title: Selective recognition of protein tetraserine motifs with a cell-permeable, pro-fluorescent bis-boronic acid.
abstract: There is considerable interest in novel cell imaging tools that avoid the use of fluorescent proteins. One widely used class of such reagents are "pro-fluorescent" biarsenical dyes such as FlAsH, ReAsH, CrAsH, and Cy3As. Despite their utility, biarsenicals are plagued by high background labeling and cytotoxicity and are challenging to apply in oxidizing cellular locale. Here we demonstrate that [(3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthene]-3',6'-diyl)bis(iminomethylene-2,1-phenylene)]bis-(9CI), a rhodamine-derived bisboronic acid (RhoBo) described initially as a monosaccharide sensor, functions as a cell-permeable, turn-on fluorescent sensor for tetraserine motifs in recombinant proteins. RhoBo binds peptides or proteins containing Ser-Ser-Pro-Gly-Ser-Ser with affinities in the nanomolar concentration range and prefers this sequence to simple monosaccharides by >10,000-fold. RhoBo fails to form fluorescent complexes with constituents of the mammalian cell surface, as judged by epifluorescent, confocal, and TIRF microscopy, but fluoresces brightly within the Ser-Ser-Pro-Gly-Ser-Ser-rich cell interior. These results suggest that current efforts to identify optimal serine-rich sequences for RhoBo will allow it to function effectively as a selective small-molecule label for appropriately tagged proteins either upon or within living cells.
authors.affiliation: Department of Chemistry, Yale University, New Haven, Connecticut 06520, USA.
authors.forename: Tiffany L
authors.initials: TL
authors.lastname: Halo
authors.forename: Jacob
authors.initials: J
authors.lastname: Appelbaum
authors.forename: Elissa M
authors.initials: EM
authors.lastname: Hobert
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Balkin
authors.forename: Alanna
authors.initials: A
authors.lastname: Schepartz
mesh_terms: D020816:Amino Acid Motifs; D001897:Boronic Acids / Q000737:chemistry*; D002463:Cell Membrane Permeability; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006601:Hexoses / Q000737:chemistry; D006801:Humans; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D012235:Rhodamines / Q000737:chemistry*; D014966:Xanthenes / Q000737:chemistry
chemical_list: D001897:Boronic Acids; D005456:Fluorescent Dyes; D006601:Hexoses; D010455:Peptides; D012235:Rhodamines; D014966:Xanthenes; C027794:polyserine; D019158:N-Acetylneuraminic Acid