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- Cholinium-Based Good’s Buffers Ionic Liquids as Remarkable Stabilizers and Recyclable Preservation Media for Recombinant Small RNAsPublication . Pedro, Augusto; Pereira, Patrícia; Quental, Maria J.; Carvalho, André P.; Santos, Sérgio M.; Queiroz, João; Sousa, Fani; Freire, Mara G.RNA is a biopolymer of high relevance in the biopharmaceuticals field and in fundamental and applied research; however, the preservation of the RNA stability is still a remarkable challenge. Herein, we demonstrate the enhanced potential of aqueous solutions of self-buffering cholinium-based Good's buffers ionic liquids (GB-ILs), at 20 and 50 % (w/w), as alternative preservation media of recombinant small RNAs. The thermal stability of RNA is highly enhanced by GB-ILs, with an increase of 14 °C in the biopolymer melting temperature - the highest increase observed up to date with ILs. Most GB-ILs investigated improve the stability of RNA at least up to 30-days, both at 25 °C and at 4 °C, without requiring the typical samples freezing. Molecular dynamics simulations were applied to better understand the molecular-level mechanisms responsible for the observed RNA improved stability. The number of IL cations surrounding the RNA chain is similar, yet with differences found for the IL anions, which are responsible for the overall charge of the biopolymer first solvation sphere. No cytotoxicity of the studied solutions containing RNA and ILs at 20 % (w/w) was observed onto two distinct human cell lines, reinforcing their potential to act as preservation media when foreseeing biopharmaceutical applications. Finally, RNA was successfully recovered from the ILs aqueous solutions, without changes in its structural integrity, and the ILs successfully recycled and reused.
- Multimodal ionic liquid-based chromatographic supports for an effective RNA purificationPublication . Carapito, Ana Rita Mugeiro; Bernardo, Sandra C.; Pereira, Matheus M.; Neves, Márcia C.; Freire, Mara G.; Sousa, FaniNucleic acids have been considered interesting molecules to be used as biopharmaceuticals for the treatment of various diseases, in gene therapy strategies. In particular, RNA arises as the most promising approach because it does not require access to the nucleus of cells to exert its function; however, it is quite challenging due to its labile nature. To increase the possibility of translating RNA-based technology to clinical protocols, the bio- manufacturing of RNAs has been intensively exploited in the last few years. However, the standard RNA puri- fication processes remain time-consuming and present limitations regarding recovery yield and purity. This work describes the functionalization of chromatographic silica-based supports with four ionic liquids (ILs) composed of functional moieties that can promote distinct interactions with nucleic acids. After an initial screening to evaluate the binding and elution behavior of nucleic acids in the IL-based supports, SSi[C3C3NH2Im]Cl has shown to be the most promising for further purification assays. This support was studied for the RNA purification from different samples (clarified or more complex) and has shown to be highly effective, for all the conditions studied. Generally, it is here presented a new method for RNA isolation in a single step, using an IL-based chromato- graphic support, able to eliminate the usage of hazardous compounds often included in standard RNA extraction protocols.