RESUMO
Today, many nucleic acid enzymes are used in gene therapy and gene regulations. However, no simple assay methods to evaluate enzymatic activities, with which we judge the enzyme design, have been reported. Here, we propose a new simple competition assay for nucleic acid enzymes of different types to evaluate the cleaving efficiency of a target RNA molecule, of which the recognition sites are different but overlapped. Two nucleic acid enzymes were added to one tube to make a competition of these two enzymes for one substrate. The assay was used on two ribozymes, hammerhead ribozyme and hairpin ribozyme, and a DNA-enzyme. We found that this assay method is capable of application to those enzymes, as a powerful tool for the selection and designing of RNA-cleaving enzymes.
Assuntos
RNA Catalítico/química , RNA Catalítico/metabolismo , Animais , Sequência de Bases , Ligação Competitiva , Desenho de Fármacos , Técnicas In Vitro , Cinética , Mutação , Conformação de Ácido Nucleico , RNA Catalítico/antagonistas & inibidores , RNA Catalítico/genética , Ratos , Especificidade por SubstratoRESUMO
Two ribozymes, hammerhead ribozyme and hairpin ribozyme, and a DNA-enzyme were designed to cleave a same RNA target, the same site of the rat complement regulatory factor 512 antigen mRNA. The kinetic properties of these RNA-cleaving enzymes were measured and compared under the same conditions, using multiple turnover kinetics and competition kinetics. The catalytic efficiencies of these enzymes, and also the order of these enzymes will be discussed.
Assuntos
RNA Catalítico/metabolismo , RNA Mensageiro/metabolismo , Animais , Antígenos de Superfície , Sequência de Bases , RNA Polimerases Dirigidas por DNA/metabolismo , Cinética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Catalítico/química , RNA Catalítico/genética , Ratos , Receptores de Superfície Celular , Receptores de Complemento/genética , Transcrição Gênica , Proteínas ViraisRESUMO
Mammalian cells are usually protected from the complement-mediated injury by a number of complement regulatory proteins. A rat protein designated as 512 antigen is thought to be such a complement regulatory protein. A cDNA of 512 antigen has been cloned and analyzed. To investigate the function of 512 antigen, we plan to construct a ribozyme system against 512 antigen expression. We have designed two hammerhead ribozymes targeted to the 512 antigen mRNA and tested the ribozyme activities in vitro. Both hammerhead ribozymes efficiently cleaved the 512 antigen mRNA in vitro. We have also designed a hairpin ribozyme against this mRNA. The activity of the hairpin ribozyme will also be discussed.
