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

Abstract

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.

Fluoromodules: Fluorescent Dye-Protein Complexes for Genetically Encodable Labels

“…Following work by Toutchkine and Hahn, 34,35 we designed a substituted version of the red fluorogen DIR in which an electron-withdrawing cyano group was attached to the methine carbon adjacent (i.e., alpha) to the dimethylindole heterocycle. 36 Although we have not verified this for DIR, it is known that cyanine photobleaching can occur at least partly through oxidative attack by singlet oxygen (generated by the fraction of the dye excited state that intersystem crosses to the triplet state). 35,37 The new fluorogen, α-CN-DIR, bound to the promiscuous FAP K7 (K D = 60 nM and ϕ f = 0.16), so new FAPs were not needed.…”

Breaking the color barrier – a multi-selective antibody reporter offers innovative strategies of fluorescence detection

“…Furthermore, in order to increasingly accelerate the off-rates of signal detection using multiselective FAPs, the future focus will be placed on isolating weakaffinity fluorogens. Here, two distinct engineering strategies may rapidly facilitate this: the site-directed mutagenesis of the protein scaffold or chemical alterations of fluorogen sub-groups (Rastede et al, 2015;Shank et al, 2009;Yates et al, 2013;Zanotti et al, 2011).…”

“…1B). On the other hand, when measuring the same fluorine-modified fluorogens using an activating medium, such as 90% glycerol, the fluorescence output of the fluorogens remained comparable (Shank et al, 2009). This shows that the fluorogenic modifications themselves are not intrinsically disruptive.…”

New Ideas for in Vivo Detection of RNA

“…Cyanine dyes appear p r o m i s i n g . I n a d d i t i o n t o D I R , a n u m b e r 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.…”