Sulfonated rhodamines as impermeable labelling substrates for cell surface protein visualization

Abstract

Sulfonated rhodamines that endow xanthene dyes with cellular impermeability are presented. We fuse charged sulfonates to red and far-red dyes to obtain Sulfo549 and Sulfo646, respectively, and further link these to SNAP- and Halo-tag substrates for protein self-labelling. Cellular impermeability is validated in live cell imaging experiments in transfected HEK cells and neurons derived from induced pluripotent stem cells (iPSCs). Lastly, we show that Sulfo646 is amenable to STED nanoscopy by recording membranes of SNAP/Halo-surface-labelled human iPSC-derived neuronal axons. We therefore provide an avenue for rendering dyes impermeable for exclusive extracellular visualization via self-labelling protein tags.

Structural and compositional diversity in the kainate receptor family

“…We fused GluK2 and GluK5 with an N-terminal Halo-tag ( Figure S5A ), which recognizes chloroalkane (CA)-conjugated fluorophores, and preserved normal receptor function ( Figure S5B ). To again limit labeling to surface receptors, we used modified sulfonated rhodamine fluorophores (“Sulfo549”), which are bright and membrane-impermeable ( Birke et al, 2021 ). We then co-expressed either HA-SNAP-GluK1 or HA-SNAP-GluK2 with Halo-GluK5 at either a 1:1 or 1:10 DNA ratio and labeled with SBG-OG and CA-Sulfo549 ( Figure S5E ).…”

Photoactivatable Fluorescent Dyes with Hydrophilic Caging Groups and Their Use in Multicolor Nanoscopy

“…Recently, sulfonated rhodamine- and silicon-rhodamine-derived probes, internalizing upon conjugation to their molecular targets, have been described as cell-membrane-impermeant fluorescent substrates for SNAP-tag- and HaloTag-fused cell surface proteins . Here, following an entirely different approach, we propose the introduction of polar sulfonate groups onto the lipophilic 2-nitrobenzyl carbamate protecting groups, rendering the caged dyes highly water-soluble and allowing photocontrol over their membrane permeability.…”

Reagents and models for detecting endogenous GLP1R and GIPR

“…A number of complementary fluorescent SNAP-tag and HaloTag labels exist, spanning most colors in the visible range, as well as different imaging modalities. Most recently, we have developed a number of SNAP-tag and HaloTag fluorescent labels, which allow surface GLP1R populations to be selectively visualized using conventionally cell permeable dyes [ 85 , 86 ]. Before this, surface labeling depended on the chemical properties of the dye itself, limiting the number of colors that could be used [86] .…”