RESUMEN
Nucleotide excision repair (NER) removes a wide range of DNA lesions, including UV-induced photoproducts and bulky base adducts. XPA is an essential protein in eukaryotic NER, although reports about its stoichiometry and role in damage recognition are controversial. Here, by PeakForce Tapping atomic force microscopy, we show that human XPA binds and bends DNA by â¼60° as a monomer. Furthermore, we observe XPA specificity for the helix-distorting base adduct N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene over non-damaged dsDNA. Moreover, single molecule fluorescence microscopy reveals that DNA-bound XPA exhibits multiple modes of linear diffusion between paused phases. The presence of DNA damage increases the frequency of pausing. Truncated XPA, lacking the intrinsically disordered N- and C-termini, loses specificity for DNA lesions and shows less pausing on damaged DNA. Our data are consistent with a working model in which monomeric XPA bends DNA, displays episodic phases of linear diffusion along DNA, and pauses in response to DNA damage.
Asunto(s)
ADN/química , ADN/metabolismo , Imagen Individual de Molécula/métodos , Proteína de la Xerodermia Pigmentosa del Grupo A/química , Proteína de la Xerodermia Pigmentosa del Grupo A/metabolismo , Biofisica/métodos , Aductos de ADN/química , Aductos de ADN/metabolismo , Daño del ADN/fisiología , Reparación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Humanos , Microscopía de Fuerza Atómica , Unión Proteica , Rayos UltravioletaRESUMEN
Effective and controlled drug delivery systems with on-demand release and targeting abilities have received enormous attention for biomedical applications. Here, we describe a novel enzyme-based cap system for mesoporous silica nanoparticles (MSNs) that is directly combined with a targeting ligand via bio-orthogonal click chemistry. The capping system is based on the pH-responsive binding of an aryl-sulfonamide-functionalized MSN and the enzyme carbonic anhydrase (CA). An unnatural amino acid (UAA) containing a norbornene moiety was genetically incorporated into CA. This UAA allowed for the site-specific bio-orthogonal attachment of even very sensitive targeting ligands such as folic acid and anandamide. This leads to specific receptor-mediated cell and stem cell uptake. We demonstrate the successful delivery and release of the chemotherapeutic agent Actinomycin D to KB cells. This novel nanocarrier concept provides a promising platform for the development of precisely controllable and highly modular theranostic systems.
Asunto(s)
Sistemas de Liberación de Medicamentos , Nanopartículas , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Transporte Biológico Activo , Anhidrasa Carbónica II/química , Anhidrasa Carbónica II/genética , Anhidrasa Carbónica II/metabolismo , Línea Celular , Dactinomicina/administración & dosificación , Dactinomicina/farmacocinética , Preparaciones de Acción Retardada , Liberación de Fármacos , Células HeLa , Humanos , Células KB , Ratones , Nanopartículas/química , Ingeniería de Proteínas , Receptores de Droga/química , Receptores de Droga/genética , Receptores de Droga/metabolismo , Dióxido de SilicioRESUMEN
We here report the construction of an E. coli expression system able to manufacture an unnatural amino acid by artificial biosynthesis. This can be orchestrated with incorporation into protein by amber stop codon suppression inside a living cell. In our case an alkyne-bearing pyrrolysine amino acid was biosynthesized and incorporated site-specifically allowing orthogonal double protein labeling.
