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Abstract(s)
As terapia baseadas em RNA têm emergido como estratégias promissoras no tratamento de diversas doenças, como cancro, doenças autoimunes e neuro-degenerativas. Contudo, o estabelecimento de terapias com base no RNA requer a preparação de elevada quantidade de RNA mantendo a sua integridade, pureza e atividade biológica. Este é o maior objetivo do uso de metodologias de purificação de RNA. Os líquidos iónicos (LI) são um grupo de sais orgânicos de variada estrutura, com grande versatilidade para manipular o design de catião/anião e apresentam características físico-químicas particulares. Mais ainda, alguns LIs conseguem o estatuto de solventes “green” e têm sido observados como uma alternativa aos solventes orgânicos usados num vasto leque de aplicações.
O principal objetivo deste trabalho é o desenvolvimento de novas matrizes superporosas funcionalizadas com ILs para cromatografia liquida preparativa com a purificação de RNA em vista.
Diferentes LIs foram ligados covalentemente em sílica (S-Sil), usada como fase estacionária, para avaliação inicial das interações dos ILs com o RNA. Esta técnica revela ser reprodutível e mais económica para a pré-avaliação antes de usar uma coluna cromatográfica. A capacidade de diferentes tipos de S-Sils (SilPrMImCl; SilPrMImArg; SilPrPyCl; SilPrArgCl; SilPrBenzCl; SilPrNEt3Cl; SilPrN(C8)3Cl; SilPrLyCl) para reconhecer e ligar ao RNA foi avaliada em modo batch e, de seguida, foram realizados diferentes testes de modo a provar a reprodutibilidade, a robustez e a resistência à regeneração. Em geral, os S-Sils mostraram a possibilidade de promover múltiplas interações incluindo hidrofóbicas, iónicas, pontes de hidrogénio e interações dipolo-dipolo com o RNA. Em particular, S-SilPrMImCl, S-SilPrNEt3Cl e S-SilPrMet2ButCl mostram ser ligandos promissores para aplicação na purificação do RNA.
Inicialmente foi realizada a funcionalização da matriz cromatográfica Sp-SilPrMImCl e, após confirmação da funcionalização, foram realizados ensaios de purificação de RNA a partir de lisados de E. coli. Neste estudo foi confirmada a completa separação entre o RNA e o DNA num único passo cromatográfico. Foi ainda avaliada a reprodutibilidade da coluna onde é visível a separação do RNA sem contaminação. O novo suporte demonstrou ainda apresentar ainda uma elevada capacidade de ligação dinâmica comprovando assim a robustez da matriz para purificar uma grande quantidade de amostra, considerando até a potencial aplicação na purificação de vários tipos de biomoléculas.
RNA-based therapies have emerged as a promising strategy in the treatment of diverse human diseases, such as cancer, autoimmune and neurodegenerative diseases. However, the establishment of RNA-based therapeutics requires large quantities of the target RNA with high integrity, purity and biological activity. This has been mostly accomplished using RNA purification methodologies that rely on using organic solvents or imply the structural modification of RNAs. Ionic liquids (ILs) are a group of organic salts of varying structure, with great versatility to manipulate the cation/anion design, and presenting particular physicochemical characteristics. Moreover, some ILs earned the statute of ‘‘green solvents’’ and have been seen as an alternative to organic solvents used in a wide range of applications. Therefore, the main aim of this work was the development of new superporous-based matrices functionalized with ILs for preparative liquid chromatography, envisaging RNA purification. First, different ILs were covalently attached on silica (Sils), used as the stationary phase, for the screening of ILs interacting with RNA. This approach revealed as a reproducible and more economical method for a pre-evaluation before using the chromatographic column. The ability of different Sils (SilPrMImCl; SilPrMImArg; SilPrPyCl; SilPrArgCl; SilPrBenzCl; SilPrNEt3Cl; SilPrN(C8)3Cl; SilPrLyCl) to selectively capture RNA was evaluated in a batch mode and then, different experiments were designed to prove the reproducibility, robustness and resistance to regeneration of the supports. In general, the obtained SILs showed the possibility to promote a multitude of interactions including hydrophobic, ionic, hydrogen bond and dipole–dipole interactions with RNA. In particular, SilPrMImCl and SilPrNEt3Cl seem to be the most promising ligands to be further explored in preparative column liquid chromatography of different biomolecules. Initially, the Sp-SilPrMImCl chromatographic matrix functionalization was performed and, after functionalization confirmation, some RNA purification assays were performed trying to isolate RNA from E. coli lysates. In this study the complete separation between RNA and DNA was achieved in a single chromatographic step. It was also evaluated the reproducibility of the column where it is visible the separation of the RNA without contamination. The results of the dynamic binding capacity were also very interesting, as it was verified a high binding capacity for this new column. Thus, it was confirmed the robustness of the matrix and the potential application in the purification of several types of biomolecules.
RNA-based therapies have emerged as a promising strategy in the treatment of diverse human diseases, such as cancer, autoimmune and neurodegenerative diseases. However, the establishment of RNA-based therapeutics requires large quantities of the target RNA with high integrity, purity and biological activity. This has been mostly accomplished using RNA purification methodologies that rely on using organic solvents or imply the structural modification of RNAs. Ionic liquids (ILs) are a group of organic salts of varying structure, with great versatility to manipulate the cation/anion design, and presenting particular physicochemical characteristics. Moreover, some ILs earned the statute of ‘‘green solvents’’ and have been seen as an alternative to organic solvents used in a wide range of applications. Therefore, the main aim of this work was the development of new superporous-based matrices functionalized with ILs for preparative liquid chromatography, envisaging RNA purification. First, different ILs were covalently attached on silica (Sils), used as the stationary phase, for the screening of ILs interacting with RNA. This approach revealed as a reproducible and more economical method for a pre-evaluation before using the chromatographic column. The ability of different Sils (SilPrMImCl; SilPrMImArg; SilPrPyCl; SilPrArgCl; SilPrBenzCl; SilPrNEt3Cl; SilPrN(C8)3Cl; SilPrLyCl) to selectively capture RNA was evaluated in a batch mode and then, different experiments were designed to prove the reproducibility, robustness and resistance to regeneration of the supports. In general, the obtained SILs showed the possibility to promote a multitude of interactions including hydrophobic, ionic, hydrogen bond and dipole–dipole interactions with RNA. In particular, SilPrMImCl and SilPrNEt3Cl seem to be the most promising ligands to be further explored in preparative column liquid chromatography of different biomolecules. Initially, the Sp-SilPrMImCl chromatographic matrix functionalization was performed and, after functionalization confirmation, some RNA purification assays were performed trying to isolate RNA from E. coli lysates. In this study the complete separation between RNA and DNA was achieved in a single chromatographic step. It was also evaluated the reproducibility of the column where it is visible the separation of the RNA without contamination. The results of the dynamic binding capacity were also very interesting, as it was verified a high binding capacity for this new column. Thus, it was confirmed the robustness of the matrix and the potential application in the purification of several types of biomolecules.
Description
Keywords
Ácidos Nucleicos Cromatografia Líquidos Iónicos Rna