Enhanced Photostability of Genetically Encodable Fluoromodules Based on Fluorogenic Cyanine Dyes and a Promiscuous Protein Partner

Department of Chemistry, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213, USA.
Journal of the American Chemical Society (Impact Factor: 12.11). 10/2009; 131(36):12960-9. DOI: 10.1021/ja9016864
Source: PubMed


Fluoromodules are discrete complexes of biomolecules and fluorogenic dyes. Binding of the dyes to their cognate biomolecule partners results in enhanced dye fluorescence. We exploited a previously reported promiscuous binding interaction between a single-chain, variable fragment antibody protein and a family of cyanine dyes to create new protein-dye fluoromodules that exhibit enhanced photostability while retaining high affinity protein-dye binding. Modifications to the dye structure included electron-withdrawing groups that provide resistance to photo-oxidative damage. Low nanomolar equilibrium dissociation constants were found for the new dyes. Fluorescence microscopy illustrates how yeast can be surface-labeled with three different colors based on a single protein and appropriately chosen dyes.

Download full-text


Available from: Peter Berget
  • Source
    • "Further efforts were made to select FAPs that can activate an oxazole thiazole blue derived blue-fluorescing cyanine dye (Zanotti et al., 2011). Moreover, DIR has been modified with an electron-withdrawing cyano group and showed significantly improved photostability while retaining a high binding affinity for the FAP (Shank et al., 2009). Saunders et al. took advantage of the fact that MG fluorogens emit around 670 nm where there is little cellular autofluorescence and developed a bifunctional fusion protein composed of a fluorescein binding and quenching scFv and a high-affinity MG-binding FAP (Saunders et al., 2014). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The pursuit of quantitative biological information via imaging requires robust labeling approaches that can be used in multiple applications and with a variety of detectable colors and properties. In addition to conventional fluorescent proteins, chemists and biologists have come together to provide a range of approaches that combine dye chemistry with the convenience of genetic targeting. This hybrid-tagging approach amalgamates the rational design of properties available through synthetic dye chemistry with the robust biological targeting available with genetic encoding. In this review, we discuss the current range of approaches that have been exploited for dye targeting or for targeting and activation and some of the recent applications that are uniquely permitted by these hybrid-tagging approaches.
    Full-text · Article · Mar 2015 · Cell and Tissue Research
  • Source
    • "Additionally, improved photostability may be attained via molecular alterations of the fluorogens. As previously shown, fluorogen chemical substitutions using electron withdrawing groups, such as cyano and fluoro, demonstrate improved stability against photo-induced damage, and also prolonged fluorescence detection as FAP complexes (Shank et al., 2009). Taken together, FAP technology as affinity reagents provides enhanced photostability, which is a favorable attribute for multiplicity of assays requiring longevity of signal. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Fluorogen-activating-proteins (FAPs) are a novel platform of fluorescence biosensors utilized for protein discovery. The technology currently demands molecular manipulation methods that limit its application and adaptability. Here, we highlight an alternative approach based on universal affinity reagents for protein detection. The affinity reagents were engineered as bi-partite fusion proteins, where the specificity moiety is derived from IgG-binding proteins -Protein-A or Protein-G - and the signaling element is a FAP. In this manner, primary antibodies provide the antigenic selectivity against a desired protein in biological samples, while FAP affinity reagents target the constant region (Fc) of antibodies and provide the biosensor component of detection. Fluorescence results using various techniques indicate minimal background and high target specificity for exogenous and endogenous proteins in mammalian cells. Additionally, FAP-based affinity reagents provide enhanced properties of detection previously absent using conventional affinity systems. Distinct features explored in this report include: (1) unfixed signal wavelengths (excitation and emission) determined by the particular fluorogen chosen, (2) real-time user controlled fluorescence on-set and off-set, (3) signal wavelength substitution while performing live analysis, and (4) enhanced resistance to photobleaching. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.
    Full-text · Article · Mar 2014 · Biotechnology and Bioengineering
  • Source
    • "Cyanine dyes appear promising. In addition to DIR, a number of cyanine dyes with reduced affinity to nonspecific nucleic acids and substantial light-up signal have been synthesized by Armitage's group and tuned to emit across the visible spectrum and even into the nearinfrared (Ozhalici-Unal et al., 2008; Shank et al., 2009). Second, a challenge for future work is to expand the available choices of light-up pairs by selection of new aptamers that specifically bind new improved fluorogenic dyes, including near-IR emitters. "
    [Show abstract] [Hide abstract]
    ABSTRACT: To date, remarkable progress has been made to visualize specific RNA molecules with high spatial and temporal resolution in living cells. These studies have begun to reveal detailed mechanisms of RNA trafficking, localization and turnover (Bratu et al., 2003, Lange et al., 2008; Zimyanin et al., 2008). However, significant challenges remain to render these methods more generally applicable and to overcome shortcomings of currently used methods. The ideal RNA biosensing system should be easy to deliver to living cells, nontoxic and non-perturbing to cellular functions. It should penetrate all parts of the cell homogeneously and be chemically stable in different cellular environments. The fluorescent
    Full-text · Chapter · Feb 2010
Show more