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Abstract(s)
Até há pouco tempo, a molécula de RNA era ainda negligenciada em comparação com o DNA e proteínas, sendo descrito como um simples intermediário no fluxo da informação genética. No entanto, ao longo dos últimos anos o RNA tem sido associado a uma multiplicidade de processos biológicos, sendo hoje em dia, intitulado como um componente celular surpreendente. A investigação em torno da molécula de RNA tem levado a inúmeras descobertas biologicamente importantes e ao desenvolvimento ou aperfeiçoamento de novas abordagens terapêuticas. Um tipo de RNA que tem sido amplamente estudado, devido à potencial aplicação terapêutica, é o microRNA (miRNA). Este RNA pertence à classe dos RNAs de baixo peso molecular que regulam a expressão génica, e a sua desregulação tem sido associada a diversas patologias.
Ainda assim, a necessidade de obter o RNA puro e intacto é extremamente relevante considerando esta vertente de aplicação clínica, e quer o seu isolamento quer a etapa de purificação e armazenamento apresentam-se como passos críticos devido à possível degradação do RNA, o que pode prejudicar a sua estabilidade estrutural e consequentemente a sua atividade biológica. Desta forma, torna-se fundamental garantir a estabilidade da molécula após todas as etapas do processo, visando a sua aplicação.
Deste modo, o objetivo global deste estudo consiste no desenvolvimento e implementação de uma nova metodologia que permita manter a conformação nativa e atividade biológica do RNA, após todas as etapas críticas do seu processamento. Inicialmente foram realizados estudos cromatográficos baseados em cromatografia de afinidade com ligandos de aminoácidos, nomeadamente a matriz de L-metionina-agarose, para obter o pre-miR-29 com elevado grau de pureza. Como estes resultados não foram conclusivos, e não foi possível a separação eficiente do pre-miR-29, os estudos de estabilização foram realizados com RNA total.
Assim, foram realizados ensaios de dicroísmo circular (DC) para avaliar a estabilidade estrutural do RNA, após o seu isolamento e incubação com diferentes classes de excipientes, nomeadamente sais (EDTA, citrato de sódio e acetato de sódio) e aminoácidos (histidina, prolina e arginina) nas concentrações de 1, 25 e 100 mM, e açúcares (sorbitol, trealose e sacarose) a 295 e 595 mM. O estudo de estabilidade inclui ainda a avaliação da estabilidade em função do tempo (7, 15 e 30 dias) e temperatura de armazenamento (4, -20 e -80ºC). De uma maneira geral, estes testes preliminares indicaram que o RNA na presença de EDTA ou arginina na concentração de 1 mM, conservados a 4ºC e -80ºC, mantém a sua estabilidade conformacional.
Considerando os resultados obtidos, e para confirmar a ação estabilizadora dos aditivos na manutenção da atividade biológica do RNA foi usado como modelo o pre-miR-29. Estudos recentes apontam para uma alteração dos níveis deste miRNA na doença de Alzheimer (DA). Em condições fisiológicas, o pre-miR-29 pode atuar diretamente sobre a enzima ß-secretase 1 (BACE 1) responsável pela clivagem da proteína percursora amilóide (APP) e desta forma diminuir os depósitos extracelulares dos péptidos ß-amilóides (Aß). Estes estudos revelam a possibilidade da aplicação do pre-miR-29b como um alvo terapêutico, já que a reposição dos seus níveis nas células neuronais poderá induzir o silenciamento da BACE 1 e redução dos níveis dos péptidos Aß.
Assim, no âmbito deste trabalho, o pre-miR-29 recombinante foi preparado e armazenado nas condições que apresentaram melhores resultados de estabilização, no dicroísmo circular, para posteriormente ser avaliada a sua atividade biológica, nomeadamente no que se refere à capacidade de reduzir os níveis de expressão da BACE 1. Após a produção do pre-miR-29, este foi purificado com a matriz de L-arginina-Sepharose, e foram realizados estudos de transfeção das células N2a695. Os resultados obtidos demonstraram que o pre-miR-29 incubado com arginina a -80ºC durante 30 dias apresenta uma diminuição da expressão do mRNA da proteína hBACE 1 de cerca de 90-100%, quando comparado com as outras condições de armazenamento testadas.
Until recently, RNA molecule was still neglected comparing to DNA and proteins, being described as a mere intermediate in the flow of genetic information. However, over the past few years, RNA has been associated to a multiplicity of biological processes, being nowadays, entitled as a surprising cellular component. Researches around the RNA molecule have led to numerous biologically important discoveries and to the development or improvement of new therapeutic approaches. A type of RNA that has been widely studied due to its potential therapeutic application is microRNA (miRNA). This RNA belongs to the class of small RNAs that regulates gene expression, and its dysregulation has been linked to various diseases. Still, the need to obtain pure and intact RNA is extremely important considering its clinical application. The isolation, purification and storage steps are presented as critical steps due to the possible RNA degradation, which can harm its structural stability and consequently its biological activity. Thus, it is essential to ensure the stability of the molecule after all stages of the process, with the view of its application. Thus, the overall aim of this study is the development and implementation of a new methodology to maintain the native conformation and biological activity of RNA, after all critical stages of processing. Initially, chromatographic studies were performed based on affinity chromatography with amino acid ligands, in particular L-methionine-agarose matrix, in order to obtain the pre-miR-29 with high purity. As these results were inconclusive, and the efficient separation of pre-miR-29 was not possible, stabilization studies were performed with total RNA. Accordingly, circular dichroism (CD) experiments were performed in order to evaluate the RNA structural stability, after its isolation and incubation with different classes of excipients, including salts (EDTA, sodium citrate and sodium acetate) and amino acids (histidine, proline and arginine) at concentrations of 1, 25 and 100 mM, and sugars (sorbitol, trehalose and sucrose) at 295 and 595 mM. The stability study also included the evaluation of different storage time (7, 15 and 30 days) and storage temperatures (4, -20 and -80ºC). In general, these preliminary tests indicated that the RNA in presence of EDTA or arginine at a concentration of 1 mM, stored at 4ºC and -80ºC, maintains its conformational stability. Considering the obtained results, and to confirm the stabilizing action of additives on maintenance of the RNA biological activity, pre-miR-29 was used as a model. Recent studies point to a change in the levels of this miRNA in Alzheimer's disease (AD). Under physiological conditions, miR-29 may act directly on ß- secretase enzyme (BACE 1) responsible for the cleavage of amyloid precursor protein (APP) and thereby decreasing the extracellular deposits of ß-amyloid peptide (Aß). These studies indicate the possibility of pre-miR-29b application as a therapeutic target, since the reinstatement of its levels in neuronal cells might induce the silencing of BACE 1, reducing levels of Aß peptide. Thus, in this work, the recombinant pre-miR-29 was prepared and stored using the conditions that have presented better stability results by circular dichroism, to later be evaluated its biological activity, in particular regarding to the ability of reducing the expression levels of BACE 1. After production of pre-miR-29, it was purified with the L-arginine-Sepharose matrix, and transfection studies were performed in N2a695 cells. The obtained results showed that pre-miR-29 incubated with arginine at -80°C for 30 days induces a decreased of about 90-100% of hBACE 1 mRNA expression, when compared to other tested storage conditions.
Until recently, RNA molecule was still neglected comparing to DNA and proteins, being described as a mere intermediate in the flow of genetic information. However, over the past few years, RNA has been associated to a multiplicity of biological processes, being nowadays, entitled as a surprising cellular component. Researches around the RNA molecule have led to numerous biologically important discoveries and to the development or improvement of new therapeutic approaches. A type of RNA that has been widely studied due to its potential therapeutic application is microRNA (miRNA). This RNA belongs to the class of small RNAs that regulates gene expression, and its dysregulation has been linked to various diseases. Still, the need to obtain pure and intact RNA is extremely important considering its clinical application. The isolation, purification and storage steps are presented as critical steps due to the possible RNA degradation, which can harm its structural stability and consequently its biological activity. Thus, it is essential to ensure the stability of the molecule after all stages of the process, with the view of its application. Thus, the overall aim of this study is the development and implementation of a new methodology to maintain the native conformation and biological activity of RNA, after all critical stages of processing. Initially, chromatographic studies were performed based on affinity chromatography with amino acid ligands, in particular L-methionine-agarose matrix, in order to obtain the pre-miR-29 with high purity. As these results were inconclusive, and the efficient separation of pre-miR-29 was not possible, stabilization studies were performed with total RNA. Accordingly, circular dichroism (CD) experiments were performed in order to evaluate the RNA structural stability, after its isolation and incubation with different classes of excipients, including salts (EDTA, sodium citrate and sodium acetate) and amino acids (histidine, proline and arginine) at concentrations of 1, 25 and 100 mM, and sugars (sorbitol, trehalose and sucrose) at 295 and 595 mM. The stability study also included the evaluation of different storage time (7, 15 and 30 days) and storage temperatures (4, -20 and -80ºC). In general, these preliminary tests indicated that the RNA in presence of EDTA or arginine at a concentration of 1 mM, stored at 4ºC and -80ºC, maintains its conformational stability. Considering the obtained results, and to confirm the stabilizing action of additives on maintenance of the RNA biological activity, pre-miR-29 was used as a model. Recent studies point to a change in the levels of this miRNA in Alzheimer's disease (AD). Under physiological conditions, miR-29 may act directly on ß- secretase enzyme (BACE 1) responsible for the cleavage of amyloid precursor protein (APP) and thereby decreasing the extracellular deposits of ß-amyloid peptide (Aß). These studies indicate the possibility of pre-miR-29b application as a therapeutic target, since the reinstatement of its levels in neuronal cells might induce the silencing of BACE 1, reducing levels of Aß peptide. Thus, in this work, the recombinant pre-miR-29 was prepared and stored using the conditions that have presented better stability results by circular dichroism, to later be evaluated its biological activity, in particular regarding to the ability of reducing the expression levels of BACE 1. After production of pre-miR-29, it was purified with the L-arginine-Sepharose matrix, and transfection studies were performed in N2a695 cells. The obtained results showed that pre-miR-29 incubated with arginine at -80°C for 30 days induces a decreased of about 90-100% of hBACE 1 mRNA expression, when compared to other tested storage conditions.
Description
Keywords
Cromatografia de Afinidade Dicroísmo Circular Estabilidade do Rna Pre-Mir-29b-1 Rna