A Rainbow of Fluoromodules: A Promiscuous scFv Protein Binds to and Activates a Diverse Set of Fluorogenic Cyanine Dyes

Abstract

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...

Bichromophoric dyes for wavelength shifting of dye-protein fluoromodules

“…This complements prior work in which promiscuous FAPs could be used to generate a range of colors. 20, 23, 41 The modularity of the technology and tolerance of the FAPs to conjugating a second dye to the original fluorogen allows these new fluoromodules to be used without necessarily requiring new proteins to be selected, although subsequent affinity maturation might be useful for enhancing affinity. In principle, different fluorogenic donor-acceptor bichromophores can be created to provide a catalogue of colors that can be used for simultaneous tracking of multiple fusion proteins through multicolor imaging/detection or temporally resolved imaging/detection of a single fusion protein through labeling at different times with mono- or bichromophoric reagents.…”

“…Expression of the soluble R1 protein in the Mach1-T1 strain was accomplished as described previously. 20 HL1.0.1-TO1 protein was secreted from yeast as described. 18 …”

“…FAPs able to enhance the fluorescence of our dyes by up to 4 orders of magnitude and binders with K D values in the low nanomolar to high picomolar range have been isolated. 18, 20 …”

“…18, 20–23 These fluorophores typically exhibit relatively small Stokes shifts, meaning that their fluorescence emission spectra are relatively close to the wavelength of light used to excite the dye. This can lead to considerable background due to cellular autofluorescence and light scattering, diminishing the quality of a microscope image.…”

Local retention of antibodies in vivo with an injectable film embedded with a fluorogen-activating protein

“…Fluorescence generated from dL5-MG can be detected optically in superficial tissues, but other FAP systems, such as far-red fluorogenic dyes currently being explored, may be used for internal imaging [62]. There are also promiscuous FAPs that can bind more than one fluorogen [63], with alternate excitation and emission wavelengths and varying affinity constants for fluorogen binding. There may be biological scenarios in which higher or lower binding affinities are called for, such as using a low affinity FAP-fluorogen combination as a more rapid but controlled release mechanism, through disassociation of the pAG-dye conjugate from the FAP itself.…”