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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 Kd 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 protein1-3 or nucleic acid4-6 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 vivo selection methods facilitate isolation of dye-binding proteins and nucleic acids. Fluoromodules are gaining increasing use as labels and sensors for bioimaging and detection.
Previously we isolated and characterized several human single-chain antibody (scFv)-based Fluorogen Activating Protein (FAP) reporters that generate fluorescence from the fluorogenic dyes thiazole orange (TO) and malachite green (MG).7 These FAPs, isolated from a yeast surface display library of human scFvs8, bind fluorogens with nanomolar affinity, increasing green or red fluorescence thousands-fold to brightness levels typical of fluorescent proteins. 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.
Dimethylindole 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 Kd = 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 DIR9 was used to enrich the complex yeast surface display scFv library composed of ca. 109 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 cytometer onto both selective growth plates (for storage and archiving) and onto plates containing galactose in the media to induce the display of scFvs on the surface of the cells. These induction plates also contained 10 μM DIR and after several days of growth, inspection of these plates under excitation illumination allowed for the direct visual selection of clones that showed the greatest amount of DIR fluorescence. Five of these clones were chosen for further analysis.
Equilibrium binding constants for DIR with the 5 surface-displayed scFvs were determined by fluorescence titration of the dye into yeast cultures expressing an scFv. Expression of the scFvs required galactose, so a negative control experiment involving yeast that were not exposed to galactose was performed in each case. Figure 1 shows the results of the titration for the scFv named K7. DIR fluorescence is enhanced by more than 100-fold and the titration data are well fit to a single site, 1:1 binding model that yields a Kd = 13.9 ± 3.2 nM. Negligible fluorescence enhancement was observed when DIR was added to the control cells or to buffer. Kd values for the other 4 clones are also shown in the inset to Figure 1; even the weakest binding clone, A8, exhibits a respectable Kd (148 nM).
The high affinities and large fluorescence enhancements exhibited by these scFvs for DIR are well suited for fluorescence microscopy. Figure 2 shows an image obtained after mixing 100 nM DIR with yeast expressing K7 on their surface. Note that because unbound dye has very low fluorescence, the cells need not be washed prior to imaging. Moreover, any dye that enters the yeast will be of similarly low fluorescence because of the weak affinity of DIR for cellular DNA and RNA. The fluorescence pattern in this confocal image indicates surface display of the DIR FAP K7 on virtually all of the cells.
To verify that the high affinity binding of DIR to K7 is not an artifact of the surface display of the protein, we next expressed a soluble version of K7 in E. coli. A fluorescence titration experiment with the soluble protein yielded Kd = 10.3 nM and confirmed the 1:1 stoichiometry of the complex (Figure S2).
The fluorescence quantum yield for DIR bound to the soluble K7 protein is 0.33, which is significantly higher than the quantum yield for the dye in a 90% glycerol solution (ϕf = 0.15 at 24°C) and in aqueous buffer (ϕf ≈ 0.002). Since the fluorescence enhancement of fluorogenic cyanines arises from conformational constraints, this result indicates a tight fit between the dye and its binding site on the protein and is consistent with the high affinity of the dye-protein complex. The quantum yield also compares favorably with that of the commonly used Cy5 (ϕf = 0.27).
We next explored the cross-reactivity between DIR and the structurally related TO1-2p for binding to their respective scFvs. Binding of DIR to surface-displayed HL1-TO1, an scFv previously selected for binding to TO1-2p7, was ca. 70-fold weaker than binding to K7 (Kd = 442 nM vs 13.9 nM). This is not surprising, given the larger size of the DIR chromophore relative to TO1. Interestingly however, TO1-2p actually bound 3-fold stronger to K7 than to HL1-TO1, its cognate scFv: Kd = 134 nM (K7) vs 360 nM (HL1-TO1). The presumably larger binding site in K7 appears to readily accommodate the smaller TO dye.
The cross-reactivity of K7 for TO1-2p motivated us to investigate a number of other unsymmetrical cyanines as binding partners for this protein. As shown in Figure 3, K7 exhibits considerable promiscuity, binding with low nanomolar Kds to dyes having methine bridge lengths ranging from 1-5 and with dimethylindole, benzothiazole or benzoxazole heterocycles. (Note that D1 is the trimethylammonium analogue of DIR). Affinity only falls off when the quinoline ring is truncated to a pyridine (compare YO-PRO-1 to PO-PRO-1). Figure 3 also shows that the fluorescence spectra for these dye-scFv fluoromodules cover most of the visible and near-IR region of the spectrum.
We next compared binding of DIR and TO1-2p to HL1.0.1-TO1, a TO1-2p-binding scFv that is a derivative of HL1-TO1 isolated for its improved binding of TO1.7 As reported previously, the Kd of TO1-2p for the improved scFv is 3 nM compared to 360 nM for the original scFv.7 In contrast, the affinity of this improved clone for DIR remains relatively low (Kd = 418 nM). Thus, isolation of improved binding scFv-based FAPs may improve selectivity as well. This suggests such derivatives of a promiscuous DIR-binding scFv may have improved selectivity for a particular dye, possibly generating a family of scFvs that selectively bind different colored fluorogens; this will be the subject of future investigations.
In conclusion, the new fluoromodules reported here add to the catalogue of fluorescence imaging tools, including inherently fluorescent proteins such as GFP11 as well as proteins labeled by exogenous dyes, as in the biarsenical FlAsH/ReAsH technology2 or the stilbene-binding antibodies1, but with added versatility. A single scFv protein “apomodule”, K7, provides access to emission wavelengths ranging from the blue (450 nm) to near IR (750 nm) using dyes with sub-micromolar Kd values. Future work will focus on structural and biophysical characterization of these fluoromodules.
DIR dyes were synthesized by G.L.S.; scFv selections were performed by C.L.P., S.A.M., and L.D.J.; H.O.-U., C.L.P. and NS performed spectroscopic experiments. We thank Yehuda Creeger for assistance with flow cytometry. This work was supported by the NIH (U54 RR022241) and by the Howard Hughes Medical Institute (HHMI Professorship to E.W.J. and Undergraduate Education Grant 52005865 to Carnegie Mellon University).
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.