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Metal-catalyzed uncaging of DNA-binding agents in living cells†Electronic supplementary information (ESI) available: Synthesis and characterization of the studied molecules and required precursors. NMR, UV, and fluorescence spectra, titrations, control experiments, and detailed procedures for cell uptake and co-staining experiments. See DOI: 10.1039/c3sc53317dClick here for additional data file.

ABSTRACT: Attachment of alloc protecting groups to the amidine units of fluorogenic DNA-binding bisbenzamidines or to the amino groups of ethidium bromide leads to a significant reduction of their DNA affinity. More importantly, the active DNA-binding species can be readily regenerated by treatment with ruthenium catalysts in aqueous conditions, even in cell cultures. The catalytic chemical uncaging can be easily monitored by fluorescence microscopy, because the protected products display both different emission properties and cell distribution to the parent compounds.

SUBMITTER: Sanchez MI

PROVIDER: S-EPMC4304260 | biostudies-other | 2014 May

REPOSITORIES: biostudies-other

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Metal-catalyzed uncaging of DNA-binding agents in living cells†Electronic supplementary information (ESI) available: Synthesis and characterization of the studied molecules and required precursors. NMR, UV, and fluorescence spectra, titrations, control experiments, and detailed procedures for cell uptake and co-staining experiments. See DOI: 10.1039/c3sc53317dClick here for additional data file.

Sánchez Mateo I MI Penas Cristina C Vázquez M Eugenio ME Mascareñas José L JL

Chemical science 20140227 5

Attachment of alloc protecting groups to the amidine units of fluorogenic DNA-binding bisbenzamidines or to the amino groups of ethidium bromide leads to a significant reduction of their DNA affinity. More importantly, the active DNA-binding species can be readily regenerated by treatment with ruthenium catalysts in aqueous conditions, even in cell cultures. The catalytic chemical uncaging can be easily monitored by fluorescence microscopy, because the protected products display both different e ...[more]

PMID: 25632343

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Project description:Thiol-ene 'click' chemistries have been widely used in biomaterials applications, including drug delivery, tissue engineering, and controlled cell culture, owing to their rapid, cytocompatible, and often orthogonal reactivity. In particular, hydrogel-based biomaterials formed by photoinitiated thiol-ene reactions afford spatiotemporal control over the biochemical and biomechanical properties of the network for creating synthetic materials that mimic the extracellular matrix or enable controlled drug release. However, the use of charged peptides functionalized with cysteines, which can form disulfides prior to reaction, and vinyl monomers that require multistep syntheses and contain ester bonds, may lead to undesired inhomogeneity or degradation under cell culture conditions. Here, we designed a thiol-ene hydrogel formed by the reaction of allyloxycarbonyl-functionalized peptides and thiol-functionalized poly(ethylene glycol). Hydrogels were polymerized by free radical initiation under cytocompatible doses of long wavelength ultraviolet light in the presence of water-soluble photoinitiators (lithium acylphosphinate, LAP, and 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methyl-1-propanone, Irgacure 2959). Mechanical properties of these hydrogels were controlled by varying the monomer concentration to mimic a range of soft tissue environments, and hydrogel stability in cell culture medium was observed over weeks. Patterns of biochemical cues were created within the hydrogels post-formation and confirmed through the incorporation of fluorescently-labeled peptides and Ellman's assay to detect free thiols. Human mesenchymal stem cells remained viable after encapsulation and subsequent photopatterning, demonstrating the utility of the monomers and hydrogels for three-dimensional cell culture. This facile approach enables the formation and characterization of hydrogels with well-defined, spatially-specific properties and expands the suite of monomers available for three-dimensional cell culture and other biological applications.

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