Purification of pre-miRNA-29 with ionic liquids-based supports

• Autor(a) principal: Nunes, Sérgio Almeida
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/13328
• topic: Desenho Experimental; Líquidos Iónicos Suportados; Pre-Mirna-29b; Processos de Purificação; Domínio/Área Científica::Engenharia e Tecnologia::Biotecnologia

MicroRNAs (miRNAs) have been studied regarding their biological role in gene expression regulation at post-transcriptional level, envisioning the establishment of new treatment strategies. Pre-miRNA-29 is the precursor form of microRNA-29, a small noncoding RNA that plays an important role in gene silencing, and proved to be involved in several cellular mechanisms, namely in neurodegenerative diseases. To consider the therapeutic application, efficient, and economically feasible manufacturing processes are required for obtaining these biopharmaceuticals, assuring their integrity, purity, stability, and bioactivity. Thus, in this work it is proposed an alternative purification method using Ionic Liquids (ILs)-based chromatographic supports. ILs are described as ionic compounds that present a melting point below 100 °C and are normally composed of large and asymmetric organic cations and organic or inorganic anions. These types of compounds can be tailored in terms of their properties, such as polarity, density, and viscosity to achieve their effectiveness regarding the purification and stabilization of nucleic acids. The application of Supported ILs (SILs) in RNA purification is highly innovative and can lead to the establishment of advantageous purification methods. Herein, the SIL under study resulted from the functionalization of silica with the 1-(3- aminopropyl)imidazole chloride, which was named as SilPrImPrACl. First, a screening of the best experimental conditions, in terms of pH and ionic strength, to promote binding and elution of RNA was carried out. Moreover, in order to develop an Experimental Design, key factors were optimized at 3 levels, using the Central Composite Design (CCD) as model. The statistical analysis of the results showed that this model was not the best fit for the optimization of the purification of pre-miRNA-29b, however further analysis was carried out in order to understand the influence of the factors studied in the recovery, purification factor and removal of impurities. Thus, it was possible to establish some conditions conducting to a recovery and purification factor of 35.1% and 1.55, respectively. Furthermore, it was verified that the process was able to remove 78% of proteins and 70% of gDNA present in the initial sample, proving the effectiveness of this SIL for the purification of RNA and particularly the pre-miRNA-29b.