RESUMO
AIM: Physical exercise prevents cardiovascular risk and atherosclerosis lesions. However, the molecular aspects are still unknown. Vascular peroxisome proliferator-activated receptors (PPARs) exert anti-atherogenic effects. The aim of this study was to determine whether exercise-induced anti-atherosclerotic effect is associated with change in PPARs vascular expression in apolipoprotein E-deficient (ApoE(-/-) ) mice. METHODS: Male ApoE(-/-) mice were fed with a high-fat diet and randomized into two groups: one trained group undergoing swimming training for 3 months and one sedentary group. Sedentary and trained C57BL/6J mice were used as control. mRNA of PPAR-α, PPAR-ß/δ and PPAR-γ was measured in aorta by quantitative PCR. mRNA of pro- (TNF-α, IL-1ß) and anti-inflammatory (IL-10, IL-1Ra) cytokines was also measured. RESULTS: Atherosclerotic lesion size was significantly reduced in trained ApoE(-/-) mice compared to sedentary ones. In contrast, reduction of atherosclerotic lesion size was not observed in trained ApoE(-/-) mice supplied with BADGE, an antagonist of PPAR-γ. Exercise training significantly increased PPAR-γ expression in aorta. PPAR-γ expression was inversely correlated with the atherosclerotic plaque area. Aortic PPAR-α and PPAR-ß/δ mRNA expressions were not changed in response to exercise training. Atherosclerosis increased the aortic mRNA expression of TNF-α, IL-1ß, IL-10 and IL-1Ra. Exercise training decreased aortic IL-1ß mRNA expression in ApoE(-/-) mice, but did not change expression of TNF-α, IL-10 and IL-1Ra. IL-1ß mRNA expression was also significantly lower in atherosclerosis lesions from trained ApoE(-/-) compared with those from sedentary ones. CONCLUSIONS: Exercise training increases vascular PPAR-γ expression in ApoE(-/-) mice that could potentially underlie training-related beneficial effects on atherosclerosis.
Assuntos
Aterosclerose/patologia , PPAR gama/biossíntese , Condicionamento Físico Animal , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Dieta Hiperlipídica , Modelos Animais de Doenças , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Cellular and organellar membranes are dynamic materials that underlie many aspects of cell biology. Biological membranes have long been thought of as elastic materials with respect to bending deformations. A wealth of theory and experimentation on pure phospholipid membranes provides abundant support for this idea. However, biological membranes are not composed solely of phospholipids--they also incorporate a variety of amphiphilic molecules that undergo rapid transbilayer flip-flop. Here we describe several experimental systems that demonstrate deformation-induced molecular flip-flop. First we use a fluorescence assay to track osmotically controlled membrane deformation in single component fatty acid vesicles, and show that the relaxation of the induced bending stress is mediated by fatty acid flip-flop. We then look at two-component phospholipid/cholesterol composite vesicles. We use NMR to show that the steady-state rate of interleaflet diffusion of cholesterol is fast relative to biological membrane remodeling. We then use a Förster resonance energy transfer assay to detect the transbilayer movement of cholesterol upon deformation. We suggest that our results can be interpreted by modifying the area difference elasticity model to account for the time-dependent relaxation of bending energy. Our findings suggest that rapid interleaflet diffusion of cholesterol may play a role in membrane remodeling in vivo. We suggest that the molecular characteristics of sterols make them evolutionarily preferred mediators of stress relaxation, and that the universal presence of sterols in the membranes of eukaryotes, even at low concentrations, reflects the importance of membrane remodeling in eukaryotic cells.
Assuntos
Membrana Celular/metabolismo , Modelos Biológicos , Estresse Mecânico , 2-Naftilamina/análogos & derivados , 2-Naftilamina/metabolismo , Fenômenos Biomecânicos , Colesterol/metabolismo , Difusão , Elasticidade , Endocitose , Eucariotos/citologia , Eucariotos/genética , Evolução Molecular , Ácidos Graxos/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/metabolismo , Cinética , Lauratos/metabolismo , Movimento , Pressão Osmótica , TermodinâmicaRESUMO
The complexity of modern biological life has long made it difficult to understand how life could emerge spontaneously from the chemistry of the early earth. The key to resolving this mystery lies in the simplicity of the earliest living cells, together with the ability of the appropriate molecular building blocks to spontaneously self-assemble into larger structures. In our view, the two key components of a primitive cell are not only self-assembling, but also self-replicating, structures: the nucleic acid genome and the cell membrane. Here, we summarize recent experimental progress toward the synthesis of efficient self-replicating nucleic acid and membrane vesicle systems and discuss some of the issues that arise during efforts to integrate these two subsystems into a coherent whole. We have shown that spontaneous nucleic-acid-copying chemistry can take place within membrane vesicles, using externally supplied activated nucleotides as substrates. Thus, membranes need not be a barrier to the uptake of environmentally supplied nutrients. We examine some of the remaining obstacles that must be overcome to enable the synthesis of a complete self-replicating protocell, and we discuss the implications of these experiments for our understanding of the emergence of Darwinian evolution and the origin and early evolution of cellular life.
Assuntos
Células/metabolismo , Modelos Biológicos , Origem da Vida , Evolução Biológica , Metabolismo dos Carboidratos , Compartimento Celular , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Ácidos Graxos/metabolismo , Ácidos Nucleicos/metabolismoRESUMO
Nutrient uptake by a primitive cell would have been limited by the permeability characteristics of its membrane. We measured the permeabilities of model protocellular membranes to water, five of the six pentoses, and selected aldohexoses, ketohexoses, and three to six carbon alditols by following volume changes of vesicles after the addition of solute to the external medium. Solute hydrophobicities correlated poorly with permeability coefficients within one structural class of compounds. The permeability coefficients of diastereomeric sugars differed by as much as a factor of 10, with ribose being the most permeable aldopentose. Flexible alditols and sugars, sugars biased toward or restricted to furanose forms, and sugars having anomers with hydrophobic faces permeated more quickly than compounds lacking these features. Among the aldopentoses, only ribose possesses all of these properties. Ribose permeated both fatty acid and phospholipid membranes more rapidly than the other aldopentoses or hexoses. The enhanced permeability conferred by the unique conformational preferences of ribose would have allowed faster assimilation of ribose by primitive cells as they passively absorbed materials from the environment. The kinetic advantage of ribose over the other aldopentoses in crossing membranes may therefore have been one factor that facilitated the emergence of the RNA world.
Assuntos
Bicamadas Lipídicas , Monossacarídeos/química , Ribose , Cinética , Conformação Molecular , PermeabilidadeRESUMO
We describe the use of the SBP-tag, a new streptavidin-binding peptide, for both the one-step purification and the detection of recombinant proteins. The SBP-tag sequence is 38 amino acids long and binds to streptavidin with an equilibrium dissociation constant of 2.5 nM. We demonstrate that a single-step purification of SBP-tagged proteins from bacterial extract yields samples that are more pure than those purified using maltose-binding protein or the His-tag. The capacity of the immobilized streptavidin used to purify SBP-tagged proteins is about 0.5 mg per milliliter of matrix, which is high enough to isolate large quantities of proteins for further study. Also, the elution conditions from the streptavidin column are very mild and specific, consisting of the wash buffer plus biotin. This combination of high-affinity, high-yield, mild elution conditions, and simplicity of use makes the SBP-tag suitable for high-throughput protein expression/purification procedures, including robotically manipulated protocols with microtiter plates. Additionally, the SBP-tag can be used for detection since a wide variety of streptavidin-conjugated fluorescent and enzymatic systems are commercially available. We also present a new, rapid, method for the measurement of protein-protein, protein-peptide, or protein-small molecule equilibrium dissociation constants that require as little as 1 fmol of labeled protein. We call this method the spin-filter binding inhibition assay.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Estreptavidina/metabolismo , Marcadores de Afinidade/química , Marcadores de Afinidade/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/química , Proteínas de Transporte/genética , Quelantes/química , Escherichia coli/genética , Vetores Genéticos , Maltose/metabolismo , Dados de Sequência Molecular , Nanotecnologia , Ligação Proteica , Biossíntese de Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Sensibilidade e Especificidade , Transformação GenéticaRESUMO
The hammerhead ribozyme was originally discovered in a group of RNAs associated with plant viruses, and has subsequently been identified in the genome of the newt (Notophthalamus viridescens), in schistosomes and in cave crickets (Dolichopoda species). The sporadic occurrence of this self-cleaving RNA motif in highly divergent organisms could be a consequence of the very early evolution of the hammerhead ribozyme, with all extant examples being descended from a single ancestral progenitor. Alternatively, the hammerhead ribozyme may have evolved independently many times. To better understand the observed distribution of hammerhead ribozymes, we used in vitro selection to search an unbiased sample of random sequences for comparably active self-cleaving motifs. Here we show that, under near-physiological conditions, the hammerhead ribozyme motif is the most common (and thus the simplest) RNA structure capable of self-cleavage at biologically observed rates. Our results suggest that the evolutionary process may have been channelled, in nature as in the laboratory, towards repeated selection of the simplest solution to a biochemical problem.
Assuntos
Evolução Biológica , RNA Catalítico/metabolismo , Sequência de Bases , Catálise , DNA , Evolução Molecular Direcionada , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Catalítico/química , RNA Catalítico/genética , Alinhamento de SequênciaRESUMO
Functional primordial proteins presumably originated from random sequences, but it is not known how frequently functional, or even folded, proteins occur in collections of random sequences. Here we have used in vitro selection of messenger RNA displayed proteins, in which each protein is covalently linked through its carboxy terminus to the 3' end of its encoding mRNA, to sample a large number of distinct random sequences. Starting from a library of 6 x 1012 proteins each containing 80 contiguous random amino acids, we selected functional proteins by enriching for those that bind to ATP. This selection yielded four new ATP-binding proteins that appear to be unrelated to each other or to anything found in the current databases of biological proteins. The frequency of occurrence of functional proteins in random-sequence libraries appears to be similar to that observed for equivalent RNA libraries.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Trifosfato de Adenosina/metabolismo , Proteínas de Escherichia coli , Proteínas de Transporte de Monossacarídeos , Biblioteca de Peptídeos , Proteínas/isolamento & purificação , RNA , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Escherichia coli , Biblioteca Gênica , Proteínas Ligantes de Maltose , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Proteínas/genética , Proteínas/metabolismo , RNA/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
We report the use of "mRNA display," an in vitro selection technique, to identify peptide aptamers to a protein target. mRNA display allows for the preparation of polypeptide libraries with far greater complexity than is possible with phage display. Starting with a library of approximately 10(13) random peptides, 20 different aptamers to streptavidin were obtained, with dissociation constants as low as 5 nM. These aptamers function without the aid of disulfide bridges or engineered scaffolds, yet possess affinities comparable to those for monoclonal antibody-antigen complexes. The aptamers bind streptavidin with three to four orders of magnitude higher affinity than those isolated previously by phage display from lower complexity libraries of shorter random peptides. Like previously isolated peptides, they contain an HPQ consensus motif. This study shows that, given sufficient length and diversity, high-affinity aptamers can be obtained even from random nonconstrained peptide libraries. By engineering structural constraints into these ultrahigh complexity peptide libraries, it may be possible to produce binding agents with subnanomolar binding constants.
Assuntos
Peptídeos/metabolismo , Estreptavidina/metabolismo , Sequência de Aminoácidos , Ligação Competitiva , Biblioteca de Peptídeos , Peptídeos/síntese química , Peptídeos/química , RNA Mensageiro/genética , Análise de Sequência de Proteína , Homologia de Sequência de AminoácidosRESUMO
Two analog uridine triphosphates tethering additional functionality, one a primary amino group and the second a mercapto group, were prepared and tested for their compatibility with in vitro RNA selection procedures. 5-(3-Aminopropyl)uridine triphosphate (UNH(2)) as a uridine substitute was a more effective substrate for T7 RNA polymerase than 5-(2-mercaptoethyl)uridine triphosphate (USH). However, both functioned in transcription assays of 100 nt templates to generate RNA transcripts in quantities sufficient to initiate RNA selection procedures. Transcription of RNA pools with T7 RNA polymerase and UNH(2) or USH occurred with efficiencies of 43 and 29%, respectively, of the values obtained for native UTP transcription. In addition, the transcribed RNA containing roughly 25% UNH(2) residues exhibited better substrate properties for SuperScript(TM) II RNase H reverse transcriptase than did RNA transcripts containing approximately 25% of the USH analog. With either analog, both transcription and reverse transcription proceeded with high fidelity for insertion of the analog residue.
Assuntos
Transcrição Gênica , Uridina/análogos & derivados , Uridina/metabolismo , Sequência de Bases , Cromatografia Líquida de Alta Pressão , RNA Polimerases Dirigidas por DNA/metabolismo , Cinética , Dados de Sequência Molecular , RNA/biossíntese , RNA/genética , RNA/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Ribonuclease H/metabolismo , Especificidade por Substrato , Moldes Genéticos , Transcrição Gênica/genética , Uridina/química , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/síntese química , Uridina Trifosfato/química , Uridina Trifosfato/metabolismo , Proteínas ViraisAssuntos
Proteínas/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/química , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Códon , DNA/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Biblioteca Gênica , Genes myc/genética , Técnicas Genéticas , Magnésio/química , Modelos Genéticos , Oligonucleotídeos/química , Peptídeos/química , Fosforilação , Reação em Cadeia da Polimerase , Potássio/química , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas/química , Puromicina/química , RNA Mensageiro/química , Proteínas Recombinantes de Fusão/genética , Reticulócitos/metabolismo , Ribossomos/químicaRESUMO
We present a method that can significantly increase the complexity of protein libraries used for in vitro or in vivo protein selection experiments. Protein libraries are often encoded by chemically synthesized DNA, in which part of the open reading frame is randomized. There are, however, major obstacles associated with the chemical synthesis of long open reading frames, especially those containing random segments. Insertions and deletions that occur during chemical synthesis cause frameshifts, and stop codons in the random region will cause premature termination. These problems can together greatly reduce the number of full-length synthetic genes in the library. We describe a strategy in which smaller segments of the synthetic open reading frame are selected in vitro using mRNA display for the absence of frameshifts and stop codons. These smaller segments are then ligated together to form combinatorial libraries of long uninterrupted open reading frames. This process can increase the number of full-length open reading frames in libraries by up to two orders of magnitude, resulting in protein libraries with complexities of greater than 10(13). We have used this methodology to generate three types of displayed protein library: a completely random sequence library, a library of concatemerized oligopeptide cassettes with a propensity for forming amphipathic alpha-helical or beta-strand structures, and a library based on one of the most common enzymatic scaffolds, the alpha/beta (TIM) barrel.
Assuntos
Clonagem Molecular/métodos , Oligopeptídeos/química , Oligopeptídeos/genética , Fases de Leitura Aberta/genética , Biblioteca de Peptídeos , Sequência de Aminoácidos , Códon/genética , DNA Ligases/metabolismo , Mutação da Fase de Leitura/genética , Indol-3-Glicerolfosfato Sintase/química , Indol-3-Glicerolfosfato Sintase/genética , Dados de Sequência Molecular , Mutagênese Insercional/genética , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/genética , Oligopeptídeos/isolamento & purificação , Oligopeptídeos/metabolismo , Biossíntese de Proteínas/genética , Dobramento de Proteína , Estrutura Secundária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Distribuição Aleatória , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Seleção Genética , Sulfolobus , Moldes GenéticosRESUMO
The RNA world hypothesis implies that coded protein synthesis evolved from a set of ribozyme catalyzed acyl-transfer reactions, including those of aminoacyl-tRNA synthetase ribozymes. We report here that a bifunctional ribozyme generated by directed in vitro evolution can specifically recognize an activated glutaminyl ester and aminoacylate a targeted tRNA, via a covalent aminoacyl-ribozyme intermediate. The ribozyme consists of two distinct catalytic domains; one domain recognizes the glutamine substrate and self-aminoacylates its own 5'-hydroxyl group, and the other recognizes the tRNA and transfers the aminoacyl group to the 3'-end. The interaction of these domains results in a unique pseudoknotted structure, and the ribozyme requires a change in conformation to perform the sequential aminoacylation reactions. Our result supports the idea that aminoacyl-tRNA synthetase ribozymes could have played a key role in the evolution of the genetic code and RNA-directed translation.
Assuntos
RNA Catalítico/metabolismo , Aminoacil-RNA de Transferência/metabolismo , RNA de Transferência/metabolismo , Acilação , Sequência de Bases , Sítios de Ligação , Domínio Catalítico , Clonagem Molecular , Evolução Molecular , Código Genético/genética , Glutamina/metabolismo , Cinética , Dados de Sequência Molecular , Mutação/genética , Conformação de Ácido Nucleico , Biossíntese de Proteínas , RNA Catalítico/química , RNA Catalítico/genética , RNA de Transferência de Glutamina/metabolismo , Alinhamento de Sequência , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
In vitro selection allows rare functional RNA or DNA molecules to be isolated from pools of over 10(15) different sequences. This approach has been used to identify RNA and DNA ligands for numerous small molecules, and recent three-dimensional structure solutions have revealed the basis for ligand recognition in several cases. By selecting high-affinity and -specificity nucleic acid ligands for proteins, promising new therapeutic and diagnostic reagents have been identified. Selection experiments have also been carried out to identify ribozymes that catalyze a variety of chemical transformations, including RNA cleavage, ligation, and synthesis, as well as alkylation and acyl-transfer reactions and N-glycosidic and peptide bond formation. The existence of such RNA enzymes supports the notion that ribozymes could have directed a primitive metabolism before the evolution of protein synthesis. New in vitro protein selection techniques should allow for a direct comparison of the frequency of ligand binding and catalytic structures in pools of random sequence polynucleotides versus polypeptides.
Assuntos
Ácidos Nucleicos/química , RNA Catalítico/químicaRESUMO
BACKGROUND: In vitro selection experiments with pools of random-sequence nucleic acids have been used extensively to isolate molecules capable of binding specific ligands and catalyzing self-modification reactions. RESULTS: In vitro selection from a random pool of single-stranded DNAs has been used to isolate molecules capable of recognizing the fluorophore sulforhodamine B with high affinity. When assayed for the ability to promote an oxidation reaction using the reduced form of a related fluorophore, dihydrotetramethylrosamine, a number of selected clones show low levels of catalytic activity. Chemical modification and site-directed mutagenesis experiments have been used to probe the structural requirements for fluorophore binding. The aptamer recognizes its ligand with relatively high affinity and is also capable of binding related molecules that share extended aromatic rings and negatively charged functional groups. CONCLUSIONS: A guanosine-rich single-stranded DNA is capable of binding fluorophores with relatively high affinity and of weakly promoting a multiple-turnover reaction. A simple motif consisting of a three-tiered G-quartet stacked upon a standard Watson-Crick duplex appears to be responsible for this activity. The corresponding sequence might provide a useful starting point for the evolution of novel, improved deoxyribozymes that generate fluorescent signals by promoting multiple-turnover reactions.
Assuntos
DNA/química , Corantes Fluorescentes , Rodaminas/química , Sequência de Bases , Catálise , Sequência Conservada , DNA de Cadeia Simples/química , Guanosina/química , Ligantes , Dados de Sequência Molecular , Oxirredução , Espectrofotometria UltravioletaRESUMO
Ligand-binding RNAs and DNAs (aptamers) isolated by in vitro selection from random sequence pools provide convenient model systems for understanding the basic relationships between RNA structure and function. We describe a series of experiments that define the functional requirements for an RNA motif that specifies high-affinity binding to the carboxylation cofactor biotin. A simple pseudoknot containing an adenosine-rich loop accounts for binding in all independently derived aptamers selected to bind biotin, suggesting that it alone represents a global optimum for recognition of this particular nonaromatic, electrostatically neutral ligand. In contrast to virtually all previously identified aptamers, unpaired nucleotides make up a small fraction of the binding motif. Instead, the identity of 14 nucleotides involved in base pairing is highly conserved among functional clones and their substitution by nonidentical base pairs significantly reduces or eliminates binding. Chemical probing is consistent with the predicted pseudoknot motif and indicates that relatively little change in structure accompanies ligand binding, a strong contrast with results for other aptamers. Competition experiments suggest that the aptamer recognizes all parts of the biotin ligand, including its thiophane ring and fatty acid tail. Two alternative modes of binding are suggested by a three-dimensional model of the pseudoknot, both of which entail significant interactions with base-paired nucleotides.
Assuntos
Biotina/metabolismo , Conformação de Ácido Nucleico , RNA/química , RNA/metabolismo , Pareamento de Bases , Sequência de Bases , Ligação Competitiva/genética , Biotina/genética , Cinética , Ligantes , Dados de Sequência Molecular , Ligação Proteica/genéticaRESUMO
Most studies of the roles of catalytic metal ions in ribozymes have focused on inner-sphere coordination of the divalent metal ions to the substrate or ribozyme. However, divalent metal ions are strongly hydrated in water, and some proteinenzymes, such as Escherichia coli RNase H and exonuclease III, are known to use metal cofactors in their fully hydrated form [Duffy, T. H., and Nowak, T. (1985) Biochemistry 24, 1152-1160; Jou, R., and Cowan, J. A. (1991) J. Am. Chem. Soc. 113, 6685-6686]. It is therefore important to consider the possibility of outer-sphere coordination of catalytic metal ions in ribozymes. We have used an exchange-inert metal complex, cobalt hexaammine, to show that the catalytic metal ion in an acyl-transferase ribozyme acts through outer-sphere coordination. Our studies provide an example of a fully hydrated Mg2+ ion that plays an essential role in ribozyme catalysis. Kinetic studies of wild-type and mutant ribozymes suggest that a pair of tandem G:U wobble base pairs adjacent to the reactive center constitute the metal-binding site. This result is consistent with recent crystallographic studies [Cate, J. H., and Doudna, J. A. (1996) Structure 4, 1221-1229; Cate, J. H., Gooding, A. R., Podell, E., Zhou, K., Golden, B. L., Kundrot, C. E., Cech, T. R., and Doudna, J. A. (1996) Science 273, 1678-1685; Cate, J. H., Hanna, R. L., and Doudna, J. A. (1997) Nat. Struct. Biol. 4, 553-558] showing that tandem wobble base pairs are good binding sites for metal hexaammines. We propose a model in which the catalytic metal ion is bound in the major groove of the tandem wobble base pairs, is precisely positioned by the ribozyme within the active site, and stabilizes the developing oxyanion in the transition state. Our results may have significant implications for understanding the mechanism of protein synthesis [Noller, H. F., Hoffarth, V., and Zimniak, L. (1992) Science 256, 1416-1419].
Assuntos
Aciltransferases/metabolismo , Cobalto/metabolismo , RNA Catalítico/metabolismo , Sítios de Ligação , Catálise , Cátions Bivalentes/metabolismo , Magnésio/metabolismo , Modelos MolecularesRESUMO
BACKGROUND: In vitro selection has been shown previously to be a powerful method for isolating nucleic acids with specific ligand-binding functions ('aptamers'). Given this capacity, we have sought to isolate RNA motifs that can confer fluorescent labeling to tagged RNA transcripts, potentially allowing in vivo detection and in vitro spectroscopic analysis of RNAs. RESULTS: Two aptamers that recognize the fluorophore sulforhodamine B were isolated by the in vitro selection process. An unusually large motif of approximately 60 nucleotides is responsible for binding in one RNA (SRB-2). This motif consists of a three-way helical junction with two large, highly conserved unpaired regions. Phosphorothioate mapping with an iodoacetamide-tagged form of the ligand shows that these two regions make close contacts with the fluorophore, suggesting that the two loops combine to form separate halves of a binding pocket. The aptamer binds the fluorophore with high affinity, recognizing both the planar aromatic ring system and a negatively charged sulfonate, a rare example of anion recognition by RNA. An aptamer (FB-1) that specifically binds fluorescein has also been isolated by mutagenesis of a sulforhodamine aptamer followed by re-selection. In a simple in vitro test, SRB-2 and FB-1 have been shown to discriminate between sulforhodamine and fluorescein, specifically localizing each fluorophore to beads tagged with the corresponding aptamer. CONCLUSIONS: In addition to serving as a model system for understanding the basis of RNA folding and function, these experiments demonstrate potential applications for the aptamers in transcript double labeling or fluorescence resonance energy transfer studies.
Assuntos
Conformação de Ácido Nucleico , RNA/química , Rodaminas , Animais , Sequência de Bases , Sítios de Ligação , Fluorescência , Humanos , Dados de Sequência MolecularRESUMO
We have previously isolated, by in vitro selection, an acyl-transferase ribozyme that is capable of transferring a biotinylated methionyl group from the 3' end of a hexanucleotide substrate to its own 5'-hydroxyl. Comparison of the sequences of a family of evolved derivatives of this ribozyme allowed us to generate a model of the secondary structure of the ribozyme. The predicted secondary structure was extensively tested and confirmed by single-mutant and compensatory double-mutant analyses. The role of the template domain in aligning the acyl-donor oligonucleotide and acyl-acceptor region of the ribozyme was confirmed in a similar manner. The significance of different domains of the ribozyme structure and the importance of two tandem G:U wobble base pairs in the template domain were studied by kinetic characterization of mutant ribozymes. The wobble base pairs contribute to the catalytic rate enhancement, but only in the context of the complete ribozyme; the ribozyme in turn alters the metal binding properties of this site. Competitive inhibition experiments with unacylated substrate oligonucleotide are consistent with the ribozyme acting to stabilize substrate binding to the template, while negative interactions with the aminoacyl portion of the substrate destabilize binding.
Assuntos
Aciltransferases/química , Evolução Molecular Direcionada , Modelos Moleculares , RNA Catalítico/química , Composição de Bases , Sequência de Bases , Biotinilação , Estrutura Molecular , Mutação , Oligonucleotídeos/química , Reação em Cadeia da Polimerase , RNA Catalítico/genética , Análise de Sequência , Moldes GenéticosRESUMO
The solution structure of the ATP-binding RNA aptamer has recently been determined by NMR spectroscopy. The three-dimensional fold of the molecule is determined to a large extent by stacking and hydrogen bond interactions. In the course of the structure determination it was discovered that several highly conserved nucleotides in the binding pocket can be substituted while retaining binding under NMR conditions. These surprising findings allow a closer look at the interactions that determine stability and specificity of the aptamer as well as local structural features of the molecule. The binding properties of ATP binder mutants and modified ligand molecules are explored using NMR spectroscopy, column binding studies and molecular modeling. We present additional evidence and new insights regarding the network of hydrogen bonds that defines the structure and determines stability and specificity of the aptamer.