Percorrer por autor "Cruz, Carla Patrícia Alves Freire Madeira"
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- Highly selective capture of minicircle DNA biopharmaceuticals by a novel zinc-histidine peptide conjugatePublication . Gaspar, Vítor Manuel Abreu; Cruz, Carla Patrícia Alves Freire Madeira; Queiroz, João; Pichon, Chantal; Correia, Ilídio; Sousa, FaniThe use of minicircle DNA (mcDNA) biomolecules as a pharmaceutical product holds remarkable potential due to their improved therapeutic efficacy in comparison with standard non-viral gene expression vectors. However, mcDNA translation into clinical application is still highly restricted due to the lack of robust technologies for minicircles detection and purification. In this study, the potential of a zinc-binding histidine-based peptide to function as a novel ligand for mcDNA recovery was investigated by using high-throughput surface plasmon resonance (SPR) analysis. The histidine-based peptide successfully bound zinc cationic ions and had affinity towards mcDNA biomolecules as confirmed by the dynamic binding responses obtained in SPR experiments. Notably, the obtained results indicate that not only zinc-peptide ligands are able to bind mcDNA with very high affinity (KD = 4.21 × 10−10 M), but also that this interaction is mostly dependent on buffer type. In general, the findings indicated that Zn2+ bound peptide has high affinity to mcDNA in low ionic strength buffers, whereas with high salt buffers no binding is detected. Overall, the novel zinc-binding peptide has shown to have suitable properties for mcDNA binding and recovery under experimental conditions that assure genetic material stability. More importantly, the straightforward approach of employing simple biomimetic ligands for mcDNA capture will contribute for development of new technologies to purify DNA biopharmaceuticals.
- Sensitive Detection of Peptide–Minicircle DNA Interactions by Surface Plasmon ResonancePublication . Gaspar, Vítor Manuel Abreu; Cruz, Carla Patrícia Alves Freire Madeira; Queiroz, João; Pichon, Chantal; Correia, Ilídio; Sousa, FaniMinicircle DNA (mcDNA) is recently becoming an exciting source of genetic material for therapeutic purposes due to its exceptional biocompatibility and efficiency over typical DNA. However, its widespread use is yet restrained because of the absence of an efficient technology that allows its purification. Here, the precise conditions of mcDNA interaction with novel arginine-arginine dipeptide ligands were explored to promote binding and recovery of these biopharmaceuticals. Such interactions were investigated by taking advantage of a highly sensitive method based on surface plasmon resonance (SPR) to screen, in real-time, for ligand-coupled biomolecules, while preserving mcDNA integrity. Through this analytic approach, we detected dynamic binding responses that are dependent on buffer type, mcDNA electrokinetic potential, and temperature conditions. Remarkably, the results obtained revealed that the ligands possess high affinity to mcDNA molecules under low salt buffers, and low affinity in the presence of salt, suggesting that electrostatic interactions mainly govern ligand–analyte coupling. These findings provide important insights for an active manipulation of parameters that promote mcDNA recovery and purification. Above all, this study showed the crucial importance of SPR for future screening of other ligands that, like the one described herein, can be used to design mcDNA recovery platforms which will have significant impact in biopharmaceutical-based therapeutics.