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  • Ionic liquids for the purification and stabilization of nucleic acids
    Publication . Carapito, Ana Rita Mugeiro; Sousa, Fani Pereira de; Martins, Mara Guadalupe Freire
    Nucleic acids-based therapies have emerged as excellent approaches to treat various diseases such as cancer and neurodegenerative disorders. However, for these biomolecules to be used as biopharmaceuticals they must be obtained with high purity, presenting biological activity and structural integrity. As these are the main challenges in obtaining biopharmaceuticals, it is necessary to develop purification strategies that ensure their quality. Ionic liquids (ILs) are liquid salts at room temperature that have very interesting properties, namely the vast combination of cation and anion that can be made, thus tailoring the structures to the intended purpose. This work has as main objective the development of a new strategy for the purification of nucleic acids using functionalized supports with different ILs. In addition, we also intend to evaluate the RNA stabilizing capacity of certain ILs as well as their toxicity in human cell lines in order to ascertain the biosafety of these substances. Initially, different ILs were covalently immobilized onto spherical silica supports, used as a stationary phase, resulting in the following materials: [Si][C3C1Im]Cl, [Si][N3222]Cl, [Si][N3444]Cl, [Si][N3888]Cl and [Si][N3114]Cl. In addition to the structural characterization of the supports, and as a screening procedure, the binding and elution of low molecular weight RNA molecules was tested under ionic and hydrophobic conditions with all the synthesised supports, in order to select the most promising ligand(s) for nucleic acids purification. [Si][N3222]Cl and [Si][N3114]Cl were the chosen supports for further separation procedures between genomic DNA and RNA. Both supports showed ability for separating these two species, although [Si][N3114]Cl displayed better selectivity, thus becoming more promising for future separation assays. Additionally, RNA stabilization assays were performed with four different types of ILs, analogues to the ligands, namely [N1111]Cl, [N2222]Cl, [N3333]Cl and [N4444]Cl. Only [N1111]Cl showed to enhance RNAs thermal stability, verifying by this, a negative contribution of the alkyl chains lengths in the stabilization of this biomolecule. Besides, cytotoxicity assays with the two chosen supports and these four ILs were performed. ILimmobilized supports did not present any cytotoxicity, while liquid ILs were discovered to largely compromise cell viability. In this sense, the usage of ILs immobilized onto solid supports appears to be safer than using bulk ILs. Thus, was possible to demonstrate the importance of various ILs in purification and manipulation of nucleic acids, becoming a continuously growing area.
    2019-12-03Master thesis Open access Show more