Rapid and selective capture of nucleic acids using carbon nanotubes

• Autor(a) principal: Ferreira, Pedro Filipe Lopes
• 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/10400.6/11493
• topic: Biofármacos; Dna Plasmídico; Extração de Fase Sólida; Nanotubos de Carbono; Rna; Separação de Ácidos Nucleicos; Domínio/Área Científica::Engenharia e Tecnologia::Biotecnologia

Gene therapy is raising as one of the most promising options for treating genetic and acquired disorders. However, recombinant plasmid DNA (pDNA) must be recovered from complex extracts with a diversity of biomolecules that share similar characteristics and properties. Moreover, those biomolecules can cause immunogenicity, and their removal is a fundamental prerequisite in preparing high-purity, stable, and biological active pDNA for therapeutic application. To respond to this challenge and isolate pDNA from complex extracts, a promising alternative is proposed, based on using carbon nanotubes (CNTs). In recent years, biological applications of CNTs have been explored, motivated by their interesting size, shape, structure, biocompatibility, and high affinity for biomolecules. In this work, an effective method to isolate pDNA was developed, using multi-walled carbon nanotubes (MWCNTs) to capture proteins and other contaminating nucleic acids, like RNA and genomic DNA (gDNA). MWCNTs with different diameters were evaluated, while mixtures of RNA and pDNA were prepared to address the selectivity of the material. The results of this work evidenced the ability of MWCNTs to capture RNA from complex samples, enabling the clarification of pDNA. The results also demonstrated that MWCNTs can reduce the amount of impurities in the first cycle of extraction, reducing about half of the proteins, and significantly reducing the levels of genomic DNA in solution, with a pDNA recovery rate of 86%. When performing up to three consecutive cycles, the purity of soluble pDNA can be significantly improved. To further reduce the environmental impact of this technology, the possibility of recycling CNTs has also been confirmed, as well as it was assured the safety on their use by verifying the biocompatibility of this material. Thus, attained results demonstrate the development of a simple, efficient, and reliable method for rapid purification of pDNA biopharmaceuticals.