Molecular docking tools for the screening of affinity ligands for pre-miRNA-29b purification

• Autor(a) principal: Almeida, Paulo Ricardo Esteves
• Data de Publicação: 2023
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/14069
• topic: Afinidade; Dinâmica Molecular; Docking Molecular; Oligonucleótidos; Pre-Mirna-29b; Domínio/Área Científica::Engenharia e Tecnologia::Biotecnologia

Over the years, the interest in using nucleic acids as biopharmaceuticals to establish alternatives to traditional treatments for diseases such as cancer and neurodegenerative disorders has been increasing. The ability to regulate gene expression may lead to new forms of treatment for diseases with ineffective therapies, such as Alzheimer's disease. To further study these biomolecules and make them usable as biopharmaceuticals, they need to have a high degree of purity and preserved biological activity. Through biotechnology, the recombinant production of these biomolecules is rapid, cost-effective, and applicable on an industrial scale. However, subsequent steps related to separation and purification make the process costly. The main purification technique currently used in the biopharmaceutical industry is chromatography. But if a more specific strategy is expected, affinity chromatography must be considered, where the selection of more selective ligands is the greatest challenge. In this context, the present work aims to identify ligands for the purification of premiRNA-29b, which has potential as a biopharmaceutical for Alzheimer's disease treatment. This identification and selection of ligands was carried out through in silico methods, making the work less costly, time-saving, and environmentally friendly. Initially, 13 oligonucleotides (oligos) were designed to interact with the pre-miRNA-29b through base complementarity with one of three possible binding sites: the 5' end, the 3' end, or the hairpin structure. Each of these oligos has a carbon chain linked to the 5' end and an amino group to allow the immobilization of the ligand onto the chromatographic support. This chain can have either six or twelve carbons, resulting in two versions of each oligonucleotide, summing up to a total of 26 different ligands. After designing the oligo sequences and pre-miRNA-29b, they were submitted to a server for molecular docking analysis, and the results regarding the affinity energy and interaction capability were subsequently analyzed. Following the analysis of the docking score, the number of connections between the ligand and the target, and the site of interaction, 4 oligos were selected as the most promising. These ligands will help understand the influence of the carbon chain and the potential differences for the pre-miRNA-29b purification between versatile oligos, which can interact with more than one target site, and selective oligos, which only interact with the site they were designed for. The results indicated that approximately 80% of the interactions established between the ligand and pre-miRNA-29b are hydrogen bonds, which can provide some stability to the complex. To obtain more information about the stability of the interaction between the ligands and the receptor, molecular dynamics assays were performed, demonstrating that the formed complex is quite stable (RMSD < 1 Å). This way, it was possible to select the best ligands for the purification of pre-miRNA-29b from the initial group, as well as to confirm their stability with the target.