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Abstract(s)
O recente desenvolvimento da terapia génica surge como uma nova abordagem para o potencial tratamento de doenças consideradas incuráveis. Apesar destas estratégias se terem iniciado com DNA, investigações recentes tem avaliado o potencial interesse terapêutico do RNA. No entanto, os protocolos disponível para o isolamento do RNA são limitados, morosos e requerem o uso de solventes tóxicos, o que tornam estes procedimentos inadequados para recuperar o produto a ser biologicamente aplicado.
O principal objectivo deste estudo é investigar a aplicabilidade da cromatografia de afinidade com histidina imobilizada no isolamento do RNA. Uma vez que o RNA, tal como o DNA, tem a capacidade de se ligar à matriz de histidina-agarose, esta interacção pode ser explorada para purificar especificamente os diferentes RNAs, atendendo às suas propriedades estruturais. Para concretizar o objectivo, o RNA total foi isolado da Escherichia coli DH5α usando a extracção com tiocianato de guanidina- fenol/clorofórmio e a separação do RNA ribossómico (rRNA) e do RNA de baixo peso molecular (sRNA) foi promovida por precipitação com 2M de sulfato de amónio a 4ºC. Os estudos dos perfis cromatográficos das espécies de sRNA e de rRNA na matriz de histidina-agarose com diferentes concentrações de sulfato de amónio revelaram que para os dois tipos de RNA, as elevadas concentrações de sal promoviam a ligação do RNA à matriz, e a diminuição da força iónica promovia a sua eluição.
Com este estudo foi possível desenvolver uma estratégia para a purificação do RNA 6S aplicando um gradiente decrescente de sulfato de amónio por passos. Após a ligação dos sRNAs à matriz de histidina, a diminuição da concentração de sal permite a separação de outros tipos de RNA e a remoção do sal na eluição promove a recuperação do RNA 6S. Este tipo de RNA foi recentemente identificado como estando envolvido no processo de transcrição, pelo que a purificação é uma mais valia para a sua caracterização estrutural e funcional.
Severe diseases that currently remain untreatable are strong candidates to novel genebased treatments. Although the gene-based therapeutic strategies started to be developed using DNA, a large number of studies are in progress in which the therapeutic potential of RNA is evaluated. However, the available protocols for isolation of RNA are limited, time-consuming and require the use of toxic solvents. All these reasons make these procedures not adequate to recover a product to be biologically applied and motivate the research for an efficient, tolerable and scalable process to purify RNA. The main purpose of this study is the investigation of the applicability of a recently established method based on affinity chromatography with amino acids as specific ligands, to isolate RNA species. As the histidine-agarose support is able to bind the RNA, this interaction can be exploited to specifically purify different RNAs, considering their structural particularities. Total RNA was isolated from Escherichia coli DH5α by guanidinium-thiocyanatephenol-chloroform extraction and ribosomal RNAs (rRNAs) and small RNAs (sRNAs) were obtained by ammonium sulphate precipitation at 4ºC. The different RNAs were then applied to a histidine-agarose support with different ammonium sulphate concentrations in order to understand their retention pattern. In both RNA classes, the higher salt concentrations promoted RNA binding to histidine-agarose matrix, while lower ionic strength caused its elution. In this study, we were also able to establish a strategy for the purification of the 6S RNA, using a decreasing stepwise elution gradient. The higher salt concentrations promoted sRNAs binding to the histidine support, and the decrease of ammonium sulphate concentration caused the elution of some RNA species. Finally, the removal of ammonium sulphate from the elution buffer allowed the recovery of 6S RNA. This type of RNA was recently recognized in the transcription process and its purification is of great importance for further structural and functional studies.
Severe diseases that currently remain untreatable are strong candidates to novel genebased treatments. Although the gene-based therapeutic strategies started to be developed using DNA, a large number of studies are in progress in which the therapeutic potential of RNA is evaluated. However, the available protocols for isolation of RNA are limited, time-consuming and require the use of toxic solvents. All these reasons make these procedures not adequate to recover a product to be biologically applied and motivate the research for an efficient, tolerable and scalable process to purify RNA. The main purpose of this study is the investigation of the applicability of a recently established method based on affinity chromatography with amino acids as specific ligands, to isolate RNA species. As the histidine-agarose support is able to bind the RNA, this interaction can be exploited to specifically purify different RNAs, considering their structural particularities. Total RNA was isolated from Escherichia coli DH5α by guanidinium-thiocyanatephenol-chloroform extraction and ribosomal RNAs (rRNAs) and small RNAs (sRNAs) were obtained by ammonium sulphate precipitation at 4ºC. The different RNAs were then applied to a histidine-agarose support with different ammonium sulphate concentrations in order to understand their retention pattern. In both RNA classes, the higher salt concentrations promoted RNA binding to histidine-agarose matrix, while lower ionic strength caused its elution. In this study, we were also able to establish a strategy for the purification of the 6S RNA, using a decreasing stepwise elution gradient. The higher salt concentrations promoted sRNAs binding to the histidine support, and the decrease of ammonium sulphate concentration caused the elution of some RNA species. Finally, the removal of ammonium sulphate from the elution buffer allowed the recovery of 6S RNA. This type of RNA was recently recognized in the transcription process and its purification is of great importance for further structural and functional studies.
Description
Keywords
RNA 6S RNA Cromatografia Histidina Geometografia de afinidade
Citation
Publisher
Universidade da Beira interior