Fluoromodules Consisting of a Promiscuous RNA Aptamer and Red or Blue Fluorogenic Cyanine Dyes: Selection, Characterization, and Bioimaging Xiaohong Tan, Tudor P. Constantin, Kelly L. Sloane, Alan S. Waggoner, Marcel P. Bruchez, and Bruce A. Armitage* Departments of Chemistry and Biological Sciences, Molecular Biosensor and Imaging Center, and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, Pennsylvania 15213-3890, United States * S Supporting Information ABSTRACT: An RNA aptamer selected for binding to the fluorogenic cyanine dye, dimethylindole red (DIR), also binds and activates another cyanine, oxazole thiazole blue (OTB), giving two well-resolved emission colors. The aptamer binds to each dye with submicromolar K D values, and the resulting fluoromodules exhibit fluorescence quantum yields ranging from 0.17 to 0.51 and excellent photostability. The aptamer was fused to a second aptamer previously selected for binding to the epidermal growth factor receptor (EGFR) to create a bifunctional aptamer that labels cell-surface EGFR on mammalian cells. The fluorescent color of the aptamer-labeled EGFR can be switched between blue and red in situ simply by exchanging the dye in the medium. The promiscuity of the aptamer can also be used to distinguish between cell-surface and internalized EGFR on the basis of the addition of red or blue fluorogen at different times. ■ INTRODUCTION Biological imaging and detection has been revolutionized by the availability of fluorescent modules, or fluoromodules, that can be used to label and track specific biomolecules such as proteins and RNA. Protein-based fluoromodules have made a bigger impact on the field, beginning with inherently fluorescent proteins such as green fluorescent protein (GFP), which can be genetically encoded as fusion constructs. 1,2 Semisynthetic fluoromodules based on chemical (e.g., FlAsH/ReAsH 3 ) or enzymatic incorporation of fluorescent dyes (e.g., SNAP 4 and HaloTag 5 technology) or noncovalent binding of fluorogenic dyes (e.g., TMP 6 and scFv 7 modules) are increasingly used with genetically encoded protein apomodules as the addition of an exogenous dye affords greater control over when the fluorescence appears during an experiment as well as versatility with respect to the actual color of the fluorescence. RNA-based fluoromodule development is less advanced than its protein-based counterpart. This is due to in part to the lack of an inherently fluorescent RNA module analogous to GFP, requiring all RNA fluoromodules to include an exogenous dye. RNA aptamers have been selected for binding numerous fluorescent or fluorogenic dyes, with the earliest example of an RNA fluoromodule consisting of an RNA aptamer that binds and activates fluorescence from malachite green (MG). 8 Later reports demonstrated fluorescence-activating aptamers for a variety of dyes, including cyanines 9,10 and Hoechst-like polyheterocycles. 11 The most significant advance in RNA fluoromodules was reported by Jaffrey and co-workers, who selected aptamers that bind to synthetic dyes modeled on the GFP chromophore, creating fluorescent RNA-dye complexes that mimic GFP spectroscopically, even though the chromo- phore is not covalently bound to the RNA. 12 The so-called Spinach fluoromodule and its improved versions have been used in a variety of bioimaging applications. 13-15 The work described herein concerns an RNA aptamer that binds to the fluorogenic cyanine dye, dimethylindole red (DIR). 9 In a separate project, a single-chain antibody fragment (scFv) that was selected for binding and activating fluorescence from DIR was found to be promiscuous, binding to several other cyanines to give fluorescent colors spanning the visible region of the spectrum. 16 The aptamer we report here is also moderately promiscuous, activating not only DIR to give red emission but also a blue-emitting fluorogen called oxazole thiazole blue (OTB) 17,18 . The aptamer was fused to a second aptamer that recognizes the epidermal growth factor receptor (EGFR) 19 at cell surfaces in order to create a modular imaging reagent. Moreover, the ability to label EGFR with either red (DIR) or blue (OTB) fluorogens allowed us to follow cell surface expression and internalization of the endogenous receptor, distinguishing between the two populations on the basis of which dye was present at different times during the experiment. Received: April 25, 2017 Article pubs.acs.org/JACS © XXXX American Chemical Society A DOI: 10.1021/jacs.7b04211 J. Am. Chem. Soc. XXXX, XXX, XXX-XXX