RESUMO
Aptamers are small-sized RNA or ssDNA ligands with a unique structure, which have high specificity and affinity to their cognate targets. Thus, in addition to the extensive values in various bio-medical fields, aptamers can also be alternatively used as affinity ligands in the bioprocess, such as for protein purification. In the present study, a hexahistidine specific aptamer named AptHis-C, was developed through the SELEX methodology, which has high affinity to hexahistidine, and its dissociation constant was as low as 20.8 nM. The structural prediction revealed that AptHis-C contains two connected stem-loop conformations. AptHis-C can only specifically recognize recombinant proteins with the hexahistidine-tag in simple or complex situations, and not to those with other tags. When immobilized on magnetic beads, AptHis-C can be used as a tool for hexahistidine-tagged recombinant protein purification. Its effectiveness is as good as traditional Ni-based beads. Besides, due to the intrinsic characteristics of nucleic acids, such as high thermal/chemical stability, immobilized aptamer-magnetic beads can be reused many times without an obvious decrease of purification effectiveness. This aptamer may represent a novel method for the detection and purification of hexahistidine-tagged recombinant proteins.
Assuntos
Aptâmeros de Nucleotídeos/química , DNA de Cadeia Simples/química , Histidina/química , Oligopeptídeos/química , Proteínas Recombinantes/isolamento & purificação , Antígeno B7-H1/genética , Antígeno B7-H1/isolamento & purificação , Cromatografia de Afinidade , Escherichia coli/química , Escherichia coli/genética , Fator 15 de Diferenciação de Crescimento/genética , Fator 15 de Diferenciação de Crescimento/isolamento & purificação , Imãs/química , Microesferas , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/isolamento & purificação , Proteínas Recombinantes/genética , Técnica de Seleção de Aptâmeros , Propriedades de SuperfícieRESUMO
Most antimicrobials treat wound infections by an oxidation effect, which is induced by the generation of reactive oxygen species (ROS). However, the potential harm of the prolonged high level of ROS should not be ignored. In this study, we presented a novel cascade-reaction nanoparticle, Ir@Cu/Zn-MOF, to effectively regulate the ROS level throughout the healing progress of the infected wound. The nanoparticles consisted of a copper/zinc-modified metal-organic framework (Cu/Zn-MOF) serving as the external structure and an inner core composed of Ir-PVP NPs, which were achieved through a process known as "bionic mineralization". The released Cu2+ and Zn2+ from the shell structure contributed to the production of ROS, which acted as antimicrobial agents during the initial stage. With the disintegration of the shell, the Ir-PVP NP core was gradually released, exhibiting the property of multiple antioxidant enzyme activities, thereby playing an important role in clearing excessive ROS and alleviating oxidative stress. In a full-layer infected rat wound model, Ir@Cu/Zn-MOF nanoparticles presented exciting performance in promoting wound healing by clearing the bacteria and accelerating neovascularization as well as collagen deposition. This study provided a promising alternative for the repair of infected wounds.
Assuntos
Cobre , Estruturas Metalorgânicas , Nanopartículas , Espécies Reativas de Oxigênio , Cicatrização , Zinco , Espécies Reativas de Oxigênio/metabolismo , Cicatrização/efeitos dos fármacos , Animais , Estruturas Metalorgânicas/química , Estruturas Metalorgânicas/farmacologia , Cobre/química , Cobre/farmacologia , Zinco/química , Nanopartículas/química , Nanopartículas/uso terapêutico , Ratos , Infecção dos Ferimentos/tratamento farmacológico , Infecção dos Ferimentos/microbiologia , Infecção dos Ferimentos/patologia , Infecção dos Ferimentos/metabolismo , Ratos Sprague-Dawley , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/uso terapêutico , Masculino , Staphylococcus aureus/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Antioxidantes/farmacologia , Antioxidantes/químicaRESUMO
Colorectal cancer (CRC) is a prevalent malignancy with poor prognosis and survival. As a Ca2+ binding protein, S100P plays a role in calcium-dependent signal transduction pathways that involve in diverse biological processes. Our previous studies have shown that S100P is overexpressed in CRC tissues and regulates cell growth, invasion, and metastasis in CRC. Therefore, S100P is expected to be an effective target for CRC therapy. Aptamers are short single-stranded oligonucleotides that could serve as specific and high-affinity probes to a wide range of target molecules for therapeutic purposes. In this study, we generated a novel DNA aptamer against S100P (AptS100P-1) by way of the SELEX process and high-throughput sequencing. The binding assay showed that AptS100P-1 had a high affinity for S100P protein. Further experiments indicated that AptS100P-1 is relatively stable in a cell culture system and could be used in flow cytometry analysis, dot blot assay, and fluorescence microscopy analysis to detect S100P. Moreover, AptS100P-1 was capable of binding to cells and had an inhibitory effect on CRC cell growth in vitro and in vivo. Also, AptS100P-1 inhibited the migration and epithelial-mesenchymal transition of CRC cells expressing S100P. These results indicate a novel DNA aptamer targeting S100P, which might be a potential therapeutic strategy for targeting S100P against S100P-expressing CRC.