resumo
High quality nucleic acids (with high integrity, purity, and biological activity) have become indispensable products of modern society, both in molecular diagnosis and to be used as biopharmaceuticals. As the current methods available for the extraction and purification of nucleic acids are laborious, time-consuming, and usually rely on the use of hazardous chemicals, there is an unmet need towards the development of more sustainable and cost-effective technologies for nucleic acids purification. Accordingly, this study addresses the preparation and evaluation of silica-based materials chemically modified with chloride-based ionic liquids (supported ionic liquids, SILs) as potential materials to effectively isolate RNAs. The investigated chloride-based SILs comprise the following cations: 1-methyl-3-propylimidazolium, triethylpropylammonium, dimethylbutylpropylammonium, and trioctylpropylammonium. All SILs were synthesized by us and characterized by solid-state C-13 Nuclear Magnetic Resonance (NMR), Scanning Electron Microscopy (SEM), elemental analysis, and zeta potential measurements, confirming the successful covalent attachment of each IL cation with no relevant changes in the morphology of materials. Their innovative application as chromatographic supports for the isolation of recombinant RNA was then evaluated. Adsorption kinetics of transfer RNA (tRNA) on the modified silica-based materials were investigated at 25 & DEG;C. Irrespective to the immobilized IL, the adsorption experimental data are better described by a pseudo first-order model, and maximum tRNA binding capacities of circa 16 mu mol of tRNA/g of material were achieved with silica modified with 1-methyl-3-propylimidazolium chloride and dimethylbutylpropylammonium chloride. Furthermore, the multimodal character displayed by SILs was explored towards the purification of tRNA from Escherichia coli lysates, which in addition to tRNA contain ribosomal RNA and genomic DNA. The best performance on the tRNA isolation was achieved with SILs comprising 1-methyl-3-propylimidazolium chloride and dimethylbutylpropylammonium chloride. Overall, the IL modified silica-based materials represent a more efficient, sustainable, and cost-effective technology for the purification of bacterial RNAs, paving the way for their use in the purification of distinct biomolecules or nucleic acids from other sources.
palavras-chave
GENE-THERAPY; PLASMID DNA; PURIFICATION; EXTRACTION; STABILITY; CHROMATOGRAPHY
categoria
Biology; Microbiology
autores
Pereira, P; Pedro, AQ; Neves, MC; Martins, JC; Rodrigues, I; Freire, MG; Sousa, F
nossos autores
Augusto Pedro
Investigador Júnior
Mara Guadalupe Freire Martins
Investigador Coordenador
Márcia Carvalho Neves
Investigador AuxiliarProjectos
CICECO - Aveiro Institute of Materials (UIDB/50011/2020)
agradecimentos
This work was developed within the scope of the project CICECO-Aveiro Institute of Materials, UIDB/50011/2020 & UIDP/50011/2020, the CICS-UBI project UIDB/00709/2020, and the CEMMPRE project UIDB/00285/2020, financed by national funds through the Portuguese Foundation for Science and Technology/MCTES. This work was financially supported by the project PUREmiRSILs-PTDC/BII-BBF/29496/2017, co-financed by FEDER, through POCI-Operational Programme Competitiveness and Internationalization, and National Funds by the Portuguese Foundation for Science and Technology (FCT). The authors additionally acknowledge the financial support from the European Union Framework Programme for Research and Innovation HORIZON 2020, under the TEAMING Grant No. 739572-The Discoveries CTR. The NMR spectrometers used in this work are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project No. 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL, and FCT through PIDDAC). Augusto Q. Pedro and Marcia C. Neves acknowledge FCT respectively, for the research contracts CEECIND/00383/2017 and CEECIND/02599/2020 under the Scientific Employment Stimulus-Individual Call.