RESUMO
Chromatin modification and higher-order chromosome structure play key roles in gene regulation, but their functional interplay in controlling gene expression is elusive. We have discovered the machinery and mechanism underlying the dynamic enrichment of histone modification H4K20me1 on hermaphrodite X chromosomes during C. elegans dosage compensation and demonstrated H4K20me1's pivotal role in regulating higher-order chromosome structure and X-chromosome-wide gene expression. The structure and the activity of the dosage compensation complex (DCC) subunit DPY-21 define a Jumonji demethylase subfamily that converts H4K20me2 to H4K20me1 in worms and mammals. Selective inactivation of demethylase activity eliminates H4K20me1 enrichment in somatic cells, elevates X-linked gene expression, reduces X chromosome compaction, and disrupts X chromosome conformation by diminishing the formation of topologically associating domains (TADs). Unexpectedly, DPY-21 also associates with autosomes of germ cells in a DCC-independent manner to enrich H4K20me1 and trigger chromosome compaction. Our findings demonstrate the direct link between chromatin modification and higher-order chromosome structure in long-range regulation of gene expression.
Assuntos
Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica , Cromossomo X/química , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/genética , Mecanismo Genético de Compensação de Dose , Embrião não Mamífero/metabolismo , Histona Desmetilases com o Domínio Jumonji/química , Histona Desmetilases com o Domínio Jumonji/metabolismo , Modelos Moleculares , Mutação , Piperidinas/metabolismo , Alinhamento de Sequência , Tiofenos/metabolismoRESUMO
The three-dimensional organization of a genome plays a critical role in regulating gene expression, yet little is known about the machinery and mechanisms that determine higher-order chromosome structure. Here we perform genome-wide chromosome conformation capture analysis, fluorescent in situ hybridization (FISH), and RNA-seq to obtain comprehensive three-dimensional (3D) maps of the Caenorhabditis elegans genome and to dissect X chromosome dosage compensation, which balances gene expression between XX hermaphrodites and XO males. The dosage compensation complex (DCC), a condensin complex, binds to both hermaphrodite X chromosomes via sequence-specific recruitment elements on X (rex sites) to reduce chromosome-wide gene expression by half. Most DCC condensin subunits also act in other condensin complexes to control the compaction and resolution of all mitotic and meiotic chromosomes. By comparing chromosome structure in wild-type and DCC-defective embryos, we show that the DCC remodels hermaphrodite X chromosomes into a sex-specific spatial conformation distinct from autosomes. Dosage-compensated X chromosomes consist of self-interacting domains (â¼1 Mb) resembling mammalian topologically associating domains (TADs). TADs on X chromosomes have stronger boundaries and more regular spacing than on autosomes. Many TAD boundaries on X chromosomes coincide with the highest-affinity rex sites and become diminished or lost in DCC-defective mutants, thereby converting the topology of X to a conformation resembling autosomes. rex sites engage in DCC-dependent long-range interactions, with the most frequent interactions occurring between rex sites at DCC-dependent TAD boundaries. These results imply that the DCC reshapes the topology of X chromosomes by forming new TAD boundaries and reinforcing weak boundaries through interactions between its highest-affinity binding sites. As this model predicts, deletion of an endogenous rex site at a DCC-dependent TAD boundary using CRISPR/Cas9 greatly diminished the boundary. Thus, the DCC imposes a distinct higher-order structure onto X chromosomes while regulating gene expression chromosome-wide.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mecanismo Genético de Compensação de Dose/fisiologia , Complexos Multiproteicos/metabolismo , Cromossomo X/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Mecanismo Genético de Compensação de Dose/genética , Feminino , Regulação da Expressão Gênica , Hibridização in Situ Fluorescente , Masculino , Ligação Proteica , Análise de Sequência de RNA , Cromossomo X/genéticaRESUMO
Transposons are mobile genetic elements that are a major constituent of most genomes. Organisms regulate transposable element expression, transposition, and insertion site preference, mitigating the genome instability caused by uncontrolled transposition. A recent burst of research has demonstrated the critical role of small non-coding RNAs in regulating transposition in fungi, plants, and animals. While mechanistically distinct, these pathways work through a conserved paradigm. The presence of a transposon is communicated by the presence of its RNA or by its integration into specific genomic loci. These signals are then translated into small non-coding RNAs that guide epigenetic modifications and gene silencing back to the transposon. In addition to being regulated by the host, transposable elements are themselves capable of influencing host gene expression. Transposon expression is responsive to environmental signals, and many transposons are activated by various cellular stresses. TEs can confer local gene regulation by acting as enhancers and can also confer global gene regulation through their non-coding RNAs. Thus, transposable elements can act as stress-responsive regulators that control host gene expression in cis and trans.
Assuntos
Metilação de DNA/genética , Elementos de DNA Transponíveis/genética , Regulação da Expressão Gênica , Pequeno RNA não Traduzido/genética , Animais , Epigênese Genética , Genoma , Plantas , Transdução de Sinais/genéticaRESUMO
Characterizing how genomic sequence interacts with trans-acting regulatory factors to implement a program of gene expression in eukaryotic organisms is critical to understanding genome function. One means by which patterns of gene expression are achieved is through the differential packaging of DNA into distinct types of chromatin. While chromatin state exerts a major influence on gene expression, the extent to which cis-acting DNA sequences contribute to the specification of chromatin state remains incompletely understood. To address this, we have used a fission yeast sequence element (L5), known to be sufficient to nucleate heterochromatin, to establish de novo heterochromatin domains in the Schizosaccharomyces pombe genome. The resulting heterochromatin domains were queried for the presence of H3K9 di-methylation and Swi6p, both hallmarks of heterochromatin, and for levels of gene expression. We describe a major effect of genomic sequences in determining the size and extent of such de novo heterochromatin domains. Heterochromatin spreading is antagonized by the presence of genes, in a manner that can occur independent of strength of transcription. Increasing the dosage of Swi6p results in increased heterochromatin proximal to the L5 element, but does not result in an expansion of the heterochromatin domain, suggesting that in this context genomic effects are dominant over trans effects. Finally, we show that the ratio of Swi6p to H3K9 di-methylation is sequence-dependent and correlates with the extent of gene repression. Taken together, these data demonstrate that the sequence content of a genomic region plays a significant role in shaping its response to encroaching heterochromatin and suggest a role of DNA sequence in specifying chromatin state.
Assuntos
Genoma Fúngico , Heterocromatina/química , Schizosaccharomyces/genética , DNA Fúngico/genética , DNA Fúngico/metabolismo , Dosagem de Genes , Regulação Fúngica da Expressão Gênica , Heterocromatina/genética , Heterocromatina/metabolismo , Estrutura Terciária de Proteína , Schizosaccharomyces/química , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Centromeres are essential for genetic inheritance-they prevent aneuploidy by providing a physical link between DNA and chromosome segregation machinery. In many organisms, centromeres form at sites of repetitive DNAs that help establish the chromatin architecture required for centromere function. These repeats are often rapidly evolving and subject to homogenization, which causes the expansion of novel repeats and sequence turnover. Thus, centromere sequence varies between individuals and across species. This variation can affect centromere function. We utilized Schizosaccharomyces pombe to assess the relationship between centromere sequence and chromatin structure and determine how sensitive this relationship is to genetic variation. In S. pombe, nucleating sequences within centromere repeats recruit heterochromatin via multiple mechanisms, which include RNA-interference (RNAi) . Heterochromatin, in turn, contributes to centromere function through its participation in three essential processes; establishment of a kinetochore, cohesion of sister chromatids, and suppression of recombination. Here, we show that a centromere element containing RevCen, a target of the RNAi pathway, establishes heterochromatin and gene silencing when relocated to a chromosome arm. Within this RevCen-containing element (RCE), a highly conserved domain is necessary for full heterochromatin nucleation but cannot establish heterochromatin independently. We characterize the 10 unique RCEs in the S. pombe centromere assembly, which range from 60% to 99.6% identical, and show that all are sufficient to establish heterochromatin. These data affirm the importance of centromere repeats in establishing heterochromatin and suggest there is flexibility within the sequences that mediate this process. Such flexibility may preserve centromere function despite the rapid evolution of centromere repeats.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Centrômero/genética , Proteínas Cromossômicas não Histona/genética , Variação Genética , Heterocromatina/genética , Humanos , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Normal pituitary gland development requires coordination between maintenance of progenitor cell pools and selection of progenitors for differentiation. The spatial and temporal expression of Notch2 during pituitary development suggested that it could control progenitor cell differentiation in the pituitary. Consistent with this idea, Notch2 is not expressed in Prop1 mutants, and anterior pituitary progenitors in Prop1 mutants appear to be unable to transition from proliferation to differentiation properly, resulting in anterior lobe failed cell specification and evolving hypoplasia. To test the function of Notch2 directly, we used the alphaGSU subunit promoter to express activated NOTCH2 persistently in pre-gonadotropes and pre-thyrotropes of transgenic mice. At birth, there is a small reduction in the population of fully differentiated thyrotropes and almost no fully differentiated gonadotropes. The temporal and spatial expression of Hey1 suggests that it could be a mediator of this effect. Gonadotropes complete their differentiation program eventually, although expression of LH and FSH is mutually exclusive with NOTCH2 transgene expression. This demonstrates that activated Notch2 is sufficient to delay gonadotrope differentiation, and it supports the hypothesis that Notch2 regulates progenitor cell differentiation in the pituitary gland.
Assuntos
Diferenciação Celular/fisiologia , Gonadotrofos/metabolismo , Gonadotrofos/fisiologia , Receptor Notch2/metabolismo , Animais , Linhagem da Célula , Embrião de Mamíferos/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Subunidade alfa de Hormônios Glicoproteicos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Hipófise/embriologia , Hipófise/metabolismo , Pró-Opiomelanocortina/metabolismo , Tireotrofos/metabolismo , Tireotropina/metabolismo , TransgenesRESUMO
Changes in chromosome number impair fitness by disrupting the balance of gene expression. Here we analyze mechanisms to compensate for changes in gene dose that accompanied the evolution of sex chromosomes from autosomes. Using single-copy transgenes integrated throughout the Caenorhabditis elegans genome, we show that expression of all X-linked transgenes is balanced between XX hermaphrodites and XO males. However, proximity of a dosage compensation complex (DCC) binding site (rex site) is neither necessary to repress X-linked transgenes nor sufficient to repress transgenes on autosomes. Thus, X is broadly permissive for dosage compensation, and the DCC acts via a chromosome-wide mechanism to balance transcription between sexes. In contrast, no analogous X-chromosome-wide mechanism balances transcription between X and autosomes: expression of compensated hermaphrodite X-linked transgenes is half that of autosomal transgenes. Furthermore, our results argue against an X-chromosome dosage compensation model contingent upon rex-directed positioning of X relative to the nuclear periphery.
Assuntos
Caenorhabditis elegans/genética , Dosagem de Genes , Expressão Gênica , Cromossomos Sexuais/metabolismo , Animais , Animais Geneticamente Modificados , Feminino , Perfilação da Expressão Gênica , Genes Reporter , MasculinoRESUMO
This review describes the application of a natural defense mechanism to develop effective agents for the post-transcriptional control of gene expression. 2-5A is a unique 2',5'-phosphodiester bond linked oligoadenylate, (pp)p5'A2'(p5'A)(n), that is elaborated in virus-infected interferon-treated cells. The 2-5A system is an RNA degradation pathway that is an important mechanistic component of interferon's action against certain viruses. It may also play a role in the anticellular effects of interferon and in general RNA decay. A major player in the 2-5A-system is the latent and constitutive 2-5A-dependent ribonuclease (RNase L) which upon activation by 2-5A, degrades RNA. This RNase L enzyme can be recruited for antisense therapeutics by linking it to an appropriate oligonucleotide targeted to a chosen RNA. Syntheses of 2-5A, its analogues, 2-5A-antisense, and its modifications are detailed herein. Applications of 2-5A-antisense to particular targets such as HIV, PKR, chronic myelogenous leukemia, telomerase, and respiratory syncytical virus are described.
Assuntos
Antineoplásicos/farmacologia , Antivirais/farmacologia , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Interferons/farmacologia , Oligorribonucleotídeos Antissenso/metabolismo , Oligorribonucleotídeos Antissenso/farmacologia , Vírus de RNA/efeitos dos fármacos , Nucleotídeos de Adenina/química , Nucleotídeos de Adenina/metabolismo , Animais , Antivirais/química , Antivirais/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proteínas de Transporte/síntese química , Proteínas de Transporte/química , Endorribonucleases/metabolismo , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Oligorribonucleotídeos/química , Oligorribonucleotídeos/metabolismo , Oligorribonucleotídeos Antissenso/síntese química , Oligorribonucleotídeos Antissenso/química , Vírus Sinciciais Respiratórios/efeitos dos fármacos , Homologia de Sequência do Ácido Nucleico , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
An unsolved question in steroid hormone action is why the amount of agonist activity displayed by antisteroids is not constant but varies with the assay conditions. Receptor mutations have provided insight into hormone action, presumably due to changes in the tertiary structure of the receptor that alter its interaction surfaces with the transcriptional machinery or/and co-factors. We have now employed two mechanistically different induction assays to determine whether disparate transactivation processes are similarly altered by receptor mutations. The two activation assays studied were (i) the standard induction of GREtkLUC in transiently transfected CV-1 cells and (ii) a novel modulation of endogenous receptor activity by transiently transfected receptors in HeLa cells. Five different mutations in the ligand binding and DNA binding domains of the rat glucocorticoid receptor (CS1, CS1/CD, 451/9, C656G, and R732Q) and seven steroids of varied structures (five antagonists and two agonists) were selected for use. The results in both induction assays were the same. However, no generalizations regarding steroid structure and activity emerged. Neither of two potent glucocorticoids were active with GR-CS1, or GR-CS1/CD, while RU 486 was the only antisteroid with appreciable agonist activity. With the GR-451/9 mutant, three antagonists afforded partial agonist activity. We confirmed that the C656G mutant is both "super-sensitive" and "super-selective" for transactivation. In contrast, the R732Q mutation caused significant decreases in activity with both antagonists and subsaturating concentrations of agonists. This inability to generalize about the behavior of any class of steroids with mutant receptors may reflect an induced fit for each receptor steroid complex. Nevertheless, the activity of a given steroid appeared to be constant in two different transactivation assays for a given mutant receptor. Thus, disparate transactivation processes may utilize identical receptor surfaces, even in the expression of partial agonist activity for specific antiglucocorticoids.
Assuntos
Glucocorticoides/farmacologia , Antagonistas de Hormônios/farmacologia , Receptores de Glucocorticoides/metabolismo , Transcrição Gênica , Ativação Transcricional , Substituição de Aminoácidos , Animais , Linhagem Celular , Chlorocebus aethiops , Desoxicorticosterona/farmacologia , Dexametasona/farmacologia , Glucocorticoides/metabolismo , Células HeLa , Antagonistas de Hormônios/farmacocinética , Humanos , Cinética , Mifepristona/farmacologia , Mutagênese Sítio-Dirigida , Pregnatrienos/farmacologia , Ratos , Receptores de Glucocorticoides/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Fully automated solid-phase synthesis gave access to a hybrid in which 5'-phosphorylated-2'-5'-linked oligoadenylate (2-5A) is connected to the 5'-terminus of DNA which, in turn, is linked at the 3'-end to PNA [2-5A-(5')-DNA-(3')-PNA chimera]. This novel antisense molecule retains full RNase L activation potency while suffering only a slight reduction in binding affinity.
Assuntos
Endorribonucleases/síntese química , Oligonucleotídeos Antissenso/metabolismo , Ácidos Nucleicos Peptídicos , Cromatografia Líquida de Alta Pressão , Endorribonucleases/metabolismo , Ativação Enzimática , Humanos , Ligação ProteicaRESUMO
Many essential aspects of genome function, including gene expression and chromosome segregation, are mediated throughout development and differentiation by changes in the chromatin state. Along with genomic signals encoded in the DNA, epigenetic processes regulate heritable gene expression patterns. Genomic signals such as enhancers, silencers, and repetitive DNA, while required for the establishment of alternative chromatin states, have an unclear role in epigenetic processes that underlie the persistence of chromatin states throughout development. Here, we demonstrate in fission yeast that the maintenance and inheritance of ectopic heterochromatin domains are independent of the genomic sequences necessary for their de novo establishment. We find that both structural heterochromatin and gene silencing can be stably maintained over an ~10-kb domain for up to hundreds of cell divisions in the absence of genomic sequences required for heterochromatin establishment, demonstrating the long-term persistence and stability of this chromatin state. The de novo heterochromatin, despite the absence of nucleation sequences, is also stably inherited through meiosis. Together, these studies provide evidence for chromatin-dependent, epigenetic control of gene silencing that is heritable, stable, and self-sustaining, even in the absence of the originating genomic signals.
Assuntos
Epigênese Genética , Genoma Fúngico , Heterocromatina/metabolismo , Schizosaccharomyces/genética , Divisão Celular , Regulação Fúngica da Expressão Gênica , Ordem dos Genes , Inativação Gênica , Loci Gênicos , Genômica , Meiose , Modelos Biológicos , Schizosaccharomyces/citologia , Schizosaccharomyces/metabolismo , Transcrição GênicaRESUMO
This paper reviews the research in progress in Oxford on the chemical, spectroscopic and redox properties of bis(thiosemicarbazonato) complexes of zinc and copper in the context of the hypoxia selectivity of the copper(II) complex. Also, covered are synthetic strategies for modified complexes with a range of functional substituents and the in vitro and in vivo characteristics of two of these derivatives are described. Finally, the synthesis of some new bifunctional macrocyclic ligands is described and some of these give Cu(II) derivatives which cannot be reduced and are therefore resistant to reductive loss of copper in vivo.
Assuntos
Cobre/química , Radioisótopos/química , Marcação por Isótopo/métodos , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/químicaRESUMO
A series of chemical in vitro assays are described to provide a rapid initial assessment of the in vivo stability and biological behaviour of potential new copper(II) based radiopharmaceutical agents. Chemical challenges using an excess of cysteine, glutathione (GSH) and histidine, which are models of S- and N-donor molecules found in vivo, are used to provide a measure of the potential for loss of the copper(II) ion from the radiopharmaceutical as a result of ligand dissociation. In addition, thiol containing molecules such as cysteine and GSH provide a redox challenge, whereby the copper(II) complex may be reduced to give a copper(I) species. The stability of the copper(I) species toward oxidation, protonation, and ligand dissociation may be crucial in determining the biodistribution, the biological half-life and excretion mechanisms of a potential radiopharmaceutical. Further evaluation of the redox stability is assessed using the ubiquitous biological reductant ascorbic acid. The relative stability of a complex with respect to ligand dissociation in human serum provides one of the most important experiments assessing the potential of a complex to be used in vivo. Further challenge experiments with serum proteins such as thioredoxin and serum albumin can be used to provide more detailed information on the probable fate of the complex in serum. Evaluation of complex stability and speciation over a range of pH values may also be used to obtain information on potential biodistribution.
Assuntos
Cobre/química , Compostos Radiofarmacêuticos/química , Proteínas Sanguíneas/química , Cobre/metabolismo , Cristalografia por Raios X/métodos , Cisteína/química , Glutationa/química , Histidina/química , Técnicas In Vitro , Íons , Modelos Químicos , Conformação Molecular , Oxirredução , Preparações Farmacêuticas/química , Potenciometria/métodos , Espectrofotometria/métodos , Compostos de SulfidrilaRESUMO
Reaction of [MoV(TpMe,Me)(O)Cl2] with 1,3-, 1,5-, 1,6-, 2,6-, and 2,7-dihydroxynaphthalene affords the dinuclear complexes [[Mo(TpMe,Me)(O)Cl]2(mu-C10H6O2)], abbreviated as 1,3-Mo2, 1,5-Mo2, 1,6-Mo2, 2,6-Mo2, and 2,7-Mo2, according to the substitution pattern of the bridging ligand. Electrochemical, UV-vis/NIR spectroscopic, and variable-temperature magnetic susceptibility studies have been used to probe the effects of the bridging-ligand topology on the metal-metal electronic and magnetic interactions. The complexes can be split into two classes according to the properties of the bridging ligands. Complexes 1,3-Mo2, 1,6-Mo2, and 2,7-Mo2 all have bridging ligands that are topologically equivalent to meta-substituted bridging ligands such as 1,3-dihydroxybenzene, in that (i) there is an odd number of C atoms separating the two oxygen atoms, regardless of the pathway that is taken through the ligand skeleton, and (ii) the doubly oxidized from of the bridging ligand is a diradical. These complexes are classified as being "T-meta" (= topologically equivalent to meta). Complexes 1,5-Mo2 and 2,6-Mo2 have bridging ligands that are topologically equivalent to para-substituted groups such as 1,4-dihydroxybenzene, in that (i) there is an even number of C atoms separating the two oxygen atoms, whichever pathway is taken through the ligand skeleton, and (ii) the doubly oxidized form of the bridging ligand is a diamagnetic quinone. These complexes are classified as "T-para". Electrochemical studies show that the comproportionation constants for the Mo(V)/Mo(IV) mixed-valence states of the T-meta complexes are smaller than those for the T-para complexes. Spectroelectrochemical studies show that the Mo(V)/Mo(IV) mixed-valence states of the T-para complexes show pronounced Mo(IV)-->Mo(V) IVCT transitions, whereas those of the T-meta complexes do not show these transitions. Magnetic susceptibility studies show that the T-meta complexes all display ferromagnetic exchange between the metal centers, whereas the T-para complexes all display antiferromagnetic exchange. Thus, both the electronic and the magnetic properties of these complexes show a clear demarcation into two sets according to the bridging-ligand topology.
RESUMO
2-5A was conjugated to N-(2-aminoethyl)-glycyl PNA by periodate oxidization, followed by coupling with amino-derivatized PNA and final cyanoborohydride reduction. An adduct of 2-5A pentamer with tetrameric thymine PNA activated RNase L with the same potency as earlier versions of 2-5A-PNA or 2-5A-DNA.
Assuntos
Nucleotídeos de Adenina/química , Endorribonucleases/metabolismo , Oligorribonucleotídeos/química , Ácidos Nucleicos Peptídicos/síntese química , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/metabolismoRESUMO
RNase L is a latent endonuclease found in reptiles, birds, and mammals. It is activated by the 2',5'-phosphodiester-linked oligoadenylates called 2-5A and has been implicated in the mechanism of action of interferon, as well as in a variety of other biological phenomena such as apoptosis. Covalent linkage of 2-5A to antisense oligonucleotides permits recruitment of RNase L for enhancement of antisense action. The purification of RNase L described herein and the assays for its detection and activation will help to provide further mechanistic details on how this unique nuclease functions and what its biochemical roles may be. In addition, such assays will facilitate the screening of 2-5A-antisense congeners for exploration of the potential therapeutic applications of RNase L.
Assuntos
Endorribonucleases/isolamento & purificação , Animais , Western Blotting , Catálise , Cromatografia Líquida/métodos , Endorribonucleases/análise , Endorribonucleases/metabolismo , Ativação Enzimática , Camundongos , Marcadores de Fotoafinidade , Poli U/metabolismo , Testes de Precipitina , Ligação Proteica , RNA Ribossômico/metabolismo , Especificidade por SubstratoRESUMO
2-Methyladenosine-substituted analogues of 2-5A, p5'A2'p5'A2'p5'(me2A), p5'(me2A)2'p5'A2'p5'A, and p5'(me2A) 2'p5'(me2A)2'pS'(me2A), were prepared via a modification of a lead ion-catalyzed ligation reaction. These 5'-monophosphates were subsequently converted into the corresponding 5'-triphosphates. Both binding and activation of human recombinant RNase L by various 2-methyladenosine-substituted 2-5A analogues were examined. Among the 2-5A analogues, p5'A2'p5'A2'p5'(me2A) showed the strongest binding affinity and was as effective as 2-5A itself as an activator of RNase L. The CD spectra of both p5'(me2A)2'p5'A2'p5'A and p5'A2'p5'A2'p5'(me2A) were superimposable on that of p5'A2'p5'A2'p5'A, indicative of an anti orientation about the base-glycoside bonds as in naturally occurring 2-5A.
Assuntos
Monofosfato de Adenosina/química , Monofosfato de Adenosina/farmacologia , Adenosina/análogos & derivados , Endorribonucleases/efeitos dos fármacos , Oligorribonucleotídeos/química , Oligorribonucleotídeos/farmacologia , Adenosina/química , Adenosina/farmacologia , Monofosfato de Adenosina/análogos & derivados , Sítios de Ligação , Catálise/efeitos dos fármacos , Dicroísmo Circular , Endorribonucleases/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , Ligação Proteica , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
To potentiate the 2-5A (2',5'-oligoadenylate)-antisense and peptide nucleic acid (PNA) approaches to regulation of gene expression, composite molecules were generated containing both 2-5A and PNA moieties. 2-5A-PNA adducts were synthesized using solid-phase techniques. Highly cross-linked polystyrene beads were functionalized with glycine tethered through a p-hydroxymethylbenzoic acid linker and the PNA domain of the chimeric oligonucleotide analogue was added by sequential elongation of the amino terminus with the monomethoxytrityl protected N-(2-aminoethyl)-N-(adenin-1-ylacetyl)glycinate. Transition to the 2-5A domain was accomplished by coupling of the PNA chain to dimethoxytrityl protected N-(2-hydroxyethyl)-N-(adenin-1-ylacetyl)glycinate. Finally, (2-cyanoethyl)-N,N-diisopropyl-4-O-(4,4-dimethoxytrityl)butylphosphor amidite and the corresponding (2-cyanoethyl)-N,N-diisopropylphosphoramidite of 5-O-(4,4'-dimethoxytrityl)-3-O-(tert-butyldimethylsilyl)-N6-benzoyladeno sine were the synthons employed to add the 2 butanediol phosphate linkers and the four 2',5'-linked riboadenylates. The 5'-phosphate moiety was introduced with 2-[[2-(4,4'-dimethoxytrityloxy)ethyl]sulfonyl]ethyl-(2-cyanoethyl) -N,N-diisopropylphosphoramidite. Deprotection with methanolic NH3 and tetraethylammonium fluoride afforded the desired products, 2-SA-pnaA4, 2-5A-pnaA8 and 2-5A-pnaA12. When evaluated for their ability to cause the degradation of two different RNA substrates by the 2-5A-dependent RNase L, these new 2-5A-PNA conjugates were found to be potent RNase L activators. The union of 2-5A and PNA presents fresh opportunities to explore the biological and therapeutic implications of these unique approaches to antisense.
Assuntos
Nucleotídeos de Adenina/química , Endorribonucleases/metabolismo , Oligorribonucleotídeos/química , Ácidos Nucleicos Peptídicos/farmacologia , Linhagem Celular , Ativação Enzimática , Humanos , Hidrólise , Ácidos Nucleicos Peptídicos/química , Ensaio Radioligante , Proteínas Recombinantes/metabolismoRESUMO
This paper presents the fully automated solid phase synthesis of 2-5A-PNA hybrids. These stable antisense probes cause RNase L mediated hydrolysis of target RNA sequences.