Combined magnetic and fluorescence cell sorting were used to select Fluorogen Activating Proteins (FAPs) from a yeast surface-displayed library for binding to the fluorogenic cyanine dye Dimethyl Indole Red (DIR). Several FAPs were selected that bind to the dye with low nanomolar K d values and enhance fluorescence more than 100-fold. One of these FAPs also exhibits considerable promiscuity, binding with high affinity to several other fluorogenic cyanine dyes with emission wavelengths covering most of the visible and near-IR regions of the spectrum. This significantly expands the number and wavelength range of scFv-based fluoromodules.Fluoromodules are specific combinations of fluorogenic dyes and cognate protein 1-3 or nucleic acid 4-6 partners. Separately, neither component is fluorescent, but when reconstituted, strong fluorescence is observed. Rational design and straightforward synthesis allow access to fluorogenic dyes spanning the visible and near IR wavelengths while powerful in vitro and in berget@cmu.edu. army@cmu.edu. † Present address: Department of Plant Biology, University of Georgia, Athens, GA 30602.Supporting Information Available: Binding titration curves for yeast-displayed K7 with various cyanine dyes and for soluble K7 with DIR; sequence information for scFvs; experimental details for scFv selection, fluorescence microscopy and binding titrations. These reagents have already been useful in visualizing cell surface elements and certain membrane proteins within the secretory apparatus of mammalian cells. 7 Some spectral variation was generated by combining a limited set of these scFvs and fluorogen derivatives. However, the spectral range of fluorescence emission is constrained by the chromophores of the fluorogenic dyes and the methods used to select the FAPs. NIH Public AccessDimethylindole Red (DIR, Chart 1) is a fluorogenic cyanine dye. It was designed to have low nonspecific binding to DNA and RNA by using the bulky dimethylindole heterocycle to suppress intercalation and the anionic propyl sulfonate group to introduce nonspecific electrostatic repulsions from nucleic acids. 9 An RNA aptamer that was selected for binding to DIR exhibited K d = 86 nM and enhanced the fluorescence of the dye ca. 60-fold. 9 Given our earlier success in selecting scFvs for binding to the related unsymmetrical cyanine TO1-2p (Chart 1), we next subjected DIR to the two-step scFv selection procedure.A biotin analogue of DIR 9 was used to enrich the complex yeast surface display scFv library composed of ca. 10 9 clones of synthetically recombined human heavy and light chain variable regions. 8 This enrichment was accomplished by two rounds of sequential selection using streptavidin magnetic microbeads followed by anti-biotin magnetic microbeads. 10 The resulting yeast sub-library was further enriched by 3 rounds of fluorescence activated cell sorting, gating the cell sorter for cells that directly activated DIR fluorescence. Individuals from these enriched populations were automatically cloned by the cyto...
Progress in fluorescence detection and imaging technologies depends on the availability of fluorescent labels with strong light absorption/emission characteristics. We have synthesized intercalator dye arrays on a compact 3-dimensional DNA-tetrahedron nanostructure. The template tolerates the structural distortions introduced by intercalation and allows concentration of multiple fluorophores within a small volume, resulting in brightly fluorescent nanotags with effective extinction coefficients in the order of 10(6) M(-1) cm(-1). Efficient energy transfer from intercalated donor dyes to covalently attached acceptor dyes in the nanotags allows the emission wavelength to be shifted to the red relative to the excitation light, providing wavelength tunability. The compact nature of the supramolecular DNA tetrahedron also provides a protective medium for the fluorophores, leading to improved photostability and enhanced resistance to nuclease digestion, relative to one- or two-dimensional nanotags described previously.
The use of chemical pesticides is limited by several public health concerns regarding their toxicity levels and indiscriminate use. Nevertheless, they are still vital components of agricultural industry since no other competitive equivalents to chemical pesticides still exist in terms of efficiency. This study describes the preparation and biological assessment of an insecticide releasing plastic film for agricultural covering purposes. The formulation was prepared by incorporation of deltamethrin loaded, nano-sized halloysite nanotubes into polymeric films. Thermal, morphological, and mechanical properties of films were characterized by Differential Scanning Calorimetry (DSC), Scanning Electron Microscopy (SEM) and Universal Testing Machine UTM. Sustained release profiles of the films were evaluated by Thermogravimetric Analysis (TGA). Results reveal that deltamethrin was successfully loaded into halloysite nanotubes and nanotube incorporation enhances the elastic modulus of linear-low density polyethylene (LLDPE) films. In addition, films exhibit controlled release function of the active agent for 32 days. Bioassays of the nanocomposite films with varying deltamethrin doses tested on grasshoppers showed that the LD50 values of the films are 1.85 10 !! g/cm 2 . Insecticidal activities of films were tested in greenhouse on Medicago Sativa plants contaminated with thrips and aphid. Nanocomposites are observed to repel mature aphids and kill young aphids and thrips.
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