RESUMO
The widely used green fluorescent protein (GFP) decarboxylates upon irradiation; this involves removal of the acidic function of the glutamic acid at position 222, thereby resulting in the irreversible photoconversion of GFP. To suppress this phenomenon, the photostable, non-photoconvertible histidine was introduced at position 222 in GFP. The variant E222H shows negligible photodynamic processes and high expression yield. In addition, the stable and bright fluorescence over a wide pH range makes the E222H protein an alternative for GFP in fluorescence imaging and spectroscopy. Other fluorescent proteins are predicted to benefit from replacement of the catalytic glutamic acid by histidine.
Assuntos
Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Substâncias Luminescentes/química , Cristalografia por Raios X , Escherichia coli/genética , Histidina/química , Histidina/genética , Isomerismo , Substâncias Luminescentes/metabolismo , Modelos Moleculares , Fotólise , Mutação Puntual , Engenharia de Proteínas , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometria de FluorescênciaRESUMO
Cationic polyrotaxanes, obtained by temperature activated threading of cationic cyclodextrin derivatives onto water-soluble cationic polymers (ionenes), form metastable nanometric polyplexes with pDNA and combinations of siRNA with pDNA. Because of their low toxicity, the polyrotaxane polyplexes constitute a very interesting system for the transfection of polynucleotides into mammalian cells. The complexation of Cy3-labeled siRNA within the polyplexes was demonstrated by fluorescence correlation spectroscopy. The uptake of the polyplexes (red) was imaged by confocal fluorescence microscopy using the A549 cell line as a model (blue: nuclei, green: membranes). The results prove the potential of polyrotaxanes for further investigations involving knocking down genes of therapeutic interest.
Assuntos
Ciclodextrinas/química , Portadores de Fármacos/química , Polinucleotídeos/administração & dosagem , Rotaxanos/química , Cátions , Linhagem Celular , Ciclodextrinas/síntese química , DNA/administração & dosagem , DNA/genética , Portadores de Fármacos/síntese química , Endocitose , Inativação Gênica , Humanos , Luciferases/genética , Espectroscopia de Ressonância Magnética , Microscopia Confocal , Microscopia de Fluorescência , Tamanho da Partícula , Plasmídeos/administração & dosagem , Plasmídeos/genética , Polinucleotídeos/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Rotaxanos/síntese química , Espectrometria de FluorescênciaRESUMO
Gold nanoclusters prepared with a controlled amount of Ag exhibit intense fluorescence with a quantum yield of ~16% and a "quasi-monoexponential" long lifetime of >200 ns. Characterization of the luminescent probes indicates high photostability and easy detection in cells. Additionally, fluorescence enhancement in the presence of proteins was found.