RESUMO
Multivalent surface display of biomolecules is crucial to study and utilize multivalent biological interactions. However, precise valency control of surface-displayed ligands remains extremely difficult. Now a series of new oligomeric avidin proteins were fabricated that allow facile control of surface multivalency of biotinylated ligands. Naturally dimeric rhizavidin (RA) was engineered to form a mixture of oligomeric avidin assemblies, and discrete RA oligomers from the dimer to octamer of RA, were homogeneously prepared. These oligomeric avidins are in polygonal forms with expected numbers of stable biotin binding sites. Upon immobilization on low-density biotin-coated gold surfaces, RA dimer, trimer, and tetramer scaffolds provided accurate mean residual valencies of 2, 3, and 4, respectively, for biotinylated proteins. Valency-controlled display of antibody binding protein G on these RA surfaces showed clear valency-dependent enhancement of antibody capturing stability.
Assuntos
Proteínas de Bactérias/metabolismo , Ligantes , Polímeros/metabolismo , Animais , Proteínas de Bactérias/química , Biotina/química , Biotina/metabolismo , Biotinilação , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Camundongos , Polímeros/química , Ligação Proteica , Coelhos , Ressonância de Plasmônio de Superfície , Propriedades de SuperfícieRESUMO
Human ferritins are emerging platforms for non-toxic protein-based drug delivery, owing to their intrinsic or acquirable targeting abilities to cancer cells and hollow cage structures for drug loading. However, reliable strategies for high-level drug encapsulation within ferritin cavities and prompt cellular drug release are still lacking. Ferritin nanocages were developed with partially opened hydrophobic channels, which provide stable routes for spontaneous and highly accumulated loading of FeII -conjugated drugs as well as pH-responsive rapid drug release at endoplasmic pH. Multiple cancer-related compounds, such as doxorubicin, curcumin, and quercetin, were actively and heavily loaded onto the prepared nicked ferritin. Drugs on these minimally modified ferritins were effectively delivered inside cancer cells with high toxicity.
Assuntos
Antineoplásicos/administração & dosagem , Curcumina/administração & dosagem , Preparações de Ação Retardada/química , Doxorrubicina/administração & dosagem , Ferritinas/química , Quercetina/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacocinética , Curcumina/química , Curcumina/farmacocinética , Doxorrubicina/química , Doxorrubicina/farmacocinética , Liberação Controlada de Fármacos , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Células MCF-7 , Modelos Moleculares , Quercetina/química , Quercetina/farmacocinéticaRESUMO
Poor specificity has been a lingering problem in many microRNA profiling methods, particularly surface hybridization-based methods such as microarrays. Here, we carefully investigated surface hybridization and dissociation processes of a number of sequentially similar microRNAs against nucleic acid capture probes. Single-base mismatched microRNAs were similarly hybridized to a complementary DNA capture probe and thereby poorly discriminated during conventional stringent hybridization. Interestingly, however, mismatched microRNAs showed significantly faster dissociation from the probe than the perfectly matched microRNA. Systematic analysis of various washing conditions clearly demonstrated that extremely high specificity can be obtained by releasing non-specific microRNAs from assay surfaces during a stringent and controlled dissociation step. For instance, compared with stringent hybridization, surface dissociation control provided up to 6-fold better specificity for Let-7a detection than for other Let-7 family microRNAs. In addition, a synthetically introduced single-base mismatch on miR206 was almost completely discriminated by optimized surface dissociation of captured microRNAs, while this mismatch was barely distinguished from target miR206 during stringent hybridization. Furthermore, a single dissociation condition was successfully used to simultaneously measure four different microRNAs with extremely high specificity using melting temperature-equalized capture probes. The present study on selective dissociation of surface bound microRNAs can be easily applied to various hybridization based detection methods for improved specificity.
Assuntos
MicroRNAs/análise , Hibridização de Ácido Nucleico/métodos , Humanos , MicroRNAs/genética , Hibridização de Ácido Nucleico/genética , Propriedades de SuperfícieRESUMO
This article describes the preparation of GeTe-based alloy films using a solution-based technique. The dissolution behavior of GeTe was initially examined by comparing the weight loss of GeTe powder in different solvents, and it was found that, unlike in the cases of n-butylamine and NH4OH, KOH fully dissolved GeTe to form an agglomerate-free solution. X-ray diffraction analysis revealed that the reaction between GeTe and KOH resulted in the formation of rhombohedral GeTe, cubic GeTe4, and hexagonal Te structures after drying. GeTe-based alloy films were then prepared by the spin coating of the GeTe-containing solutions on a silicon substrate. The surface morphology and reflectance properties of the prepared films were found to be highly dependent on the spin speed, with optimization of the spin coating parameter resulting in the deposition of a continuous and smooth film.
RESUMO
Copper is indispensable in most aerobic organisms although it is toxic if unregulated as illustrated in many neurodegenerative diseases. To elucidate the mechanisms underlying copper release from cells, a membrane-targeting reporter which can compete with extracellular copper-binding molecules is highly desirable. However, engineering a reporter protein to provide both high sensitivity and selectivity for copper(ii) has been challenging, likely due to a lack of proper copper(ii)-chelating strategies within proteins. Here, we report a new genetically encoded fluorescent copper(ii) reporter by employing a copper-binding tripeptide derived from human serum albumin (HSA), which is one of the major copper-binding proteins in extracellular environments. Optimized insertion of the tripeptide into the green fluorescent protein leads to rapid fluorescence quenching (up to >85% change) upon copper-binding, while other metal ions have no effect. Furthermore, the high binding affinity of the reporter enables reliable copper detection even in the presence of competing biomolecules such as HSA and amyloid beta peptides. We also demonstrate that our reporter proteins can be used to visualize dynamic copper fluctuations on living HeLa cell surfaces.