RESUMO
Genomic SELEX is a method for studying the network of nucleic acid-protein interactions within any organism. Here we report the discovery of several interesting and potentially biologically important interactions using genomic SELEX. We have found that bacteriophage MS2 coat protein binds several Escherichia coli mRNA fragments more tightly than it binds the natural, well-studied, phage mRNA site. MS2 coat protein binds mRNA fragments from rffG (involved in formation of lipopolysaccharide in the bacterial outer membrane), ebgR (lactose utilization repressor), as well as from several other genes. Genomic SELEX may yield experimentally induced artifacts, such as molecules in which the fixed sequences participate in binding. We describe several methods (annealing of oligonucleotides complementary to fixed sequences or switching fixed sequences) to eliminate some, or almost all, of these artifacts. Such methods may be useful tools for both randomized sequence SELEX and genomic SELEX.
Assuntos
Bacteriófagos , Proteínas do Capsídeo , Capsídeo/metabolismo , Genoma Bacteriano , RNA Bacteriano/metabolismo , Proteínas de Ligação a RNA/metabolismo , Artefatos , Sequência de Bases , Sítios de Ligação , Biologia Computacional , Sequência Consenso , Genes Bacterianos/genética , Biblioteca Genômica , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Reação em Cadeia da Polimerase , Ligação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Sensibilidade e Especificidade , Especificidade por Substrato , Transcrição GênicaRESUMO
The factors that lead to high-affinity binding are a good fit between the surfaces of the two molecules in their ground state and charge complementarity. Exactly the same factors give high specificity for a target. We argue that selection for high-affinity binding automatically leads to highly specific binding. This principle can be used to simplify screening approaches aimed at generating useful drugs.
Assuntos
Receptores de Droga/química , Animais , Desenho de Fármacos , Humanos , Ligação Proteica , Propriedades de SuperfícieRESUMO
In this paper, we describe the synthesis of a series of trans-N-[2-(methylamino)cyclohexyl]benzamides possessing morphine-like pharmacological properties. The affinity of the compounds for the agonist and antagonist states of the mu opioid receptor has been established by means of an in vitro binding assay. We have investigated the geometry and electronic structure of the molecules using molecular mechanics and an ab initio SCF-MO procedure with FSGO basis sets. Comparison to naloxone reveals properties of possible importance in receptor association. We have considered both the S,S and R,R isomers in the binding model. Statistical analyses imply that three factors play a significant role in binding: (1) membrane-water partitioning, (2) the capacity of the aromatic ring and amine N-substituent to act as electron acceptors, (3) the conformational energy required to attain the binding configuration.
Assuntos
Benzamidas/metabolismo , Cicloexilaminas/metabolismo , Receptores Opioides/metabolismo , Animais , Benzamidas/síntese química , Benzamidas/farmacologia , Encéfalo/metabolismo , Fenômenos Químicos , Físico-Química , Cicloexilaminas/síntese química , Cicloexilaminas/farmacologia , Eletroquímica , Isomerismo , Masculino , Camundongos , Conformação Molecular , Morfina/metabolismo , Naloxona/metabolismo , Ratos , Relação Estrutura-Atividade , TermodinâmicaAssuntos
Simulação por Computador , Modelos Moleculares , Conformação de Ácido Nucleico , RNA/química , RNA/metabolismo , Sequência de Bases , Sequência Consenso , Fator 2 de Crescimento de Fibroblastos/metabolismo , Transcriptase Reversa do HIV/metabolismo , HIV-1/enzimologia , Ligantes , Dados de Sequência Molecular , Ligação Proteica , RNA/classificação , Proteínas de Ligação a RNA/metabolismo , Alinhamento de Sequência/métodos , Análise de Sequência , Software , Relação Estrutura-AtividadeRESUMO
A new paradigm for drug discovery and biological research has developed from technologies that integrate combinatorial chemistry with rounds of selection and amplification, a technique called in vitro selection or systematic evolution of ligands by exponential enrichment (SELEX). This overview unit discusses nucleic acid libraries that can be used, affinity probability distributions, an equilibrium model for SELEX, and optimal conditions including concentrations and signal-to-noise ratios.
Assuntos
Aptâmeros de Nucleotídeos/isolamento & purificação , Técnicas de Química Combinatória/métodos , Técnica de Seleção de Aptâmeros/métodos , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Escherichia coli/genética , Modelos Biológicos , Dados de Sequência MolecularRESUMO
We present a computer-aided approach for identifying and aligning consensus secondary structure within a set of functionally related oligonucleotide sequences aligned by sequence. The method relies on visualization of secondary structure using a generalization of the dot matrix representation appropriate for consensus sequence data sets. An interactive computer program implementing such a visualization of consensus structure has been developed. The program allows for alignment editing, data and display filtering and various modes of base pair representation, including co-variation. The utility of this approach is demonstrated with four sample data sets derived from in vitro selection experiments and one data set comprising tRNA sequences.
Assuntos
Sequência Consenso , Apresentação de Dados , Conformação de Ácido Nucleico , RNA/química , Alinhamento de Sequência/métodos , Algoritmos , Sequência de Bases , Simulação por Computador , Modelos Moleculares , Dados de Sequência Molecular , Oligorribonucleotídeos/químicaRESUMO
In vitro selection techniques provide a means of isolating nucleic acid ligands for binding to particular protein targets. Although most aptamers have quite high affinities for their target proteins, it has been shown that post-SELEX modification can result in further enhancement of binding affinity, as well as other desired properties. This has led to the current development of a more systematic approach to aptamer optimization using a combinatorial screening methodology.