Design of Deep Eutectic Solvents to improve RNA stabilization and storage

• Autor(a) principal: Ferro, Ana Isabel da Silva Pereira
• Data de Publicação: 2021
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/11652
• topic: Des; Dicroísmo Circular; Estabilidade de Rna; Rnase; Domínio/Área Científica::Engenharia e Tecnologia::Biotecnologia

Until recently, the RNA molecule was not particularly studied from the point of view of therapeutic application, being essentially considered an intermediary in the transfer of genetic information from DNA to the expression of proteins. However, advances in research related to RNA led this biomolecule to be now considered a versatile and dynamic molecule, with a clear recognition of new biological functions, and potential application as a therapeutic agent. More specifically, with the use of mRNAs and microRNAs, new therapies have emerged, such as the vaccines against COVID-19. Still, the use of these vaccines and their global distribution has been much discussed, as it is necessary to maintain the ideal storage conditions, such as temperature, to guarantee the safety and efficacy of the vaccine. For this reason, the need to identify new compounds with the ability to stabilize the RNA molecule in the storage and distribution process, ensuring the biological activity of the molecule, is evidenced. In the present work, the ability of Deep Eutectic Solvents (DES) to stabilize and protect RNA was tested. The DES mixtures under study are composed of choline chloride and different amino acids, such as arginine, glycine, methionine, tryptophan, tyrosine, phenylalanine, alanine, and cysteine, prepared in concentrations of 1 and 4 mM. In a first approach, the toxicity of DES in a human cell line was evaluated after an exposure period of 48 hours. These did not demonstrate significant toxicity, however it was noticeable that there is a relationship between the concentration of DES and the level of safety. In the next step, the structural stability of the RNA was evaluated when placed in contact with the different excipient candidates, the DES. Circular dichroism analysis allowed to verify the stability of RNA samples stored for 0, 7, 15, 30, 60, 120 and 180 days at room temperature or 4 °C. In general, the DES used promoted the stabilization of the RNA molecule, in some cases for both temperatures. Additionally, the potential of DES to protect RNA when exposed to RNases was studied. For this, preliminary assays were performed with different concentrations of RNase (0.33 and 0.5 µg/mL), and after selecting the most appropriate concentrations, the premir-9 samples were incubated with DES for 0, 16 and 24 hours at room temperature and 4 °C. The results obtained indicate that for room temperature the best protection results were achieved with a mixture of ChCl:Glycine at a temperature of 4 °C, followed by a mixture of ChCl:Cysteine and finally ChCl:Alanine. Note that in the latter case, protection was achieved for storage at both temperatures. Subsequently, some tests were carried out, with RNA exposure to each of the elements that composed DES (choline chloride and free amino acids) in order to assess the partial contribution of each element to the global stabilization/protection of RNA. The results obtained indicate that at both temperatures cysteine, phenylalanine, glycine, methionine and choline chloride demonstrated to have a stabilizing effect on the molecule. Thus, with the results obtained, it was possible to confirm the stabilization of the RNA molecule with a new class of compounds, candidate excipients, after storage at room temperature or at 4 ºC for a period of time. Within the scope of the project, it was also possible to verify the ability of DES to induce the protection of the molecule when it is exposed to a more aggressive condition that can lead to its degradation, namely by exposure to nucleases. Overall, this work allows us to bring new perspectives for the design and study of DES for the medium/long term stabilization of biopharmaceuticals.