuntos
Endotélio Vascular/metabolismo , Molécula 1 de Adesão Intercelular/biossíntese , Oligonucleotídeos Antissenso/farmacologia , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , RNA Mensageiro/biossíntese , Transcrição Gênica/efeitos dos fármacos , Células Cultivadas , Selectina E/biossíntese , Etil-Éteres , Citometria de Fluxo , Humanos , Cinética , Biossíntese de Proteínas , Capuzes de RNA , RNA Mensageiro/química , Relação Estrutura-Atividade , Veias UmbilicaisRESUMO
Biophysical and pharmacokinetic properties of five analogs of ISIS 3082, a 20-mer phosphorothioate oligodeoxynucleotide that inhibits the expression of mouse intercellular adhesion molecule 1, were evaluated. Compared to the parent compound, ISIS 3082, the 2'-propoxy modified phosphodiester, ISIS 9044 and the 2'-propoxy phosphorothioate, ISIS 9045, had greater affinity for complementary RNA and were more lipophilic. A chimeric oligonucleotide comprised of 2'-propoxy diester wings and a phosphorothioate deoxy center (ISIS 9046) had equal affinity. It was also more lipophilic than ISIS 3082, but less so than the other 2'-propoxy modified analogs. The two analogs with 5'-lipophilic conjugates, ISIS 9047 (5'-octadecylamine) and ISIS 8005 (5'-(2'-O-hexylamino-carbonyl-oxycholesterol) were more lipophilic than ISIS 3082 (3- and 7-fold, respectively) but had similar affinity for complementary RNA. Binding of ISIS 3082 to bovine serum albumin was salt-dependent and, at physiological concentration (320 mOsmol), the dissociation constant (Kd) was 140 microM. Similarly, the 2'-propoxy phosphodiester, ISIS 9044, displayed salt-dependent bovine serum albumin binding, but not binding was measurable at physiological salt conditions. In contrast, the more lipophilic phosphorothioate analogs displayed much higher affinity to bovine serum albumin at 320 mOsmol than ISIS 3082. After bolus injection to mice, the initial volumes of distribution of the more lipophilic phosphorothioate analogs, ISIS 9045, ISIS 9047 and ISIS 8005, were less and the initial clearance from plasma was slower than ISIS 3082. The pharmacokinetics of the other analogs was similar to ISIS 3082. Distribution of ISIS 3082 into peripheral tissues was similar to that reported for other phosphorothioates with liver and kidney accumulating the highest fraction of the dose. The only modification to markedly influence distribution was the very lipophilic cholesterol conjugate (ISIS 8005), which increased substantially the fraction of the dose accumulated by the liver. Little intact drug was found in urine or feces for any analog, and the patterns of metabolites suggested that for all analogs the principal metabolic pathway was due to 3'-exonuclease activity. The metabolism of ISIS 3082 was similar to that reported for other phosphorothioates. After 2 hr, most of the radioactivity in plasma represented metabolites but, in tissues, intact ISIS 3082 was present for much longer periods of time and metabolites accumulated more slowly. The 24-hr exposure to ISIS 3082 of liver and kidney was 20.7 and 67.9 microM/hr, respectively. The rates of metabolism in plasma, liver and kidney of the two 5'-conjugates, ISIS 9047 and ISIS 8005, were similar to ISIS 3082, as was the pattern of metabolism. The rate of metabolism of ISIS 9044 (2'-propoxy phosphodiester oligonucleotide) was much more rapid in liver and plasma, but surprisingly much slower in the kidney. ISIS 9045 (full 2-propoxy phosphorothioate) was much more stable than ISIS in all tissues, the enhanced stability of ISIS 9045 resulted in increased exposure of liver and kidney to the drug, whereas the exposure of the liver to the two more lipophilic analogs, ISIS 9047 and ISIS 8005, was greater because a higher fraction of the dose was distributed to the liver. The exposure of the kidney to ISIS 9044 was also greater than that to ISIS 3082 due to the surprising stability of the drug in the kidney.
Assuntos
Molécula 1 de Adesão Intercelular/genética , Oligonucleotídeos Antissenso/farmacocinética , Tionucleotídeos/farmacocinética , Animais , Sequência de Bases , Bovinos , Cromatografia Líquida de Alta Pressão , Rim/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Ligação Proteica , Distribuição TecidualRESUMO
Gel shift assays were used to examine the binding of the lactose (lac) repressor to polyoperator DNA molecules. Specific binding was differentiated from nonspecific DNA association by (i) equilibrating repressor-operator complexes below the nonspecific association constant and (ii) demonstrating the effects of the inducer isopropyl beta-D-thiogalactoside (IPTG) on the formation of repressor-operator complexes. With the linear polyoperator molecules, all eight operator sites could be simultaneously bound by distinct repressors. However, with circular molecules, the eight operator sites were saturable by repressor only in the nicked circular state and not in the covalently closed circular form. Under the experimental conditions used, there was no evidence of bifunctional repressor binding or loop formation. The results suggest that the conformational perturbation of DNA that occurs upon specific repressor binding was retained in topologically closed molecules and could modify other operator sites so as to make them unavailable for specific binding.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Bacteriano/metabolismo , DNA Circular/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Regiões Operadoras Genéticas , Proteínas Repressoras/metabolismo , Sequência de Bases , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Isopropiltiogalactosídeo/farmacologia , Repressores Lac , Dados de Sequência Molecular , Sequências Repetitivas de Ácido NucleicoRESUMO
We have analyzed lac repressor binding in vivo and in vitro to several symmetric lac operator sequences. Two features of the operator appear to be important for repressor binding: sequence, both of the operator and of its extended regions, and the spacing of the operator halves. Host mutations that alter DNA superhelical density (topA, gyrB) did not change the relative affinity of cloned symmetric operator sequences for repressor. Analysis by dimethylsulfate methylation and DNaseI digestion of repressor-operator complexes indicated that repressor makes symmetric contacts with the symmetric operator, in contrast to its contacts with the two halves of the natural operator.
Assuntos
Escherichia coli/genética , Óperon Lac , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , DNA Super-Helicoidal/genética , Dados de Sequência Molecular , MutaçãoRESUMO
16 single-site mutations and a 1-bp deletion in the lac operator have been cloned and examined with regard to repressor binding. A 13-bp, central 'core' operator sequence, bp 5-17 of the natural operator, was also synthesized and cloned. Repressor affinity was assessed in vivo by quantitating the level of beta-galactosidase activity resulting from chromosomal operon derepression and in vitro by measuring the stability of repressor-operator complexes. Our results support the general conclusion that the repressor-operator interaction is asymmetric, particularly across the center of the operator sequence, with little or no specific contact at position 12. Some sequence changes in the right side of the operator markedly reduced repressor affinity, indicating that although binding to this half of the sequence has been suggested to be less important than the left half, it still significantly contributes to the binding affinity.
Assuntos
Óperon Lac , Regiões Operadoras Genéticas , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Clonagem Molecular , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Técnicas In Vitro , Metilação , Mutação , Conformação de Ácido Nucleico , Relação Estrutura-AtividadeRESUMO
Using a Hirsch classification maze, selection was made for positive and negative geotactic behaviors in three different strains of Drosophila pseudoobscura. Hybridization studies were then carried out with flies from the diverged strains. The geotactic scores of the parents and F1 flies indicate that both negative and positive geotactic behaviors in these strains are strongly influenced by genes in the X chromosome. Additional hybridization studies using flies from strains with similar phenotypes suggest that the diverged strains contain similar alleles and the number of loci in the X chromosomes responsible for these types of behavior is limited. The loci may be highly organized in the X chromosome.